The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tesar, B. M.
Right arrow Articles by Goldstein, D. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tesar, B. M.
Right arrow Articles by Goldstein, D. R.
The Journal of Immunology, 2004, 173: 4377-4386.
Copyright © 2004 by The American Association of Immunologists

Direct Antigen Presentation by a Xenograft Induces Immunity Independently of Secondary Lymphoid Organs1

Bethany M. Tesar*, Geetha Chalasani{dagger}, Lonnette Smith-Diggs{dagger}, Fady K. Baddoura§, Fadi G. Lakkis{dagger},{ddagger} and Daniel R. Goldstein2,*

Sections of * Cardiovascular Medicine and {dagger} Nephrology, Department of Internal Medicine, and {ddagger} Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06520; and § Department of Pathology, Veterans Affairs Medical Center, State University of New York, Buffalo, NY 14215


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The location of immune activation is controversial during acute allograft rejection and unknown in xenotransplantation. To determine where immune activation to a xenograft occurs, we examined whether splenectomized alymphoplastic mice that possess no secondary lymphoid organs can reject porcine skin xenografts. Our results show that these mice rejected their xenografts, in a T cell-dependent fashion, at the same tempo as wild-type recipients, demonstrating that xenograft rejection is not critically dependent on secondary lymphoid organs. Furthermore, we provide evidence that immune activation in the bone marrow did not take place during xenograft rejection. Importantly, immunity to xenoantigens was only induced after xenotransplantation and not by immunization with porcine spleen cells, as xenografted mutant mice developed an effector response, whereas mutant mice immunized by porcine spleen cells via i.p. injection failed to do so. Moreover, we provide evidence that antixenograft immunity occurred via direct and indirect Ag presentation, as recipient T cells could be stimulated by either donor spleen cells or recipient APCs. Thus, our data provide evidence that direct and indirect Ag presentation by a xenograft induces immunity in the absence of secondary lymphoid organs. These results have important implications for developing relevant xenotransplantation protocols.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Presentation of foreign Ag by the immune system is a multistep process. At the site of injury, APCs, for example immature dendritic cells (DCs), 3 capture foreign Ags and process them (1). This initiates a maturation program that includes up-regulation of chemokine receptors on the surface of DCs, allowing their effective migration to the draining lymph nodes (2). It is here that mature DCs present processed foreign peptides in the groove of MHC molecules to naive T cells via the TCR (signal 1) (3). Delivery of signal 2 via costimulatory molecules on the DCs allows the T cells to become activated and differentiate into effectors (3). These effectors can then mount an adaptive immune response against the invading pathogen, leading to elimination of the noxious stimulus and the development of immunological memory (4). The importance of secondary lymphoid organs in this process has been demonstrated by several studies that have established that infections and tumors are ignored if APCs are prevented from accessing the draining lymph nodes (5, 6, 7, 8). Furthermore, impairing DC migration by inhibiting the CCR7-CCL21 pathway impedes T and B cell priming (9, 10). However, the location of immune activation during solid organ transplantation is less clearly defined.

An important difference in Ag presentation of transplant Ags is the ability of passenger, donor APCs, with allo- or xeno-derived MHC molecules, to directly activate naive, recipient T cells (direct presentation) (11). In addition to this pathway, foreign peptides derived from the transplant can be processed and presented by recipient APCs (indirect presentation). Both of these pathways are sufficient to induce acute allograft rejection (12, 13). Early studies suggested that priming of the alloimmune response could take place within the graft itself without the need of secondary lymphoid organs, and a recent study has demonstrated that donor endothelium can activate naive T cells (14, 15, 16). This is in contrast to other studies, using mutant mice that lack Peyer’s patches and lymph nodes (alymphoplastic mouse, aly/aly) that failed to reject skin allografts (17). When these mice were rendered completely devoid of all secondary lymphoid organs via splenectomy, the alloantigen was ignored and rejection of vascularized allografts was prevented (18). However, there remains considerable controversy as the use of alternate mutant mice (lymphotoxin {alpha} and lymphotoxin {beta} receptor deficient) that also lack lymph nodes and Peyer’s patches demonstrated that although rejection of vascularized allografts was severely retarded, rejection ultimately occurred, suggesting that secondary lymphoid organs may not be critically involved in allorecognition (19). The difference in these two studies may be due to confounding mutations present within each specific recipient (20).

Solid organ transplantation remains an important therapeutic modality for end stage organ dysfunction. However, due to the limited number of available donors, thousands of candidates die while waiting on transplant lists. Xenotransplantation would be an obvious solution to the limitation in donor availability because transgenic porcine donors can be easily bred. Over the last two decades, considerable advances have been made in our understanding of the mechanisms responsible for xenograft rejection (21). Most of these investigations have focused on hyperacute and acute vascular rejection, although prior studies have demonstrated that an intact adaptive immune system is required for the cellular rejection of xenografts and both direct and indirect recognition of xenoantigens occurs (22, 23). However, it is unknown where immune activation to xenografts is located. We chose to investigate this question by using a porcine skin xenograft model using splenectomized aly/aly mice as recipients. Our results demonstrate that these mutant mice are able to reject porcine xenografts at the same tempo as wild-type mice, providing evidence that secondary lymphoid organs are not critical for xenograft rejection. Furthermore, we demonstrate that an antixenograft effector response occurs after direct and indirect presentation of xenoantigens.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Skin transplantation, splenectomy, and phlebotomy

Porcine skin was obtained from outbred Yorkshire pigs from a local abattoir and cleaned at the time of harvest with betadine solution. Split skin xenografts (<3 mm in depth) were prepared using a 0.016-gauge dermatome (Pilling Surgical, Fort Washington, PA) and cleaned using 70% ethanol and betadine solutions. Rat skin was harvested via a similar procedure. Xenografts (1 cm x 1 cm area) were sutured using metallic clips onto the recipient left thoracic area after a suitable wound area was created. Full thickness syngeneic skin transplants were harvested from 6- to 8-wk-old donor mice and sutured onto the recipients’ L thoracic area, as already described (24). Rejection was defined as a scab covering >90% of the graft area. Splenectomy was performed, as previously described (25). Mice were bled via a retro-orbital eye bleed or via direct cardiac puncture.

Rodents

B6.ALY-Map3k14aly/NscJc1 (designated as aly/aly) and wild-type controls (aly+) were purchased from CLEA Japan (Tokyo, Japan). B6.129S7-Rag1Tm1Mom (designated RAG–/–), B6.129P2-Tcrbtm1MomTcrdtm1Mom (designated T cell deficient), B6.SJL-Ptprca Pep3b/BoyJ (designated CD45.1), and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). STOCK-PrkdcscidLystbg (designated SCID/Bg) mice were generously provided by J. Pober, Yale University. Sprague-Dawley rats were generously provided by R. Russell, Yale University. All rodents were kept in pathogen-free conditions.

Histology and immunohistochemistry

Graft tissue was fixed in B5 solution and 10% formalin, embedded in paraffin, sectioned, and stained with H&E or anti-mouse CD3 (BD Pharmingen, San Diego, CA), followed by peroxidase-conjugated secondary Ab staining. Percentage of CD3 infiltration was calculated as the proportion of T cells divided by the total number of cells.

Cell preparation and T cell purification before adoptive transfer

Spleen cells were harvested from CD45.1 gender-matched mice. Red cells were lysed after incubation with hypotonic ammonium chloride solution. Cells were resuspended in cold (4°C) 1% PBS-BSA, and then T cell enriched via negative selection by first incubating the suspension with biotin-Ab-conjugated mixture (containing mAbs against CD11b, B220, DX5, and Ter-119) for 15 min at 4°C. The solution was then washed and incubated with anti-biotin magnetic microbeads, according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). The labeled cells were then removed on an automatic magnetic cell separating system (AutoMACS; Miltenyi Biotec), as previously described (24). APCs were purified via positive selection (>95% purity) from recipient spleens using magnetic microbeads conjugated to an anti-murine MHC class II mAb (Miltenyi Biotec) using the AutoMACs system. Purified, untouched T cells that were used in adoptive transfer experiments (>98% purity) were then CFSE (Molecular Probes, Eugene, OR) incorporated by incubating cells with 5 µM for 10 min at 4°C. A total of 2.5 x 107 T cells was transferred via i.p. injection. Donor porcine spleen cells were harvested and resuspended in complete RPMI 1640 solution. After RBC lysis, donor cells were either used directly or stored in liquid nitrogen at –70°C in 70% RPMI 1640, 20% DMSO, and 10% FBS solution. Note that for all experiments that used both in vivo and in vitro immunization with porcine spleen cells, the porcine cells were matched with the skin xenograft (i.e., both porcine spleen cells and skin originated from the same donor). All results were repeated in triplicate.

CD4, CD8, and NK1.1 monoclonal-depleting Abs

GK 1.5 and 2.43 (both IgG2) were generously provided by J. George (University of Alabama, Birmingham, AL). Animals received 150 µg via i.p. injection on days –1 and +2 posttransplantation, and then at weekly intervals until the third week posttransplantation. NK1.1 (PK136, IgG2) was kindly provided by K. Kokko (Emory University, Atlanta, GA), and 100 µg was administered on days –3, –2, and –1 pretransplantation and weekly posttransplantation. Prior studies have demonstrated that these reagents specifically inhibit CD4, CD8, and NK cells, respectively (26, 27, 28). Nevertheless, we performed preliminary studies that demonstrated >95% depletion measured by flow cytometry at the above stated doses.

Flow cytometric staining

All of the following Abs were purchased from BD Pharmingen: FITC-conjugated rat anti-mouse IgM, IgG, and CD4; PE rat anti-mouse CD8; and biotinylated rat anti-mouse CD45.1. Incubation was at 4°C for 30 min, except for CD45.1, which was 30 min, followed by 15-min staining with streptavidin PerCP. Isotype controls were used in every experiment. To measure anti-porcine Abs, serum was collected from recipient mice, and heat inactivated by incubation at 56°C for 1 h. Serum dilutions were then incubated with donor porcine spleen cells (2.5 x 106) for 15 min at 37°C. After washing three times, the suspension was incubated with either rat anti-mouse IgM or IgG. Preliminary experiments established that a serum dilution of 1/5 gave the optimum results. Results were read as median fluorescent intensity by flow cytometry, and results in each group were compared with that obtained using isotype control Abs. Data were acquired on a FACSCalibur flow cytometer (BD Immunocytometry Systems, San Diego, CA) and analyzed using CellQuest Pro software. All results were repeated in triplicate.

Preparation of porcine xenoantigens

Whole protein was extracted from a suspension of porcine spleen cells by incubating the solution with lysis buffer (containing 20 mM HEPES, 50 mM {beta}-glycerol phosphate, 2 mM EDTA, 1 mM DTT, 10 mM NaF, 1% Triton X-100, 100 µM leupeptin, and 10 µg/ml aprotonin) for 1 h. The resulting protein was precipitated from this solution by TCA acid precipitation. The protein was then solubilized in 100 µl of 8 M urea/0.4 M NH4HC03, and reduced with 45 mM dithiotheitol by incubation at 37°C for 20 min. The cysteine residues were then modified by adding 100 mM iodoacetamide and incubating the solution at room temperature for 20 min. The protein was then digested into peptides by adding 0.1 mg/ml trypsin solution and incubating at 37°C for 18 h. The peptides were consequently purified by passing the solution through a Sep-Pak column. The resulting peptide solution was dried by speed vac centrifugation and resuspended in RPMI 1640, 10% FCS, 1% PenStrep solution. The pH was checked to assure that the peptides did not alter the pH during the MLR.

Immunization and ELISPOT protocol

Mice that were immunized with porcine spleen cells received 1.5 x 107 porcine cells (via i.p. injection) 14 days before MLR. IFN {gamma} ELISPOT kits were purchased from BD Pharmingen and used per the manufacturer’s instructions. Briefly, 96-well ELISPOT plates were incubated with capture Ab for 24 h. Before the MLR, the membrane in each well was blocked with RPMI 1640, 10% FCS, 1% PenStrep solution. The MLR was performed using 2 x 106 murine splenocytes or 1 x 106 purified, untouched T cells plus 3 x 105 mitomycin C-treated porcine stimulators or the equivalent amount of xenopeptides per well (1 µg/well). Preliminary experiments determined the optimum doses in each case. Incubation was 20 h at 37°C, after which the cellular suspension was removed and biotinylated detection Ab added at the recommended concentration. After the recommended number of washes, avidin-HRP and 3-amino-9-ethylcarbazole substrate were added per manufacturer’s instructions. Con A, 2 µg/ml (optimum dose established in preliminary experiments), was added to responder splenocytes only and served as a positive, technical control. Stimulator only, responder only, and medium only controls were included in every experiment. Plates were read on a CTL automatic ELISPOT reader (CTL, Cleveland, OH) and analyzed using Immunospot 3.1 software (CTL). All results were repeated in triplicate.

Statistical analysis

Survival analysis between groups was calculated using the log rank method. Comparison of means was performed using a two-tailed t test. All results were generated using SPSS statistical software (Chicago, IL). Statistical significance was considered by a p value <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rejection of porcine xenografts is not dependent on secondary lymphoid organs

To examine the role of secondary lymphoid organs in the rejection of porcine skin xenografts, we used the alymphoplastic mouse, which lacks lymph nodes and Peyer’s patches due to a mutation in NF-{kappa}B-inducing kinase (B6.ALY-Map3k14aly/NscJc1, referred to as aly/aly mice) (29). To render these mice completely devoid of secondary lymphoid organs, the mice were splenectomized 2 wk before transplantation (mice referred to as aly/aly-SPL). The results demonstrate that aly/aly-SPL recipients were equally able to reject xenografts when compared with wild-type littermate controls (aly+) (median survival time (MST): aly/aly-SPL = 19 vs 21 days, aly+, p = 0.7) (Fig. 1a). Similar graft survival was demonstrated in nonsplenectomized aly/aly mice (MST = 21 days, p = 0.4 vs aly+) and splenectomized aly+ recipients (MST = 19 days, p = 0.5 vs aly+) (Fig. 1a). These results were supported by photographic and histological examination of the xenografts at day +14 posttransplantation that confirmed the appearance of acutely inflamed grafts and the presence of acute cellular rejection in the absence of secondary lymphoid organs, respectively (Fig. 1, b–d). Note that aly/aly-SPL recipients that received an allogeneic skin graft (BALB/c donor, H2d) did not reject their allografts (MST >100 days) in agreement with prior studies (17, 18). Hence, these results demonstrate that the presence of secondary lymphoid organs is not critical for acute xenograft rejection.



View larger version (73K):
[in this window]
[in a new window]
  		FIGURE 1. Secondary lymphoid organs are not critically required for the rejection of porcine skin xenografts. a, Splenectomized aly/aly mice (aly/aly-SPL, {blacksquare}) rejected their skin xenografts with a MST = 19 days, which was not statistically significant vs aly+ ({circ}, MST = 21 days, p = 0.7), splenectomized aly+ ({triangleup}, MST = 19 days, p = 0.5), or nonsplenectomized aly/aly mice ({diamondsuit}, MST = 21 days, p = 0.5). Note that aly/aly-SPL recipients did not reject fully allogeneic skin grafts (+). b and c, Histological analysis at day +14 posttransplantation, demonstrating similar cell infiltration in aly+ and aly/aly-SPL recipients. d, Representative photograph of an aly/aly-SPL recipient with an acutely inflamed xenograft at day +14 posttransplantation.

 
Acute cellular xenograft rejection depends on the presence of adaptive immunity and not NK cells

To investigate the role of adaptive immunity to xenografts in our model, we transplanted porcine xenograft onto two immunodeficient strains of recipients: RAG-deficient (B6.129S7-Rag1Tm1Mom, designated RAG–/–) and SCID beige mice(STOCK-PrkdcscidLystbg, designated SCID/Bg). Results show that these immunodeficient recipients accepted porcine xenografts indefinitely (Fig. 2a) consistent with a previous study (30). Photographic and histological analysis demonstrated healthy appearing skin with no evidence of cellular infiltrate (Fig. 2, b–d). As RAG–/– mice have functional NK cells, yet were still able to accept xenografts, this suggested that NK cells do not account for xenograft rejection in the absence of secondary lymphoid organs. Nevertheless, we sought to rule out the possibility that NK cells mediated xenograft rejection in the aly/aly-SPL model by administrating NK-depleting Abs (NK1.1) to recipients. These mice were able to reject their xenografts without delay (Fig. 2a), supporting the results of a previous study (28). Thus, these results demonstrate that a functional adaptive immune response that is not critically dependent on NK cells must be present for cellular xenograft rejection.



View larger version (57K):
[in this window]
[in a new window]
  		FIGURE 2. Adaptive immune responses are required for the rejection of porcine xenografts. a, Both RAG–/– ({diamondsuit}) and SCID/Bg (•) immunodeficient recipients did not manifest rejection of porcine xenografts. Addition of NK-depleting Ab did not delay rejection in aly/aly-SPL recipients ({blacksquare}) (MST = 20 days, p = 0.8 vs untreated aly/aly-SPL recipients in Fig. 1). b and c, Photographic evidence of intact xenografts in SCID/Bg (note, porcine hair) and RAG–/– recipients, respectively. d, Histological evidence of lack of xenograft cellular infiltration in a representative immunodeficient (SCID/Bg) recipient.

 
Xenograft rejection is dependent on the presence of T cells

We next examined the role of the T cell compartment in xenograft rejection. Hence, we transplanted xenografts onto T cell-deficient recipients (B6.129P2-Tcrbtm1MomTcrdtm1Mom, designated T cell deficient) and aly/aly-SPL recipients that were treated with CD4- and CD8-depleting Abs. Both groups were able to accept porcine xenografts for >80 days posttransplantation (Fig. 3a). At day 83 posttransplantation, one aly/aly-SPL recipient rejected its xenograft. Flow cytometric analysis at this time point showed recovery of circulating CD8+ and CD4+ T cells that were not present at 1 mo posttransplantation (Fig. 3b). To confirm the contribution of T cells in xenograft rejection, we analyzed xenografts from aly+ and aly/aly-SPL recipients. T cell infiltration in both groups was equivalent by day +14 posttransplantation (Fig. 3, c and d). Thus, these results demonstrate that porcine xenograft rejection is critically dependent on T cells, regardless of whether secondary lymphoid organs are present or absent.



View larger version (51K):
[in this window]
[in a new window]
  		FIGURE 3. Xenograft rejection is critically dependent on T cells. a, Both T cell-deficient ({diamondsuit}) and aly/aly-SPL recipients ({blacksquare}) treated with CD4- and CD8-depleting Abs manifested abrogated or severely retarded rejection of porcine xenografts. b, On day +83 posttransplantation, one aly/aly-SPL recipient rejected its xenograft. Peripheral blood analysis demonstrated repopulation of both CD4 and CD8 cells that were not present at 1 mo posttransplantation. c, Immunohistochemical staining of xenografts reveals equal T cell infiltration at day +14 posttransplantation in aly+ and aly/aly-SPL groups. d, There was no difference in the percentage of CD3 infiltration at day +14 posttransplantation between aly+ and aly/aly-SPL groups, although there was a reduction at day +7 in the aly/aly-SPL recipients.

 
Evidence of xenograft-induced T cell proliferation in the absence of secondary lymphoid organs

To examine whether xenotransplantation induced T cell proliferation in the aly/aly-SPL recipient, we adoptively transferred 2.5 x 107 purified congenic, CD45.1 CFSE-labeled, splenic T cells into aly/aly-SPL recipients on the day of transplantation. aly/aly-SPL mice that received syngeneic isografts and were adoptively transferred with CD45.1, CFSE-labeled T cells acted as controls. At days +7, +11, and +14 posttransplantation, blood was obtained from xenograft and control recipients, and examined by flow cytometry for evidence of T cell division. Note that there was an n = 6 for the day +7 and +14 time points, and an n = 3 for day +11. Results show that xenospecific proliferation occurred at day +7 posttransplantation (5% divided, xenografted group vs 2%, control group p = 0.0007) and increased further on days +11 and +14 (day +11, 11 vs 3%, p = 0.02, and day +14, 26 vs 13%, p = 0.001) (Fig. 4). Thus, these results provide evidence that T cell proliferation in response to the xenograft occurs independently of secondary lymphoid organs.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 4. T cells proliferate in response to xenotransplantation in a host devoid of secondary lymphoid organs and without evidence of xenograft-specific proliferation within the bone marrow. a, Examination of CD45.1-purified, CFSE-incorporated T cells at weeks 1 and 2 posttransplantation revealed significant xenograft-specific proliferation within the blood, but not the bone marrow. Note that there were insufficient adoptively transferred T cells within the bone marrow at week 2 to accurately determine the degree of proliferation. Brackets denote cells that have proliferated. b, Histograms demonstrating significant xenograft-induced proliferation on day +7 vs syngeneic grafted controls within the blood (5.3% proliferation vs 2.0%, p = 0.0007). No evidence for xenograft-specific T cell proliferation was found within the bone marrow (8% proliferation vs 10.5%, p = 0.4) at the same time point. c, Demonstration of increased T cell proliferation in the blood of xenotransplanted aly/aly-SPL recipients vs aly/aly-SPL mice transplanted with a syngeneic skin graft. At all time points, there were significantly higher amounts of proliferation in the xenotransplanted group (day +7, 5.3% proliferation vs 2.0%, p = 0.0007; day +11, 11 vs 3%, p = 0.02; and day +14, 26 vs 13%, p = 0.001). Note, there was an n = 6 for the day +7 and +14 time points and an n = 3 for day +11.

 
No evidence for immune activation within the bone marrow

Because recent evidence has demonstrated that the bone marrow can possess immune activating capabilities, we wished to determine whether activation of T cells occurred first within the bone marrow (31). Therefore, we analyzed the bone marrow for T cell proliferation at day +7 from xenotransplanted (n = 6) and syngeneic grafted aly/aly-SPL (controls, n = 6) recipients that were adoptively transferred with CFSE-incorporated, CD45.1 T cells at the time of transplantation. Our results indicate that there was no significant xenograft-specific proliferation occurring within the bone marrow during the first week posttransplantation (8% divided, aly/aly-SPL-xenografted recipients, vs 10.5% divided, controls, p = 0.4) (Fig. 4b). Examination at later time points (days +11, n = 3/group, and +14, n = 6/group) did not revealxenograft-specific proliferation within the bone marrow; however, the reduced numbers of adoptively transferred T cells noted within the bone marrow at these time points prevented quantification of the degree of proliferation (Fig. 4a). Thus, these results do not provide evidence that the bone marrow is the source of T cell activation during cellular xenograft rejection.

Xenotransplantation is superior than i.p. immunization in inducing an effector response to xenoantigens

To determine whether the xenograft itself was sufficient to induce an effector response to the xenoantigens, we compared the ability of aly/aly mice to mount an effector recall IFN-{gamma} response to the xenograft vs aly/aly mice that were immunized with porcine spleen cell via i.p. injection. Thus, spleen cells from aly/aly mice that received a xenograft 14 days earlier were compared with aly/aly mice that were immunized with porcine spleen cells at the same time point for their ability to generate IFN-{gamma} in the presence of donor-specific porcine spleen cells in a recall ELISPOT assay (response designated as an effector recall response). The results show that the xenotransplanted aly/aly recipients generated a specific IFN-{gamma} effector recall response after exposure to mitomycin C-treated donor porcine spleen cells in a one-way MLR (60 spots/1 x 106 spleen cells) that was significantly increased compared with nonstimulated responders from the xenotransplanted aly/aly mice (20 spots/1 x 106 spleen cells, p = 0.003) (Fig. 5). In contrast, aly/aly mice that were immunized with porcine spleen cells via i.p. injection failed to generate an IFN-{gamma} effector recall response when exposed in vitro by mitomycin C-treated porcine spleen cells (13 spots/1 x 106 spleen cells exposed to mitomycin C-treated porcine spleen cells, vs 10 spots/1 x 106 nonexposed responder spleen cells, p = 0.6) (Fig. 5). Importantly, the response from exposed spleen cells from xenografted aly/aly recipients was significantly superior to exposed spleen cells from immunized aly/aly mice (p = 0.001) (Fig. 5). Note that aly+ recipients that were immunized via i.p. injection were able to mount an effector recall response after exposure to mitomycin C-treated spleen cells (120 spots/1 x 106 spleen cells) vs nonexposed responder spleen cells (26 spots, 1 x 106 spleen cells, p < 0.0001), providing evidence that the dose of porcine spleen cells and route of administration were sufficient to induce an effector recall response in wild-type recipients. Additionally, both aly/aly and aly+ spleen cells were equally able to secrete IFN-{gamma} in response to nonspecific stimulation with Con A (both >300 spots/1 x 106 spleen cells, p = 0.8). Thus, these results provide evidence that xenotransplantation promotes T cell priming more effectively than immunization with porcine spleen cells.



View larger version (60K):
[in this window]
[in a new window]
  		FIGURE 5. Ag delivering via the xenograft induces an effector recall response in contrast to delivery of Ag via i.p. immunization in aly/aly recipients. a, Spleen cells from aly/aly xenotransplant recipients (designated as aly/aly xeno + stim) were able to mount an IFN-{gamma} effector recall response when stimulated in vitro with mitomycin C-treated porcine spleen cells (**, p = 0.003 vs nonstimulated spleen cells from transplanted aly/aly mice (designated as aly/aly xeno resp) and ***, p = 0.001 vs aly/aly spleen cells from mice that were immunized with porcine spleen cells via i.p. injection and stimulated in vitro (designated as aly/aly immunized + stim)). In contrast, spleen cells from immunized aly/aly mutant mice failed to generate an IFN-{gamma} effector recall response when stimulated in vitro with mitomycin C-treated porcine spleen cells ({blacktriangledown}, p = 0.6). Note that aly+ animals were able to mount an IFN-{gamma} effector recall response (*, p < 0.0001), demonstrating that the dose and route of administration of xenoantigens were sufficient to induce a response in wild-type recipients. b, Representative ELISPOT wells from specified experimental groups.

 
Effector recall response to xenoantigens occurs via the direct and indirect pathway

To determine whether the effector recall response to xenoantigens occurs via the direct or indirect pathway of Ag presentation, we examined whether this response was dependent on the presence of recipient APCs (indirect pathway) or donor APCs (direct pathway). To test the direct pathway, responder APCs were removed from xenograft aly/aly recipients by purifying responder splenic T cells to >98% purity. These responder T cells maintained their ability to produce an IFN-{gamma} effector recall response in the absence of responder APCs (116 spots/1 x 106 T cells + porcine stimulators vs 11 spots/1 x 106 nonexposed, responder T cells, p = 0.04), demonstrating that this response can be induced by the presence of donor APCs within the stimulating porcine spleen cells (i.e., direct pathway) (Fig. 6). To test the indirect pathway, donor APCs were removed by deriving porcine peptides from the quantity of donor spleen cells used to induce the effector recall response in the one-way MLR. These peptides failed to produce an effector recall response when added to responder spleen cells (containing responder T cells and APCs) from transplanted aly/aly recipients (14 spots/1 x 106 spleen cells, peptide + responders vs 20 spots/1 x 106 spleen cells, responders only) (Fig. 6). Note that spleen cells harvested from aly+ mice that were immunized with porcine stimulators generated an IFN-{gamma} response when exposed to xenopeptide in vitro (xenopeptide-stimulated responder spleen cells = 42 spots/1 x 106 spleen cells vs unstimulated responder spleen cells = 10 spots, p < 0.0001) and that the presence of the peptide did not inhibit aly/aly spleen cells to produce IFN-{gamma} in response to nonspecific stimulation with Con A (>300 spots/1 x 106 spleen cells). An additional test of the indirect pathway included an examination as to whether the addition of purified, mitomycin C-treated recipient APCs harvested from xenotransplanted recipients increased the IFN-{gamma} response of responder T cells in the presence of porcine stimulators. The results demonstrate that although there was not a significant increase in the IFN-{gamma} response when 3 x 103 and 3 x 104 APCs were added/well (76 spots/1 x 106 T cells and 70 spots/1 x 106 T cells, respectively), there was a significant augmentation of the IFN-{gamma} response after the addition of 3 x 105 APCs (284 spots/1 x 106 T cells, p = 0.004 vs T cells without APCs). Thus, these results provide evidence that both the direct and indirect pathway of Ag presentation can induce an effector recall response to xenoantigens.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 6. Both direct and indirect presentation of xenoantigens can induce an effector recall response. a, The effector recall response was not diminished by removing recipient APCs, as responder T cells (>98% purity) from xenografted aly/aly mice (designated as resp T cells + stim) produced an effector recall response when stimulated with mitomycin C-treated porcine SPCs (116 spots/1 x 106 cells, exposed T cells + porcine stimulators vs 11 spots/1 x 106, T responder cells alone, p = 0.04). However, removing donor APCs by deriving xenopeptides from stimulating porcine spleen cells failed to induce an effector recall response in responder spleen cells from xenograft aly/aly recipients (designated as Resp SPC + Peptide) (14 spots/1 x 106 cells, peptide + responders vs 20 spots/1 x 106 cells, responders only). Note that spleen cells harvested from aly+ mice that were immunized with porcine stimulators generated an IFN-{gamma} response when exposed to xenopetide in vitro (xenopeptide-stimulated responder spleen cells = 42 spots/1 x 106 spleen cells vs unstimulated responder spleen cells = 10 spots, p < 0.0001). b, ELISPOT wells demonstrating typical representatives of specified experimental groups. c, Recipient APCs enhance the IFN-{gamma} response when cocultured with responder T cells in the presence of porcine SPC ({blacksquare} denotes coculture of T cells with mitomycin C-treated APCs and porcine SPC). The addition of 3 x 105 APCs significantly increased the IFN-{gamma} response (284 spots/1 x 106 T cells, p = 0.004 vs T cells without APCs).

 
No evidence of preformed Abs or preformed effector memory T cells in naive aly/aly mice

The presence of immune mechanisms that do not depend on secondary lymphoid organs for rejection is a possible contributing explanation as to why recipients devoid of secondary lymphoid organs were able to mediate xenograft rejection at a normal tempo. Such factors include preformed Abs and preformed effector memory T cells. Although mice possess the {alpha} galactosyl transferase enzyme and hence do not have preformed Abs against porcine endothelium {alpha} gal, it is possible that mice possess preformed Abs that cross-react with other porcine epitopes. As the presence of such cross-reactive preformed Abs might explain why recipients devoid of secondary lymphoid organs were able to reject xenografts, we sought to quantify the levels of circulating anti-porcine IgM and IgG Abs in naive untransplanted mice. Our results show that naive aly+ mice do not possess any serological evidence of anti-porcine IgM or IgG Abs (Table I).


View this table:
[in this window]
[in a new window]
  		Table I. Anti-porcine Ab levelsa

 
To assess whether mice devoid of secondary lymphoid organs could generate anti-donor Ab responses after transplantation, we quantified anti-porcine Ab levels 14 days after transplantation. The results show that aly/aly-SPL recipients were not able to mount either an anti-porcine IgM or IgG response, whereas aly+ recipients had a nonsignificant elevation of IgM and a significant elevation of IgG levels in response to xenotransplantation (Table I). Thus, these results demonstrate that mice do not possess preformed Abs against porcine Ags and that aly/aly-SPL recipients have an impaired humoral response to xenotransplantation despite rejection occurring at the same tempo as wild-type mice. This finding rules out the contribution of Abs to xenograft rejection in the absence of secondary lymphoid organs.

Effector memory T cells have been shown not to require secondary lymphoid organs to mediate allograft rejection (25). A characteristic of effector memory T cells is their ability to rapidly secrete IFN-{gamma} in response to their cognate Ag (32). To investigate whether naive mice possessed anti-porcine effector memory T cells, we used a recall (20-h) ELISPOT assay with IFN-{gamma} secretion as readout. Such assays have been shown to detect the presence of effector memory T cells in both mice and humans (32). The results show that spleen cells from naive aly/aly mice failed to demonstrate an increase in the frequency of IFN-{gamma}-positive cells when exposed to mitomycin C-treated porcine spleen cells in a one-way MLR vs naive aly/aly responders only (exposed aly/aly spleen cells = 10 spots/1 x 106 spleen cells vs aly/aly responders only = 12 spots/1 x 106 spleen cells, p = 0.6) (Fig. 7). Because it is possible that cross-reactive T cells could occur at lower frequencies than can be detected using 2 x 106 unsorted spleen cells/well, we repeated the ELISPOT experiment, but used 2 x 106 purified responder T cells/well. The results do not demonstrate the presence of effector memory T cells in naive aly/aly mice at this level of sensitivity (porcine-stimulated aly/aly T cells = 36 spots/1 x 106 T cells vs responders only = 37 spots/1 x 106 T cells, p = 0.8). Furthermore, the results were not changed when responder T cells were stimulated with mitomycin-treated syngeneic spleen cells (44 spots/1 x 106 T cells, p = 0.12). Thus, these results demonstrate that there is no evidence of preformed effector memory T cells in naive aly/aly mice that recognize porcine Ag, although it is possible that effector memory T cells that are beyond the detection limits of the ELISPOT assay still exist.



View larger version (51K):
[in this window]
[in a new window]
  		FIGURE 7. No evidence of preformed xenoreactive effector memory T cells in naive aly/aly mice. a, Naive aly/aly splenocytes did not reveal any evidence of xenoreactive cross-reactive memory T cells, as IFN-{gamma} spot counts after stimulation with donor mitomycin C-treated, porcine spleen cells were not significantly different from responder only control wells (stimulated aly/aly spleen cells = 10 spots/1 x 106 spleen cells vs aly/aly responders only = 12 spots/1 x 106 spleen cells, p = 0.6). b, ELISPOT wells demonstrating typical representatives of experimental groups.

 
Rejection of less divergent xenografts occurs in the absence of secondary lymphoid organs

We used a rat to murine skin xenograft model to investigate whether secondary lymphoid organs are required for the rejection of less divergent skin xenografts. In this model, acute rejection occurred in the absence of secondary lymphoid organs, although there was a modest delay in time to rejection (MST: aly+ = 13 days, aly/aly-SPL = 18 days, p = 0.004) (Fig. 8). Hence, these data demonstrate that secondary lymphoid organs are not critically important for rejection of less divergent xenografts, although their absence delays rejection modestly.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 8. Xenograft rejection still occurs in a less divergent xenograft strain combination (rat->mouse). The aly/aly-SPL recipients ({diamondsuit}) (MST = 18 days) rejected their xenografts in a modestly delayed fashion vs aly+ ({blacksquare}) recipients (MST = 13 days, p = 0.004).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have demonstrated that recipients devoid of secondary lymphoid organs are able to reject porcine skin xenografts without delay. We provide evidence that rejection in this model is T cell dependent and occurs when the Ag is presented via the direct pathway by the xenograft. Furthermore, we did not find any evidence that rejection is reliant on immune mechanisms known to be secondary lymphoid organ independent or dependent on immune activation within the bone marrow, a site that has recently been shown to possess immune activating capabilities (31). The inferences from our work are that recognition of xenoantigens occurs within the xenograft itself, a result that is different from experimental work in certain allograft, infectious, and tumor models (5, 6, 7, 8, 18).

Secondary lymphoid organs serve as a venue in which mature DCs can prime naive T cells, leading to a productive adaptive immune response (1). This coordinated meeting is orchestrated by chemoattractant chemokines that allow effective leukocyte homing (2). Naive T cells have an up-regulated expression of CCR7 and CD62L that allows their effective migration into the T cell zone of lymph nodes (33). Immature DCs, in contrast, have down-regulated CCR7, and up-regulated chemokines CCR1, CCR5, and CCR6, allowing entry into nonlymphoid tissues, where they can encounter foreign peptides (2, 33) (34). Once this happens, DC maturation leads to a switch in expression of chemokines, with CCR7 having now been up-regulated, with consequential DC migration to the T cell zone of lymph nodes via the high endothelial venules (2). Priming of T cells by DCs initiates a T cell effector phenotype that expresses high levels of inflammatory chemokines CCR3, CCR5, and CXCR3, and a low expression of CCR7 and CD62L, allowing these effectors to migrate to the site of inflammation. The importance of CCR7 for DC and T cell interactions within secondary lymphoid organs and their ability to produce a productive adaptive immune response have been demonstrated in studies using CCR7-deficient hosts and by inhibiting the CCR7 ligand CCL21 (35, 36). Thus, these studies clearly demonstrate the importance of secondary lymphoid organs in initiating immune responses.

The role of secondary lymphoid organs in solid organ transplantation has been controversial. Early studies indicated that T cell activation could occur within the graft itself (14, 15), and more recently, a study has demonstrated that donor endothelium could activate T cells directly within the allograft (16). However, prior studies have shown that skin allograft rejection does not occur in the aly/aly mouse (17, 18), in contrast to our present results in a xenotransplantation model. Furthermore, vascularized allografts are not rejected in the splenectomized aly/aly recipient (18) and are severely retarded, but ultimately lead to rejection in a related, but different mutation that also renders recipient mice devoid of secondary lymphoid organs (19).

Our work provides evidence that immunity to the xenograft is more effectively initiated by the xenograft than by i.p. immunization with xenogeneic spleen cells. This may be explained by several mechanisms. First, immune activation at the site of inflammation, in this case the xenograft, could occur via processing within tertiary lymphoid structures that develop in response to inflammation. Such a model was proposed to explain how local DCs prime T cells within lung tissue without the need to migrate to draining lymph nodes (37). A marker of the presence of lymphoid structures is the presence of peripheral node addressin (38). However, peripheral node addressin expression was only found in one of six rejecting xenografts from aly/aly-SPL recipients (data not shown). Thus, we have not found consistent data demonstrating the presence of tertiary lymphoid organs within rejecting xenografts. Second, Ag presentation within the graft could occur via donor APCs, for example endothelial cells. This is supported by a recent study demonstrating that donor endothelial cells can activate naive T cells in the setting of allotransplantation, and by another study demonstrating that porcine endothelial cells could activate human CD8+ T cells (16, 39). Prior studies have shown that both direct and indirect Ag presentation can occur in response to xenotransplantation (22, 23). Specifically, one study showed that when the indirect response was disabled, xenograft rejection was delayed by ~1 wk, although it still occurred, providing evidence that direct presentation was also responsible for xenograft rejection (22). In support of the above studies, our results provide evidence that presentation via both the direct and indirect pathway can induce an effector recall response, supporting a role for donor and recipient APCs in initiating cellular immune responses to xenografts. The pathway in which donor APCs within the graft directly activate responder T cells would also help explain why aly/aly-SPL recipients could not generate a humoral response to the xenograft as shown in Table I. Finally, our data in the RAG–/– mice that possess normal macrophage, DC, and NK cell function provide evidence that none of these subpopulations are critical or sufficient for xenograft rejection as RAG–/– recipients accepted their skin xenografts indefinitely. Future studies are required to determine the level of participation of these subpopulations, including the role of T cell-activated macrophages, that have been shown to be important in islet xenograft rejection (40).

Previous studies have demonstrated that the T cell precursor frequency to organ allografts is significantly higher than infectious foreign Ags (41, 42). Within organ transplants, xenografts are characteristically more divergent and display a wider array of foreign peptides to the host than an allograft (21). Because our data show that the more divergent the xenograft the reduced dependency on secondary lymphoid organs, one can speculate that this increased divergency leads to a higher frequency of graft-reactive T cells that can be activated at the site of injury. However, when we compared the number of IFN-{gamma}-secreting T cells by ELISPOT and measured the level of complement-fixing anti-donor IgG2a Ab levels between wild-type recipients of fully allogeneic skin allografts and porcine skin grafts, respectively, allografted recipients demonstrated higher number of IFN-{gamma}-producing cells and IgG2a levels vs xenograft recipients (data not shown). Clearly, future studies will be required to determine the mechanism of T cell activation in response to xenotransplantation.

Although significant advances have been made in understanding the mechanisms of xenograft rejection, most studies have focused on hyperacute and acute vascular rejection, with significantly less known concerning the cellular basis for xenograft rejection (21). The implication of our work is that activation requirements for cellular immunity against xenoantigens may be significantly reduced compared with alloantigens. Hence, the ability to control cellular immunity to xenografts is likely to be harder to control than for allografts. This agrees with prior experimental studies that have demonstrated difficulty in suppressing xenograft rejection using standard clinical immunosuppressive regimens (21). Hence, our results are highly relevant for the development of clinically applicable xenograft protocols.


   Acknowledgments
 
We thank Dr. Peter Heeger (Cleveland Clinic Foundation, Cleveland, OH) for helpful comments regarding ELISPOT analysis and for initially acquiring data on the CTL analyzer.


   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 K08 AI1732 to D.R.G. Back

2 Address correspondence and reprint requests to Dr. Daniel R. Goldstein, Section of Cardiovascular Medicine, Yale University School of Medicine, 333 Cedar Street, 3 FMP, P.O. Box 208017, New Haven, CT 06520-8018. E-mail address: daniel.goldstein{at}yale.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; aly/aly, alymphoplastic mouse; MST, median survival time; SPL, splenectomized. Back

Received for publication April 15, 2004. Accepted for publication August 2, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245.[Medline]
  2. Luster, A. D.. 2002. The role of chemokines in linking innate and adaptive immunity. Curr. Opin. Immunol. 14:129.[Medline]
  3. Mellman, I., R. M. Steinman. 2001. Dendritic cells: specialized and regulated antigen processing machines. Cell 106:255.[Medline]
  4. Janeway, C. A., P. Travers, M. Walport, M. J. Shlomchik. 2001. Immunological memory. Immunobiology: the Immune System in Health and Disease 412. Garland, New York.
  5. Voehringer, D., C. Blaser, A. B. Grawitz, F. V. Chisari, K. Buerki, H. Pircher. 2000. Break of T cell ignorance to a viral antigen in the liver induces hepatitis. J. Immunol. 165:2415.[Abstract/Free Full Text]
  6. Zinkernagel, R. M., S. Ehl, P. Aichele, S. Oehen, T. Kundig, H. Hengartner. 1997. Antigen localization regulates immune responses in a dose- and time-dependent fashion: a geographical view of immune reactivity. Immunol. Rev. 156:199.[Medline]
  7. Ochsenbein, A. F., P. Klenerman, U. Karrer, B. Ludewig, M. Pericin, H. Hengartner, R. M. Zinkernagel. 1999. Immune surveillance against a solid tumor fails because of immunological ignorance. Proc. Natl. Acad. Sci. USA 96:2233.[Abstract/Free Full Text]
  8. Karrer, U., A. Althage, B. Odermatt, C. W. Roberts, S. J. Korsmeyer, S. Miyawaki, H. Hengartner, R. M. Zinkernagel. 1997. On the key role of secondary lymphoid organs in antiviral immune responses studied in alymphoplastic (aly/aly) and spleenless (Hox11–/–) mutant mice. J. Exp. Med. 185:2157.[Abstract/Free Full Text]
  9. MartIn-Fontecha, A., S. Sebastiani, U. E. Hopken, M. Uguccioni, M. Lipp, A. Lanzavecchia, F. Sallusto. 2003. Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming. J. Exp. Med. 198:615.[Abstract/Free Full Text]
  10. Dieu, M. C., B. Vanbervliet, A. Vicari, J. M. Bridon, E. Oldham, S. Ait-Yahia, F. Briere, A. Zlotnik, S. Lebecque, C. Caux. 1998. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J. Exp. Med. 188:373.[Abstract/Free Full Text]
  11. Lechler, R., W. F. Ng, R. M. Steinman. 2001. Dendritic cells in transplantation: friend or foe?. Immunity 14:357.[Medline]
  12. Valujskikh, A., C. Hartig, P. S. Heeger. 2001. Indirectly primed CD8+ T cells are a prominent component of the allogeneic T-cell repertoire after skin graft rejection in mice. Transplantation 71:418.[Medline]
  13. Valujskikh, A., P. S. Heeger. 2000. CD4+ T cells responsive through the indirect pathway can mediate skin graft rejection in the absence of interferon-{gamma}. Transplantation 69:1016.[Medline]
  14. Vetto, R., R. Lawson. 1967. The role of vascular endothelium in the afferent pathway as suggested by the alymphatic renal homotransplant. Transplantation 5:1537.
  15. Barker, C., R. Billingham. 1968. The role of afferent lymphatics in the rejection of skin homografts. Transplantation 5:962.
  16. Kreisel, D., A. Krupnick, A. Gelman, F. Engels, S. Popma, A. Krasinskas, K. Balsara, W. Szeto, L. Turka, B. Rosengard. 2002. Non-hematopoietic allograft cells directly activate CD8+ T cells and trigger acute rejection: an alternative mechanism of allorecognition. Nat. Med. 8:233.[Medline]
  17. Miyawaki, S., Y. Nakamura, H. Suzuka, M. Koba, R. Yasumizu, S. Ikehara, Y. Shibata. 1994. A new mutation, aly, that induces a generalized lack of lymph nodes accompanied by immunodeficiency in mice. Eur. J. Immunol. 24:429.[Medline]
  18. Lakkis, F. G., A. Arakelov, B. T. Konieczny, Y. Inoue. 2000. Immunologic ‘ignorance’ of vascularized organ transplants in the absence of secondary lymphoid tissue. Nat. Med. 6:686.[Medline]
  19. Zhou, P., K. Hwang, D. Palucki, O. Kim, K. Newell, Y. Fu, M. Alegre. 2003. Secondary lymphoid organs are important but not absolutely required for allograft response. Am. J. Transplant. 3:259.[Medline]
  20. Lakkis, F. G.. 2003. Where is the alloimmune response initiated?. Am. J. Transplant. 3:241.[Medline]
  21. Auchincloss, H., Jr, D. H. Sachs. 1998. Xenogeneic transplantation. Annu. Rev. Immunol. 16:433.[Medline]
  22. Chitilian, H. V., T. M. Laufer, K. Stenger, S. Shea, H. Auchincloss. 1998. The strength of cell-mediated xenograft rejection in the mouse is due to the CD4+ indirect response. Xenotransplantation 5:93.[Medline]
  23. Dorling, A., G. Lombardi, R. Binns, R. I. Lechler. 1996. Detection of primary direct and indirect human anti-porcine T cell responses using a porcine dendritic cell population. Eur. J. Immunol. 26:1378.[Medline]
  24. Goldstein, D. R., B. M. Tesar, S. Akira, F. G. Lakkis. 2003. Critical role of the Toll-like receptor signal adaptor protein MyD88 in acute allograft rejection. J. Clin. Invest. 111:1571.[Medline]
  25. Chalasani, G., Z. Dai, B. T. Konieczny, F. K. Baddoura, F. G. Lakkis. 2002. Recall and propagation of allospecific memory T cells independent of secondary lymphoid organs. Proc. Natl. Acad. Sci. USA 99:6175.[Abstract/Free Full Text]
  26. Koglin, J., T. Glysing-Jensen, J. S. Mudgett, M. E. Russell. 1998. Exacerbated transplant arteriosclerosis in inducible nitric oxide-deficient mice. Circulation 97:2059.[Abstract/Free Full Text]
  27. Trambley, J., A. W. Bingaman, A. Lin, E. T. Elwood, S. Y. Waitze, J. Ha, M. M. Durham, M. Corbascio, S. R. Cowan, T. C. Pearson, C. P. Larsen. 1999. Asialo GM1+ CD8+ T cells play a critical role in costimulation blockade-resistant allograft rejection. J. Clin. Invest. 104:1715.[Medline]
  28. Karlsson-Parra, A., A. Ridderstad, A. C. Wallgren, E. Moller, H. G. Ljunggren, O. Korsgren. 1996. Xenograft rejection of porcine islet-like cell clusters in normal and natural killer cell-depleted mice. Transplantation 61:1313.[Medline]
  29. Shinkura, R., K. Kitada, F. Matsuda, K. Tashiro, K. Ikuta, M. Suzuki, K. Kogishi, T. Serikawa, T. Honjo. 1999. Alymphoplasia is caused by a point mutation in the mouse gene encoding NF-{kappa}B-inducing kinase. Nat. Genet. 22:74.[Medline]
  30. Sawada, T., P. A. DellaPelle, J. D. Seebach, D. H. Sachs, R. B. Colvin, J. Iacomini. 1997. Human cell-mediated rejection of porcine xenografts in an immunodeficient mouse model. Transplantation 63:1331.[Medline]
  31. Feuerer, M., P. Beckhove, N. Garbi, Y. Mahnke, A. Limmer, M. Hommel, G. J. Hammerling, B. Kyewski, A. Hamann, V. Umansky, V. Schirrmacher. 2003. Bone marrow as a priming site for T-cell responses to blood-borne antigen. Nat. Med. 9:1151.[Medline]
  32. Hartig, C. V., G. W. Haller, D. H. Sachs, S. Kuhlenschmidt, P. S. Heeger. 2000. Naturally developing memory T cell xenoreactivity to swine antigens in human peripheral blood lymphocytes. J. Immunol. 164:2790.[Abstract/Free Full Text]
  33. Muller, G., U. E. Hopken, M. Lipp. 2003. The impact of CCR7 and CXCR5 on lymphoid organ development and systemic immunity. Immunol. Rev. 195:117.[Medline]
  34. Campbell, D. J., C. H. Kim, E. C. Butcher. 2003. Chemokines in the systemic organization of immunity. Immunol. Rev. 195:58.[Medline]
  35. Saeki, H., A. M. Moore, M. J. Brown, S. T. Hwang. 1999. Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes. J. Immunol. 162:2472.[Abstract/Free Full Text]
  36. Forster, M., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, M. Lipp. 1999. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99:23.[Medline]
  37. Constant, S. L., J. L. Brogdon, D. A. Piggott, C. A. Herrick, I. Visintin, N. H. Ruddle, K. Bottomly. 2002. Resident lung antigen-presenting cells have the capacity to promote Th2 T cell differentiation in situ. J. Clin. Invest. 110:1441.[Medline]
  38. Shomer, N. H., J. G. Fox, A. E. Juedes, N. H. Ruddle. 2003. Helicobacter-induced chronic active lymphoid aggregates have characteristics of tertiary lymphoid tissue. Infect. Immun. 71:3572.[Abstract/Free Full Text]
  39. Murray, A. G., M. M. Khodadoust, J. S. Pober, A. L. Bothwell. 1994. Porcine aortic endothelial cells activate human T cells: direct presentation of MHC antigens and costimulation by ligands for human CD2 and CD28. Immunity 1:57.[Medline]
  40. Yi, S., W. J. Hawthorne, A. M. Lehnert, H. Ha, J. K. W. Wong, N. van Rooijen, K. Davey, A. T. Patel, S. N. Walters, A. Chandra, P. J. O’Connell. 2003. T cell-activated macrophages are capable of both recognition and rejection of pancreatic islet xenografts. J. Immunol. 170:2750.[Abstract/Free Full Text]
  41. Lindahl, K., D. Wilson. 1977. Histocompatibility antigen-activated cytotoxic T lymphocytes. II. Estimates of the frequency and specificity of precursors. J. Exp. Med. 145:508.[Abstract/Free Full Text]
  42. Matzinger, P., M. J. Bevan. 1977. Why do so many lymphocytes respond to major histocompatibility antigens?. Cell 29:1.



This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. E. Gelman, W. Li, S. B. Richardson, B. H. Zinselmeyer, J. Lai, M. Okazaki, C. G. Kornfeld, F. H. Kreisel, S. Sugimoto, J. R. Tietjens, et al.
Cutting Edge: Acute Lung Allograft Rejection Is Independent of Secondary Lymphoid Organs
J. Immunol., April 1, 2009; 182(7): 3969 - 3973.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
M.-C. Dieu-Nosjean, M. Antoine, C. Danel, D. Heudes, M. Wislez, V. Poulot, N. Rabbe, L. Laurans, E. Tartour, L. de Chaisemartin, et al.
Long-Term Survival for Patients With Non-Small-Cell Lung Cancer With Intratumoral Lymphoid Structures
J. Clin. Oncol., September 20, 2008; 26(27): 4410 - 4417.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
W. Chen, J. Diao, S. M. Stepkowski, and L. Zhang
Both Infiltrating Regulatory T Cells and Insufficient Antigen Presentation Are Involved in Long-Term Cardiac Xenograft Survival
J. Immunol., August 1, 2007; 179(3): 1542 - 1548.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
W. E. Walker, I. W. Nasr, G. Camirand, B. M. Tesar, C. J. Booth, and D. R. Goldstein
Absence of Innate MyD88 Signaling Promotes Inducible Allograft Acceptance
J. Immunol., October 15, 2006; 177(8): 5307 - 5316.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tesar, B. M.
Right arrow Articles by Goldstein, D. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tesar, B. M.
Right arrow Articles by Goldstein, D. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS