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*Substance via MeSH
Medline Plus Health Information
*Heart Transplantation
*Organ Donation
The Journal of Immunology, 2005, 175: 3409-3416.
Copyright © 2005 by The American Association of Immunologists

Donor Lymphocyte Infusion Induces Long-Term Donor-Specific Cardiac Xenograft Survival through Activation of Recipient Double-Negative Regulatory T Cells1

Wenhao Chen*, Dejun Zhou*, Jose R. Torrealba*, Thomas K. Waddell*, David Grant* and Li Zhang2,*,{dagger}

* Department of Laboratory Medicine and Pathobiology, Multi Organ Transplantation Program, Toronto General Research Institute, University Health Network, and {dagger} Department of Immunology, University of Toronto, Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Previous studies have shown that pretransplant donor lymphocyte infusion (DLI) can enhance xenograft survival. However, the mechanism by which DLI induces xenograft survival remains obscure. Using T cell subset-deficient mice as recipients we show that CD4+, but not CD8+, T cells are necessary to mediate the rejection of concordant cardiac xenografts. Adoptive transfer of naive CD4+ T cells induces rejection of accepted cardiac xenografts in CD4–/– mice. This rejection can be prevented by pretransplant DLI in the absence of any other treatment. Furthermore, we demonstrate that DLI activates {alpha}{beta}-TCR+CD3+CD4–CD8– double-negative (DN) regulatory T (Treg) cells in xenograft recipients, and that DLI-activated DN Treg cells can inhibit the proliferation of donor-specific xenoreactive CD4+ T cells in vitro. More importantly, adoptive transfer of DLI-activated DN Treg cells from xenograft recipients can suppress the proliferation of xenoreactive CD4+ T cells and their ability to produce IL-2 and IFN-{gamma} in vivo. Adoptive transfer of DLI-activated DN Treg cells also prevents CD4+ T cell-mediated cardiac xenograft rejection in an Ag-specific fashion. These data provide direct evidence that DLI can activate recipient DN Treg cells, which can induce donor-specific long-term cardiac xenograft survival by suppressing the proliferation and function of donor-specific CD4+ T cells in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although xenotransplantation currently faces compounding biological and immunological hurdles, it still remains as one of the most attractive approaches to overcome the severe shortage of organ donors (1, 2). Recently, significant progress has been made to overcome or reduce Ab-mediated hyperacute and acute humoral xenograft rejection. Strategies such as immunoadsorption (3), complement inhibition (4), and the use of organs from donors that are transgenic for human complement regulatory proteins (5, 6) seem effective. Pigs lacking the expression of galactosyl-{alpha}1,3-galactose epitope (7, 8), which is the target for natural Abs present in humans and Old World primates, have been successfully generated by knockout of the {alpha}1,3-galactosyltransferase gene (9). Survival of heart and kidney grafts derived from {alpha}1,3-galactosyltransferase knockout pigs was shown to exceed 80 days in baboons in the presence of immunosuppressive regimens (7, 8). Whereas the success in overcoming the early stages of xenograft rejection is remarkable, there is a need for a better understanding of the cell-mediated immune responses to xenoantigens in vivo to achieve long-term xenograft survival. Induction of specific immunologic unresponsiveness, or tolerance, of xenoreactive T cells toward donor organs is an ideal approach to successful xenotransplantation (2, 10, 11). Recent studies have shown that T cell tolerance to xenoantigens can be induced through mixed hemopoietic chimerism (12), as has been demonstrated in allogeneic transplantation models (13). Whether long-term tolerance to xenografts can be induced in the periphery of recipients requires further study.

Pretransplant donor lymphocyte infusion (DLI),3 either alone or in combination with other treatments, has been shown to enhance donor-specific graft survival in various allogeneic transplantation models including rodents, primates and humans (14, 15, 16, 17, 18, 19). It has been shown that DLI is also able to prolong skin and islet xenograft survival when combined with other treatments such as anti-CD4 and anti-CD40L (20, 21). Various mechanisms, including deletion of donor-reactive T cells (22, 23), induction of clonal anergy (24, 25), and suppression by regulatory T (Treg) cells (19, 26, 27), have been shown to be involved in DLI-induced tolerance to allografts. Previously we have shown in single MHC class I locus mismatched models that pretransplant DLI can lead to permanent acceptance of donor-specific skin allograft survival (27, 29). We have identified and cloned {alpha}{beta}-TCR+CD3+CD4–CD8–NK1.1– (defined as double negative, DN) T cells from mice that permanently accepted skin allografts after DLI (27, 28, 29). These DN T cells are able to specifically suppress and kill syngeneic CD8+ alloreactive T cells in vitro and prevent allograft rejection when adoptively transferred into naive syngeneic recipients (27, 28). Similar to what has been found in murine models, human DN T cells can also kill syngeneic CD8+ T cells that are activated by the same Ags as those used to activate DN T cells (30). These studies demonstrated that both mouse and human DN T cells can function as regulatory cells to down-regulate immune responses.

Treg cells, including CD4+CD25+ (31, 32, 33), CD4+CD25– (34), CD8+ (35), DN (27), and NK (36) T cells, have been reported to play a role in the induction/maintenance of tolerance to allografts. However, the role of Treg cells in down-regulating xenoreactive T cell responses is largely unknown. We have recently demonstrated that a combination of pretransplant DLI and a short course of depleting anti-CD4 mAb can induce permanent concordant cardiac xenograft survival, whereas injection of anti-CD4 mAb alone failed to do so (37). The mechanism by which DLI induces long-term xenograft survival was not clear. We found that the DN T cells isolated from the spleens of DLI/anti-CD4-treated xenografted mice can suppress the proliferation of xenoreactive T cells in vitro (37). This finding suggests, but does not directly prove, that DN T cells are responsible for the induction and maintenance of long-term cardiac xenograft survival in this model. Furthermore, all the previous studies on the mechanism of DN Treg cell-mediated suppression used in vitro cell culture systems. In this study, we investigated the mechanisms of DN Treg cell-mediated suppression in vivo. We also studied whether adoptive transfer of DLI-activated DN Treg cells was sufficient to induce long-term xenograft survival. Our results indicate that CD4+ T cells play an essential role in rejecting concordant cardiac xenografts. This process can be prevented by pretransplant DLI, which activates recipient DN Treg cells. Furthermore, DLI-activated DN Treg cells can specifically inhibit the in vivo proliferation of xenoreactive CD4+ T cells and their cytokine production, thus preventing xenograft rejection in a donor-specific manner. These findings provide the first direct in vivo evidence that DN Treg cells are able to prevent concordant xenograft rejection, and suggest the potential of using DN Treg cells as a novel cellular therapy to induce long-term xenograft survival.


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

C57BL/6 (B6), B6.129S2-Cd4tm1Mak/J (B6 CD4–/–), and B6.129S2-Cd8atm1Mak/J (B6 CD8–/–) mice were purchased from The Jackson Laboratory. The B6 CD4–/– mice harbor a null mutation of the CD4 gene and have no mature CD4+ T cells. The development of CD8+ T cells, B lymphocytes, and myeloid components is unaltered in these mice (38). Inbred Lewis and Wistar-Furth (WF) rats were obtained from Harlan Sprague Dawley and used as heart donors between 11 and 13 days of age or for in vitro studies between 6 and 8 wk of age. All the animals were kept in specific pathogen-free conditions at the University Health Network animal facility (Toronto, Ontario, Canada). Mice were used according to the institutional guidelines.

DLI and cardiac transplantation

Lymphocytes collected from the spleens of Lewis rats were i.v. injected (4 x 107 cells/mouse) into B6 CD4–/– recipients 2 days before transplantation. Heterotopic cardiac transplantation was performed using the techniques previously described (37). The survival of the graft was monitored daily by palpation. Graft rejection was confirmed histologically.

Isolation and adoptive transfer of CD4+ effector or DN Treg cells

CD8+ T cells or CD8+ together with CD4+ T cells were in vivo depleted in naive or treated mice by i.p. injection of 400 µg/mouse of depleting anti-CD8 mAb (YTS169) (39) either alone or in combination with 400 µg/mouse of depleting anti-CD4 mAb (YTS191.1) (40). One day later, spleen cells were obtained from these mice and passed through a nylon wool column to enrich the T cell population. To purify CD4+ T cells, the non-nylon wool-binding cells that had been depleted of CD8+ T cells in vivo were further stained with biotinylated anti-CD4 mAb (BD Pharmingen), and incubated with streptavidin-conjugated magnetic MicroBeads (Miltenyi Biotec). The CD4+ T cells were then isolated by passage through a LS magnetic column (Miltenyi Biotec). To purify DN T cells, the non-nylon wool-binding cells that were in vivo depleted of both CD4+ and CD8+ T cells were stained with biotinylated anti-CD3 mAb (BD Pharmingen), labeled with streptavidin-conjugated magnetic MicroBeads, and purified on a LS column. The cell purity following these procedures was consistently (95–99%), as determined by flow cytometric analysis.

Thirty days after receiving Lewis or WF heart grafts, B6 CD4–/– recipient mice were infused with 2.5 x 106 CD4+ T cells purified from naive B6 mice, either alone or together with the same number of purified DN T cells isolated from Lewis DLI-treated and Lewis heart-grafted B6 CD4–/– recipient mice.

Cell surface marker staining and intracellular cytokine staining

Single-cell suspensions of splenocytes collected from recipient mice were triple stained with FITC-conjugated anti-CD3 mAb, PE-conjugated anti-CD4 mAb, or CyChrome-conjugated anti-CD8 mAb (BD Pharmingen). For intracellular staining of IL-2 or IFN-{gamma}, lymphocytes were stimulated for 5 h with 20 ng/ml PMA (Sigma-Aldrich) and 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of GolgiStop (BD Pharmingen). Cells were fixed and permeabilized by incubation with Cytofix/Cytoperm solution (BD Pharmingen), and then intracellularly stained with PE-conjugated anti-IL-2 or anti-IFN-{gamma} mAb (BD Pharmingen) for 30 min. Data were acquired and analyzed on an EPICS XL-MCL flow cytometer (Beckman Coulter).

CFSE labeling and in vitro suppression of CD4+ T cells

Splenic CD4+ T cells from naive B6 mice were suspended in PBS containing 1 µM CFSE (Molecular Probes) and incubated for 10 min at 37°C. CFSE-labeled CD4+ T cells were then cocultured in 24-well plates (1 x 105 cells/well) with irradiated splenocytes (3 x 106 cells/well) from Lewis or WF rats in the presence of 50 U/ml rIL-2, and 30 U/ml rIL-4 in {alpha}-MEM supplemented with 10% FCS and 0.1% 2-ME. Splenic DN T cells (1 x 105) isolated from Lewis DLI-treated and Lewis heart-grafted B6 CD4–/– recipient mice were added into each well as putative suppressor cells. After 4 days incubation, cells were collected and analyzed by flow cytometry. The in vitro proliferation of CD4+ T cells in the presence or absence of DN T cells was measured by calculating the percentage of divided cells in CD4+ T cell population.

In vivo suppression of CD4+ T cells

Thirty days after transplantation of Lewis hearts, B6 CD4–/– mice were i.v. infused with 2.5 x 106 CFSE-labeled naive B6 CD4+ T cells either alone or together with the same number of DN T cells from Lewis DLI-treated and Lewis heart-grafted B6 CD4–/– recipient mice. Nine days after adoptive transfer, splenocytes were collected and stained with PE-conjugated anti-CD4 mAb followed by flow cytometry analysis. The percentage of divided CD4+ cells and their responder frequency among CFSE-labeled CD4+ T cells in each group were calculated using methods previously described (41).

Histopathology

Cardiac xenografts from B6 CD4–/– mice were harvested and fixed in 10% buffered formalin. Tissues were then dehydrated, embedded in paraffin, and 6- to 7-micrometer sections were obtained from each paraffin block. Sections were stained with H&E and examined under light microscopy by pathologist J. R. Torrealba (University of Toronto, Toronto, Ontario, Canada) in a blinded fashion. The Heart Rejection Study Group criterion for grading of cardiac allograft rejection (42) was used. Briefly, the system identifies each category as: no rejection (grade 0), focal (grade 1A), or diffuse (grade 1B) interstitial mononuclear infiltrates without myocyte necrosis, focal aggressive cellular rejection with myocyte damage (grade 2), multifocal aggressive infiltrates with myocyte damage (grade 3A), diffuse inflammatory process with necrosis (grade 3B), and diffuse aggressive polymorphous/mononuclear infiltrate with edema, hemorrhage, vasculitis, and necrosis (grade 4).

Statistical analysis

The difference in graft survival between groups was determined by the Mann-Whitney U test. All other statistical analyses were performed using the unpaired Student’s t test. Values for p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD4+ T cells play an essential role in rejecting concordant cardiac xenografts

A robust T cell response is one of the major barriers to xenotransplantation (43). To develop a model in which T cell responses to xenografts can be monitored in vivo, we first determined which subset of T cells is critical for rejection of concordant xenografts. Baby Lewis rat hearts were transplanted into B6 mice that are deficient for either CD4+ or CD8+ T cells, and the graft survival was compared with Lewis hearts transplanted into wild-type B6 mice. As shown in Fig. 1A, B6 CD8–/– mice were able to reject concordant cardiac xenografts in a manner equivalent to wild-type mice. In contrast, Lewis cardiac grafts were permanently accepted by all B6 CD4–/– mice. To further confirm the role of CD4+ T cells in concordant cardiac xenograft rejection, we adoptively transferred varying numbers of CD4+ spleen cells from naive B6 mice into B6 CD4–/– mice that had accepted Lewis heart grafts transplanted 30 days previously. We found that infusing as few as 2.5 x 106 naive B6 CD4+ T cells/mouse was sufficient to induce concordant cardiac xenograft rejection in all B6 CD4–/– mice (Fig. 1B). These results clearly demonstrate that CD4+ T cells are necessary to mediate the rejection of Lewis cardiac xenografts in mice. Conversely, in the absence of CD4+ T cells, CD8+ T cells are not sufficient to initiate concordant cardiac xenograft rejection.



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  		FIGURE 1. CD4+, but not CD8+, T cells are critical for mediating concordant cardiac xenograft rejection. A, B6 (•; n = 6), B6 CD8–/– ({diamondsuit}; n = 4), and B6 CD4–/– ({blacksquare}; n = 10) mice were heterotopically transplanted with baby Lewis heart grafts. Graft survival was monitored by daily palpation. B, B6 CD4–/– mice were transplanted with baby Lewis heart grafts. The recipients received either no further treatment ({blacksquare}; n = 10) or were adoptively transferred with 10 x 106 ({blacktriangledown} n = 3), 2.5 x 106 (•; n = 6), or 1 x 106 ({diamondsuit}; n = 4) naive B6 splenic CD4+ T cells/mouse at 30 days after transplantation as indicated. Graft survival after CD4+ T cell transfer was monitored.

 
DLI prevents CD4+ T cell-mediated xenograft rejection in B6 CD4–/– mice

The above experiments indicate the critical role of CD4+ T cells in inducing cardiac xenograft rejection. We next focused our study on the effect of DLI on preventing CD4+ T cell-mediated cardiac xenograft rejection. B6 CD4–/– recipients were infused with Lewis splenocytes (DLI) or left untreated. Two days later, all mice were transplanted with Lewis heart grafts. Thirty days after transplantation, when all grafts were accepted by the recipients, each mouse was adoptively transferred with 2.5 x 106 naive syngeneic CD4+ T cells to initiate graft rejection. As shown in Fig. 2, the mean graft survival time (MST) after cell transfer was significantly increased in DLI-treated mice (MST = 87 days post cell transfer) compare with that of non-DLI treated mice (MST = 23 days post cell transfer, p < 0.01). In fact, four of six DLI-treated mice achieved long-term (>100 days) survival of Lewis heart grafts, whereas all six mice that received CD4+ T cells but not pretransplant DLI rejected their grafts between 9 and 46 days. These results indicate that a single DLI is sufficient to prevent CD4+ T cell-mediated rejection of concordant cardiac xenografts.



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  		FIGURE 2. Pretransplant DLI prevents CD4+ T cell-mediated Lewis heart graft rejection in B6 CD4–/– mice. B6 CD4–/– mice were given DLI and transplanted with Lewis heart grafts 2 days later. At 30 days after transplantation, each mouse was infused with 2.5 x 106 naive B6 CD4+ T cells (DLI + CD4+ cells, {diamondsuit}; n = 6). As a control, another group of mice were transplanted and were infused with CD4+ T cells without pretransplant DLI (CD4+ cells, •; n = 6). Graft survival was significantly (p < 0.01) enhanced in DLI-treated recipients when compared with those that received CD4+ T cells alone.

 
DLI increases the number and suppressive ability of DN Treg cells in xenograft recipients

We have demonstrated in allogeneic skin transplantation models that pretransplant DLI leads to activation of recipient DN Treg cells (27, 29). To determine whether DLI promotes xenograft survival through activation of DN Treg cells, the number of DN T cells in the spleens and lymph nodes of DLI-treated xenograft recipients as well as their ability to suppress xenoreactive CD4+ T cells was compared with non-DLI-treated control xenograft recipients. B6 CD4–/– mice that were given DLI 2 days before Lewis heart transplantation had a significantly higher percentage of DN T cells in their spleens and lymph nodes compared with that of non-DLI-treated B6 CD4–/– xenograft recipients (Fig. 3A, left). Likewise, the total number of DN T cells was also significantly increased in the spleens of DLI-treated xenograft recipients when compared with that of non-DLI-treated mice (Fig. 3A, right). To further determine whether the DN T cells in either DLI- or non-DLI-treated recipients have a differential ability to suppress CD4+ T cells, and whether this suppression occurs in an Ag-specific manner or not, DN T cells were isolated from the spleens of Lewis DLI-treated, as well as from non-DLI-treated Lewis heart graft recipients and used as putative regulatory cells. As shown in Fig. 3B, DN T cells isolated from Lewis DLI-treated, Lewis heart graft recipient mice are able to suppress the in vitro proliferation of CFSE-labeled naive CD4+ T cells upon stimulation with donor-specific (Lewis), but not with third-party (WF) stimulator cells (Fig. 3B). However, an equal number of DN T cells from non-DLI-treated xenograft recipients showed minimal suppression of the in vitro proliferation of xenoreactive CD4+ T cells when stimulated by either donor-specific (Lewis) or third-party (WF) stimulator cells (Fig. 3B). Together, these results demonstrate that DLI increases both the number and the regulatory function of DN Treg cells in xenograft recipients. The data also suggest that the DLI-activated DN Treg cells can suppress the in vitro proliferation of xenoreactive CD4+ T cells in an Ag-specific manner.



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  		FIGURE 3. DLI increases the number and suppressive ability of DN Treg cells in xenograft recipients. A, Increase in percentage and total number of peripheral DN T cells in DLI-treated B6 CD4–/– xenograft recipients. Cells were collected from spleens and lymph nodes of non-DLI-treated ({square}) and DLI-treated ({cjs2108}) B6 CD4–/– recipients at 30 days after Lewis heart transplantation. The cells were triple stained with CD3-FITC, CD4-PE, and CD8-CyChrome to calculate the percentage of CD3+CD4–CD8– (DN) T cells (left). The total number of DN T cells (right) was determined by multiplying the percentage of DN T cell by the total number of lymphocytes in the spleen. B, DN T cells from DLI-treated xenograft recipients can inhibit the proliferation of xenoreactive CD4+ T cells in vitro in an Ag-specific fashion. A total of 1 x 105 CFSE-labeled naive B6 CD4+ T cells was stimulated with irradiated donor-specific (Lewis, right column) or third-party (WF, left column) splenocytes. Some cultures also included the same number of DN T cells purified from the spleens of either Lewis DLI-treated (DLI-DN) or non-DLI-treated (non-DLI-DN) Lewis heart graft recipient B6 CD4–/– mice. After 4 days incubation, cells were stained with CD4-PE and analyzed by flow cytometry. CD4+ T cells are gated and their CFSE intensity is shown. The percentages of divided CD4+ T cells among total CD4+ T cells in each treatment group are indicated.

 
DN Treg cells suppress the in vivo proliferation and cytokine production of xenoreactive CD4+ T cells

Next, we addressed the question of whether DLI-activated DN Treg cells can suppress xenoreactive CD4+ T cells in vivo. We first analyzed the ability of CD4+ T cells to proliferate and produce cytokines in vivo in the presence or absence of xenoantigens. To this end, B6 CD4–/– mice were transplanted with either a syngeneic or xenogeneic heart graft. At 30 days after transplantation, mice that had accepted their heart grafts were treated with 2.5 x 106 CFSE-labeled naive CD4+ T cells. On day 2 and 9 after infusion of the cells, the proportion of adoptively transferred CD4+ T cells that had divided and were producing IL-2 and IFN-{gamma} was determined by flow cytometry. As shown in Fig. 4, A–C, 2 days after adoptive transfer, the percentage of IFN-{gamma}- and IL-2-producing cells among CFSE-labeled CD4+ T cells was significantly increased in recipients of xenografts when compared with syngeneic graft recipients (Fig. 4, A–C). Consistent with this finding, 9 days after adoptive transfer, the percentage of divided cells and responder frequency of transferred CD4+ T cells was also significantly higher in xenograft recipients compared with syngeneic graft recipients (Fig. 4, D–F). These data reveal the proliferation and cytokine production of CD4+ T cells in syngeneic graft recipients and the frequency of CD4+ T cells that are able to respond to a concordant xenograft in vivo.



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  		FIGURE 4. DN Treg cells suppress the in vivo proliferation of and cytokine production by xenoreactive CD4+ T cells. Thirty days after transplantation of Lewis heart grafts, each B6 CD4–/– mouse was adoptively transferred with 2.5 x 106 CFSE-labeled naive CD4+ T cells either alone (Xeno + CD4) or together with the same number of DN T cells isolated from DLI-treated Lewis heart-grafted B6 CD4–/– recipients (Xeno + CD4 + DN). B6 CD4–/– mice that received syngeneic heart grafts were transferred with 2.5 x 106 CFSE-labeled naive CD4+ T cells/mouse 30 days after heart transplantation (Syn + CD4). A–C, Two days after adoptive transfer, splenocytes were collected from each group and stained for intracellular IFN-{gamma} or IL-2. A, The percentages of IFN-{gamma}-producing (top row) or IL-2-producing (bottom row) cells of gated CFSE+ cells are shown. The bar graphs show the mean percentages ± SD of IFN-{gamma}- (B) and IL-2-producing (C) cells among CFSE-labeled CD4+ cells in the spleens of graft recipients. Each group comprised four mice. D–F, Nine days after adoptive transfer, splenocytes were collected from each group and stained with anti-CD4-PE. D, Dot plots show proliferation of CD4+ cells based on their CFSE intensity, and the percentages of divided CD4+ T cells among total CD4+ T cells are indicated. The bar graph represents the percentage of divided CD4+ cells (E) and the frequency of proliferating precursors (responder frequency) (F) among the CFSE-labeled CD4+ T cells in each group. The results shown are mean percentage ± SD of five mice in each group.

 
To further study the mechanism of DN Treg cell-mediated suppression of xenoreactive CD4+ T cells in vivo, 30 days after transplantation of Lewis hearts, B6 CD4–/– mice were infused with 2.5 x 106 CFSE-labeled naive B6 CD4+ T cells and an equal number of DN Treg cells isolated from B6 CD4–/– recipient mice that had been treated with Lewis DLI and given Lewis heart xenografts 25–30 days previously. We found that DN Treg cells were able to inhibit the in vivo proliferation of xenoreactive CD4+ T cells. Both the responder frequency (Fig. 4F) and the percentage of divided CD4+ T cells (Fig. 4, D and E) was significantly lower in mice that received both CD4+ and DN Treg cells when compared with those that received CD4+ T cells alone. Coinjection of DN Treg cells also inhibited IFN-{gamma} and IL-2 production by CD4+ T cells (Fig. 4, A–C). These data provided the first direct evidence that DN Treg cells can suppress the cytokine production by, and the proliferation of, xenoreactive CD4+ T cells in vivo.

Adoptive transfer of DN Treg cells from tolerant mice prevents CD4+ T cell-mediated donor-specific xenograft rejection

Next, we directly assessed whether the transfer of DLI-activated DN Treg cells is sufficient to prevent CD4+ T cell-mediated xenograft rejection. Thirty days after being transplanted with a Lewis or a WF heart, each B6 CD4–/– recipient mouse was infused with 2.5 x 106 naive B6 CD4+ T cells either alone or together with the same number of DN Treg cells isolated from Lewis DLI-treated and Lewis heart transplanted, tolerant B6 CD4–/– mice. B6 CD4–/– xenograft recipients that did not receive cell transfer served as controls. Graft survival was monitored for >100 days after cell transfer and was confirmed by histopathology. None of the CD4–/– mice from the control groups rejected their Lewis or WF heart grafts, and histopathological studies showed scattered interstitial lymphocytic cell infiltrates without myocyte damage in the grafts (Fig. 5, A and Bi-ii). All mice infused with naive B6 CD4+ cells alone rejected their Lewis or WF heart grafts before day 45 post cell transfer, and showed extensive cellular infiltrate with severe myocyte damage (Fig. 5, A and Biii-iv). Interestingly, Lewis heart grafts survived for >100 days in five of six mice infused with naive B6 CD4+ cells along with Lewis-DLI-activated DN Treg cells (Fig. 5A). Lewis heart grafts harvested between 32 and 130 days after transfer of cells showed multifocal interstitial lymphocytic infiltrates but no myocyte damage (Fig. 5, Bv and Bvii). In contrast, all mice that were infused with naive B6 CD4+ T cells and Lewis-DLI-activated DN Treg cells rejected the WF heart grafts at the same pace as those infused with naive B6 CD4+ T cells alone (Fig. 5A, Biv and Bvi). These data indicate that transfer with DLI-activated DN Treg cells can significantly reduce the histopathological lesions and enhance the overall survival of cardiac xenografts, and that the enhanced xenograft survival mediated by DLI-activated DN Treg cells occurs in an Ag-specific fashion.



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  		FIGURE 5. Cotransfer of DN Treg cells prevents CD4+ T cell-mediated xenograft rejection in B6 CD4–/– mice in an Ag-specific manner. A, Thirty days after transplantation of either Lewis (filled symbols) or WF (open symbols) heart grafts, each B6 CD4–/– mouse was infused with 2.5 x 106 naive B6 CD4+ T cells either alone (circle symbols) or together with the same number of DN T cells isolated from the spleens of Lewis DLI-treated, Lewis heart-grafted B6 CD4–/– recipients (triangle symbols). B6 CD4–/– recipients that did not receive cell transfer served as controls (square symbols). B, Representative xenograft sections from the different groups are illustrated. CD4–/– mice that received heart grafts from either Lewis or WF without injection of CD4+ T cells did not reject their grafts. Lewis at magnification x200 (i) or WF at magnification x100 (ii) grafts were harvested at 65 days posttransplant and both showed scattered interstitial lymphocytic cell infiltrates without myocyte damage (grade 1A, arrows). Mice infused with 2.5 x 106 naive B6 CD4+ T cells alone rejected both their Lewis xenograft (27 days post cell transfer; magnification, x100) (iii) and WF xenograft (35 days post cell transfer; magnification, x100) (iv), and revealed extensive cellular infiltrates with myocyte damage (arrow heads) and areas of necrosis, grade 4 (dashed arrows). Mice infused with both naive B6 CD4+ T cells and Lewis DLI-activated DN T cells had long-term Lewis heart graft survival. Grafts harvested at 37 (magnification, x200) (v) and 130 days (magnification, x100) (vii) post cell transfer showed multifocal interstitial lymphocytic infiltrates and no myocyte damage, grade 1A (arrows). In contrast, mice infused with both naive B6 CD4+ T cells and Lewis DLI-activated DN T cells, but transplanted with third-party WF hearts showed grade 4 rejection with extensive myocyte necrosis, edema and hemorrhage (dashed arrows) at 31 days (vi) post cell transfer (magnification, x100).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
T cells have been shown to be major players in the process of xenograft rejection as evidenced by the fact that xenografts survive in athymic nude mice but not in wild-type mice (44). However, the cellular mechanisms by which xenografts are rejected are not well understood. Most studies indicated that CD4+ but not CD8+ T cells play a major role in rejecting islet and pancreatic xenografts (39, 45, 46). However, several reports have also shown that CD8+ T cells are capable of rejecting xenogeneic islet (47), pancreatic (47, 48), and skin grafts (45, 49) in the absence of CD4+ T cells. In cardiac xenotransplantation, Lin et al. (50) showed that CD8+ T cell-reconstituted nude rats rejected their hamster cardiac grafts more slowly, and required a larger number of transferred cells for rejection, when compared with CD4+ T cell-reconstituted nude rats. Using CD4 or CD8 deficient mice as recipients, we demonstrate in this study that CD4+ T cells, but not CD8+ T cells, are required to induce concordant cardiac xenograft rejection (Fig. 1). Therefore, specifically down-regulating the in vivo function of xenoreactive CD4+ T cells could be a critical step in the induction of cardiac xenotransplantation tolerance.

Pretransplant DLI, either alone or in combination with other treatment, has been shown to enhance donor-specific graft survival in various allogeneic and xenogeneic transplantation models (14, 15, 16, 17, 18, 19, 20, 21). We have previously reported, in a wild-type B6 mice model, that neither pretransplant DLI nor anti-CD4 mAb alone can induce long-term Lewis cardiac xenograft survival. However, DLI when combined with a short course of depleting anti-CD4 mAb can induce permanent acceptance of rat cardiac xenografts by B6 mice (37). Perhaps the elimination of the majority of xenoreactive CD4+ T cells at the time of transplantation in wild-type mice provides a window of opportunity for recipients to implement a mechanism of tolerance, as proposed in various allotransplantation models (51, 52, 53). Consistent with this hypothesis, we demonstrate that the rejection of rat heart xenografts by adoptively transferring a small number of naive CD4+ T cells into B6 CD4–/– mice can be prevented by a single DLI before transplantation, without any other treatment (Fig. 2). These data further confirmed our previous findings in wild-type mice, and indicate that pretransplant DLI is able to induce long-term concordant cardiac xenograft survival when the number of CD4+ T cells in the recipient is limited.

Mechanisms by which DLI induces transplantation tolerance vary depending on the models used. Several studies have shown that induction of Treg cells is involved in DLI-induced tolerance to allografts (19, 26, 27, 54). For instance, DLI plus anti-CD154 mAb treatment induces fully allogeneic skin graft tolerance in mice, which has been shown to involve CD4+CD25+ Treg cells (55, 56). Similarly, pretreatment with DLI and nondepleting anti-CD4 mAb results in the generation of CD4+CD25+ Treg cells, which are capable of preventing donor-specific fully allogeneic skin graft rejection when adoptively transferred into T cell-deficient allotransplant recipient mice that have also been reconstituted with CD45RBhighCD4+ effector cells (54). Furthermore, pretreatment with multiple DLIs also generates CD4+CD25+ T cells that are as potent in immune suppression as those generated from DLI/anti-CD4 mAb treatment (19). We have previously demonstrated in single class I or class II mismatched skin transplantation models that pretransplant DLI also activates recipient-derived DN Treg cells, which can specifically suppress anti-donor immune responses (27, 57). In addition, DN Treg cells that are expanded in vitro upon stimulation with allogeneic donor lymphocytes can prolong donor-specific skin and cardiac graft survival when infused into syngeneic naive mice (27, 58). These studies indicate that pretransplant DLI facilitates the generation of Treg cells, which can then contribute to donor-specific allograft survival.

However, few studies have addressed the role of Treg cells in preventing xenograft rejection. Ikehara et al. (59) have shown that transient depletion of CD4+ T cells at the time of transplantation could enhance rat islet xenograft survival in mice, and that CD4+V{alpha}14+ NK T cells seemed to be essential for the acceptance of these concordant islet xenografts. In our xenotransplantation models, we found a significant increase in the number and percentage of peripheral DN Treg cells in both DLI/anti-CD4 treated wild-type B6 (37) and DLI-treated B6 CD4–/– xenograft recipients (Fig. 3A). Furthermore, we demonstrate in this study that DN Treg cells isolated from DLI-treated B6 CD4–/– xenograft recipients can suppress the proliferation of xenoreactive CD4+ T cells to donor-specific, but not third-party Ags in vitro (Fig. 3B). More importantly, when DN Treg cells were purified from the spleens of DLI-treated, xeno-heart-grafted B6 CD4–/– recipients and coinjected with naive CD4+ T cells into B6 CD4–/– xenograft recipients, the majority of recipient mice achieved long-term donor specific, but not third party, heart graft survival (Fig. 5). Taken together, these data indicate that DLI treatment leads to activation and expansion of recipient DN Treg cells, which can prevent CD4+ T cell-mediated cardiac xenograft rejection in an Ag-specific manner.

Extensive studies have been done to reveal the mechanisms of action mediated by Treg cells (60, 61, 62). Most studies are performed using in vitro suppression assays that may not reflect the in vivo situation (63). Recently, several groups have also studied the Treg cell function in vivo. It has been reported that coinjection of purified natural occurring CD4+CD25+ Treg cells with naive CD4+ T cells into lymphopenic or nonlymphopenic recipients can suppress naive CD4+ T cell proliferation in vivo (64, 65, 66). Sanchez-Fueyo et al. (67) also found that lymphocytes obtained from tolerant islet allograft recipients can suppress the proliferation of naive CD4+ and CD8+ T cells in vivo, and that the suppression can be abolished by removal of CD4+CD25+ T cells. In contrast, Lin et al. (68) have shown that after adoptive transfer into tolerant skin allograft recipients the CD8 effector cells proliferate normally, but they did show compromised graft rejection, IFN-{gamma} production, and cell-mediated cytotoxicity. This result suggests that in tolerant recipients, Treg cells act by inhibiting immune effector function rather than by suppressing the proliferation of alloreactive T cells. By monitoring the fate of adoptively transferred CFSE-labeled naive CD4+ T cells in B6 CD4–/– mice that have accepted their syngeneic or xenogeneic heart grafts, we found that the infused naive CD4+ T cells showed low proliferation in CD4–/– mice that received syngeneic heart grafts, which is consistent with reports from others (69, 70). However, the proportion of CD4+ T cells that are able to proliferate and produce IL-2 and IFN-{gamma} in xenograft recipients is significantly higher when compared with CD4+ T cells in syngeneic graft recipients (Fig. 4). We further demonstrate that cotransfer of DN Treg cells almost completely prevented the in vivo proliferation of naive CD4+ T cells to xenoantigens (Fig. 4, D–F), and reduced the ability of CD4+ T cells to produce IL-2 and IFN-{gamma} to the level of production seen in syngeneic heart graft recipients (Fig. 4, A–C). These data provide an in vivo mechanism by which DLI-activated DN Treg cells prevent xenograft rejection.

The molecular mechanisms involved in DN Treg cell-mediated Ag-specific suppression of xenoreactive CD4+ T cells remain to be determined. Our previous studies in rodent allogeneic transplant models indicated that DN Treg cells suppress alloresponses at least partially through directly killing of activated alloreactive T cells. DN Treg cells have also been shown to be able to acquire specific allo-MHC-peptides from APCs and present the acquired allo-MHC-peptides to other alloreactive T cells that express the same TCR specificity as the DN Treg cells. DN Treg cells can then kill the alloreactive T cells through Fas-Fas ligand interactions (27). Recently, Fischer et al. (30) have shown that human DN Treg cells can also acquire peptide-HLA-A2 complexes from APCs. Moreover, the human DN Treg cells that have acquired peptide-HLA can kill syngeneic CD8+ T cells that are activated by the same peptides (30). These findings suggest a mechanism by which DN Treg cells induce Ag-specific suppression. However, because of the low frequency of DN Treg cells in vivo, as well as the lack of knowledge of the specific xenoantigens and the TCRs that can recognizing them, we are presently unable to investigate whether DN Treg cells down-regulate xenoresponses via a similar mechanism as seen in suppression of allogeneic immune responses.

In this study we focused on the role of DLI-activated DN Treg cells in preventing cardiac xenograft rejection. In our model the B6 CD4–/– recipient mice harbor a null mutation of the CD4 gene and have no mature CD4+ T cells (38). Furthermore, infusion of nonfractionated naive CD4+ T cells, which compose both CD4+CD25– and a small fraction of natural occurring CD4+CD25+ T cells from wild-type B6 mice, caused graft rejection rather than tolerance in these mice. These results suggest that either naturally occurring CD4+CD25+ Treg cells do not play a role in the enhancement of xenotransplant survival seen in this model, or the small fraction of natural occurring CD4+CD25+ Treg cells that were adoptively transferred into xenotransplanted mice were not sufficient to induce long-term xenograft survival in this model. Although we cannot rule out the possibility that Ag-induced CD4+CD25+ Treg cells (19, 54) or other type of Treg cells such as Tr1 (71) and CD8+ Treg cells (35) may also enhance long-term xenograft survival in DLI-treated recipients, our study does show that transfer of DLI-activated DN Treg cell is able to induce long-term xenograft survival in this model (Fig. 5).

In summary, the present study provides evidence indicating that CD4+ T cells play a critical role in the rejection of concordant cardiac xenografts. Pretransplant DLI can activate recipient DN Treg cells, which are able to down-regulate the function of donor-specific xenoreactive CD4+ T cells, and prevent xenograft rejection. These findings provide new insights into the in vivo cellular immune responses to xenografts and how such a response is controlled by DN Treg cells. Further understanding of the molecular mechanisms involved in these processes may lead to novel approaches for preventing cell-mediated xenograft rejection.


   Acknowledgments
 
We thank M. S. Ford and C. W. Thomson for critically reading the manuscript.


   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 is supported by the Canadian Institutes of Health Research Grants MOP 14431 and HRP 52447. L.Z. is a Clinical Research Chair in Transplantation cosponsored by the Canadian Institutes of Health Research and Wyeth-Ayerst (Markham, Ontario, Canada). W.C. is a recipient of a Canada Graduate Scholarship Doctoral Award. Back

2 Address correspondence and reprint requests to Dr. Li Zhang, Toronto General Research Institute, Norman Urquhart Wing G-001, University Health Network, 621 University Avenue, Toronto, Ontario M5G 2C4, Canada. E-mail address: lzhang{at}uhnres.utoronto.ca Back

3 Abbreviations used in this paper: DLI, donor lymphocyte infusion; DN, double negative; Treg, regulatory T; WF, Wistar-Furth; MST, mean graft survival time. Back

Received for publication March 10, 2005. Accepted for publication June 14, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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