Donor Lymphoid Organs Are a Major Site of Alloreactive T-Cell Priming Following Intestinal Transplantation

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1 American Journal of Transplantation 2006; 6: Blackwell Munksgaard C 2006 The Authors Journal compilation C 2006 The American Society of Transplantation and the American Society of Transplant Surgeons doi: /j x Donor Lymphoid Organs Are a Major Site of Alloreactive T-Cell Priming Following Intestinal Transplantation J. Wang a,b, Y. Dong a,b, J.-Z. Sun a,b, R. T. Taylor c, C. Guo d, M.-L. Alegre e, I. R. Williams c and K. A. Newell a,b, a Emory Transplant Center, and the b Departments of Surgery and c Pathology, Emory University School of Medicine, Atlanta, Georgia, USA d Department of Medicine, Brigham and Women s Hospital, Harvard Medical School, Boston, Massachusetts, USA e Department of Medicine, University of Chicago, Chicago, Illinois, USA Corresponding author: Kenneth A. Newell, kenneth.newell@emoryhealthcare.org We hypothesized that lymphoid organs within intestinal allografts contribute to their immunogenicity. Consistent with this hypothesis recipient T cells rapidly migrated to the lymph nodes and Peyer s patches of syngeneic and allogeneic intestinal grafts such that at 24 h approximately 50% of the lymphocytes isolated from donor lymphoid organs were of recipient origin. However, only in the lymphoid organs of allografts did recipient T cells display an activated phenotype, proliferate and produce IFNc. Rejection of allogeneic intestines lacking lymphoid organs was dramatically impaired in splenectomized, lymph node-deficient recipients compared to lymph node bearing, wild-type allogeneic intestines. This demonstrates the important role of donor lymphoid organs in the rejection process. Furthermore, recipient T cells proliferated more extensively and produced more IFNc in donor lymphoid organs than in recipient lymphoid organs, indicating that donor lymphoid organs play a dominant role in initiating the recipient anti-donor immune response following intestinal transplantation. Key words: Cell trafficking, rodent, T cells, transplantation Received 2 May 2006, revised 14 June 2006 and accepted for publication 30 June 2006 Introduction The differing propensity of various transplanted organs and tissues to promote a rejection response is well recognized. Both clinical and experimental data demonstrate that intestinal allografts elicit an unusually strong recipient immune response (1,2). The potency of this immune response directly contributes to the inferior graft and patient survival of patients undergoing intestinal transplantation (3). There are several potential factors that may contribute to differences in the immunogenicity of different transplanted organs. First, the mechanism by which rejection is mediated may vary between organs. For example, rejection of heart allografts in mice is dependent upon CD4 + T cells (4) while CD8 + T cells are sufficient to mediate rejection of intestinal allografts (5). Similarly, kidney allografts in humans are rejected in a hyperacute manner in patients with preformed anti-donor alloantibodies while liver allografts are resistant to this mechanism of injury (6,7). The susceptibility of different transplanted organs to injury mediated by a given effector mechanism may also explain the differences in outcome observed after transplantation of different organs. Islets, heart, kidney and skin allografts have been shown to display different degrees of injury following adoptive transfer of TCR transgenic, allospecific CD8 + T cells (8). Unique anatomic features of the intestine in combination with specialized aspects of the intestinal immune system may also contribute to the enhanced immunogenicity of transplanted intestines. Similar to the lung, another highly immunogenic organ, the intestine interacts continuously and intimately with environmental antigens and a large variety of commensal and potentially pathogenic microorganisms. To facilitate these interactions the intestine makes use of specialized molecules that regulate cell trafficking and a highly ordered system of secondary and tertiary lymphoid tissues including mesenteric lymph nodes (MLNs), Peyer s patches (PPs), cryptopatches and isolated lymphoid follicles. Previous work has demonstrated a critical role of recipient secondary lymphoid organs for generating an immune response in naïve recipients to transplanted organs that do not contain secondary lymphoid organs. Heart allografts transplanted into splenectomized recipients congenitally lacking lymph nodes due to a spontaneous recessive point mutation in the gene encoding NFjB-inducing kinase (NIK) or knockout of the genes encoding the lymphotoxin b receptor or lymphotoxin a demonstrated 2563

2 Wang et al. dramatically prolonged survival (9,10). A potential role for lymphoid organs within intestinal allografts was suggested by the observation that, coincident with the histologic changes that define rejection, recipient T cells had migrated to the MLNs and PPs of transplanted intestines (11). Based on these observations we hypothesized that secondary lymphoid organs within the transplanted intestine are important contributors to the vigor of the recipient alloimmune response. Herein we demonstrate that recipient T cells migrate to the secondary lymphoid organs of transplanted intestines well before rejection is manifested histologically. Within intestinal allografts recipient T cells display an activated phenotype, undergo extensive proliferation and develop effector function. Allograft rejection is significantly impaired when both donor and recipient lack secondary lymphoid organs. These findings demonstrate a role for donor lymphoid organs in the rejection of transplanted intestines and suggest that strategies that interfere with the migration of recipient T cells to donor lymphoid organs may be useful as adjuncts to decrease the immunogenicity of intestinal allografts. Materials and Methods Mice C3H/HeJ, C57BL6, B6.SJL-Ptprca Pep3b/BoyJ (designated CD45.1) and lymphotoxin a knockout (LTa / ) mice on the C57BL/6 background were purchased from The Jackson Laboratories (Bar Harbor, ME, USA). Alymphoplasia (aly/aly) mice on the C57BL/6 background were obtained from F. Lakkis (University of Pittsburgh). Mice were housed in a specific pathogenfree facility. All studies were reviewed and approved by the Institutional Animal Care and Use Committee of Emory University. Intestinal transplantation and graft assessment Heterotopic intestine and heart transplantation were performed as described (12,13). Briefly, intestinal grafts were transplanted by anastomosing the graft portal vein and superior mesenteric artery to the recipient inferior vena cava and infrarenal aorta, respectively. A stoma was formed from the proximal graft and the distal graft was anastomosed to the recipient jejunum. Intestines from C3H (H2 k ), C57BL/6 (H2 b ), B6C3F2 aly/aly H2K k/k or H2K k/b ) and B6C3F2 WT (H2K k/k or H2K k/b ) donors were transplanted into C57BL/6 WT or splenectomized C57BL/6 LTa / (both H2 b ) recipients. Transplanted intestines were assessed histologically. Sections (3 lm) from formalin-fixed, paraffin-embedded graft specimens were stained with H&E and evaluated by a blinded pathologist. Rejection was graded using a modification of the system described by Ruiz et al. (14). Assigned scores ranged from 0 to 4 according to the following definitions: 0 normal architecture essentially identical to normal bowel, 1 scattered apoptotic crypt cells, 2 diffuse apoptosis of crypt cells, increased mitotic activity of crypt cells, and a mild mononuclear cell infiltrate, 3 moderate to severe mononuclear cell infiltrate with diffuse crypt destruction and 4 extensive mucosal ulceration with or without transmural necrosis. Heterotopic heart transplantation was performed by anastomosing the pulmonary artery and ascending aorta of the heart graft to the recipient s inferior vena cava and abdominal aorta, respectively. Mice were monitored daily. Rejection was defined as the cessation of palpable heart beat. Breeding and genotyping of B6C3F2 aly/aly and wild-type mice Male aly/aly mice on the C57BL/6 background were bred with female wildtype C3H mice to produce B6C3F1 aly/+ offspring which were then intercrossed to create B6C3F2 mice. B6C3F2 mice were screened for expression of the aly mutation and for expression of MHC K k and K b by PCR. The following primers were used to genotype mice for the presence of wild type and the aly mutation of NIK (modified from (15)): the sense primer for wild-type NIK (5 -AACATCCCGAGCTACTTCAACG-3 ), the sense primer for the aly mutation (5 -CACATCCCGAGCTACTTCAACAA- 3 ) and the antisense primer for both wild type and the aly mutation (5 - CCTTCGGGGACTCTACAGGC-3 ). PCR cycling parameters were: 94 Cfor 3 min; 30 cycles of 94 C for 40 s; 66.5 C for 30 s; 72 C for 30 s (GeneAmp PCR System 9600, PerkinElmer, Norwalk, CT, USA). In order to confirm that the mice generated by this breeding strategy expressed allogeneic MHC antigens all donor mice were genotyped for the MHC class I alleles H-2K k and K b as described by Hofer MJ et al. (16). Three of the 4 donors from both the B6C3F2 aly/aly experimental group and the B6C3F2 WT control group were heterozygous at the H2K locus (H2K k/b ) while the remaining donor in each group was homozygous (H2K k/k ). Consistent with our previous observations that intestines from B6C3F1 and C3H donors were promptly rejected by C57BL/6 recipients, no difference in the tempo or severity of rejection between H2K k/b and H2K k/k B6C3F2 donors was observed in the current experiments. Flow cytometric analysis Single-cell suspensions prepared from lymphoid organs or the peripheral blood were stained using antibodies specific for the following molecules: CD45.1 (A20), H-2K k (36-7-5), H-2K b (AF6-88.5), CD4 (RM4-5), CD8 (53-6.7), CD19 (1D3), CD25 (3C7), CD69 (H1.2F3) and CD62L (MEL-14, all from BD Biosciences, San Diego, CA, USA). Stained cells were analyzed using a FACSCalibur flow cytometer with either Cellquest or FlowJo software. Confocal imaging Tissue blocks were embedded in OCT and rapidly frozen in cold 2-methylbutane on dry ice. Six micron frozen sections were fixed for 10 min in cold acetone and then blocked with TNB buffer (PerkinElmer, Boston, MA). Sections were stained with directly conjugated anti-thy 1.2, anti-b220, anti-cd45.2 and anti-h-2k b (BD Biosciences). Two and three color fluorescence images were acquired using a Zeiss LSM510 confocal microscope. T-cell purification For adoptive transfer experiments splenic T cells were purified by positive selection using an AutoMACS (anti-cd90 (30-H12)-coated microbeads, Bergisch Gladbach, Germany). For ELISPOT experiments T cells were purified by negative selection using commercially available murine T-cell isolation columns (R&D Systems, Minneapolis, MN, USA). All isolations resulted in >90% purity. In vivo proliferation assay Purified T cells ( /ml) were labeled with the tracking fluorochrome 5-carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, Portland, OR) diluted to a final concentration of 5 lm in PBS. After 10 min at 37 C, cell labeling was terminated by addition of an equal volume of FCS for 1 minute. Cells were then washed twice in PBS. On the day of transplantation CFSE-labeled, purified T cells from C57BL/6 CD45.1 congenic mice were transferred i.v. into C57BL/6 recipients (CD45.2) of intestines from C3H or C57BL/6 donors (both CD45.2). Three to 4 days following cell transfer recipient mice were sacrificed and the intensity of green fluorescence by CD45.1 cells within the recipient s blood, peripheral LN, MLNs and spleen determined by flow cytometry and compared to that of CD45.1 cells within the donor/graft MLNs. ELISPOT The frequency of IFNc - producing T cells was determined after a 24 h stimulation with irradiated (2000 rads) stimulator cells using the ELISPOT 2564 American Journal of Transplantation 2006; 6:

3 Donor Lymphoid Organs assay as previously described (17). Briefly purified T cells from responder mice together with irradiated (2000 rad) syngeneic or allogeneic stimulator cells were added to each well of plates coated with a capture mab specific for murine IFNc (R46A2, PharMingen, San Diego, CA, USA). After incubation for h at 37 C, the plates were washed and developed by sequentially adding and washing away biotinylated rat anti-mouse IFNc capture mab (XMG1.2, PharMingen, San Diego, CA), an alkaline phosphatase-conjugated anti-biotin antibody (Vector Laboratories, Burlingame, CA), nitroblue tetrazolium chloride (Bio-Rad Laboratories, Hercules, CA) and 5-bromo-4-chloro-3-indolyl phosphate (Sigma Chemical Co., St. Louis, MO, USA) substrate. Individual wells were analyzed using Image- Pro Plus (Media Cybernetics, Silver Spring, MD, USA). Statistics Continuous variables were compared using the unpaired t test with Welch correction to compare two groups and the one-way ANOVA to compare three or more groups.values of p< 0.05 were considered significant. Comparison of binary data groups was performed using Fisher s exact test. Results Recipient T cells rapidly migrate to the MLNs and PPs of transplanted intestines Previous studies have demonstrated that the rejection of heart and skin allografts by naïve recipient mice is critically dependent upon the presence of recipient secondary lymphoid organs (9,10). In initial experiments we confirmed the finding that the survival of C3H heart allografts is significantly prolonged in splenectomized, C57BL/6 LN-deficient LTa / recipients (MST > 99 vs days for splenectomized and non-splenectomized LTa / recipients, respectively, N = 5 mice per group). In contrast to the prolonged survival of heart allografts in recipients lacking secondary lymphoid organs, intestinal allografts from wild type C3H donors transplanted into splenectomized C57BL/6 LTa / recipients lacking secondary lymphoid organs all displayed histologic evidence of severe rejection by day as reflected by a mean rejection score of 3.9 ± 0.3 (N = 10 mice). As the splenectomized LTa / recipients had no secondary lymphoid organs, we reasoned that the initial priming of the alloreactive recipient T cells might occur within the transplanted intestine itself. Analysis of the MLNs of C3H intestinal allografts transplanted into splenectomized C57BL/6 LTa / recipients demonstrated that by day 3 the majority of the CD8 + lymphocytes were of recipient origin (H2K k negative) and that by day 5 virtually all of the CD8 + lymphocytes were of recipient origin (Figure 1A). Both CD4 + lymphocytes and B cells displayed a similar pattern of migration to the donor MLNs (data not shown). We next asked whether this same process occurred in wild type, nonsplenectomized recipients. As previously discussed, an earlier study demonstrated that by day 5 the vast majority of T cells isolated from the MLNs and PPs of intestinal allografts were of recipient origin (11). However, by this time histologic evidence of established rejection was noted. We sought to determine whether significant infiltration of donor secondary lymphoid organs by recipient T cells preceded the development of rejection as defined by characteristic histologic changes. For these experiments C3H (H2 k ) or C57BL/6 (H2 b ) (CD45.1) intestines were transplanted into C57BL/6 (CD45.2) recipients. As shown in Figure 1B, within 24 h of transplantation large numbers of recipient T and B cells had migrated to the T and B cell areas of both syngeneic and allogeneic intestinal grafts (identified as CD and H2K b+ cells respectively). In a third set of experiments intestines from C57BL/6 or C3H donors (both CD45.2) were transplanted into C57BL/6 (CD45.1) recipients. Flow cytometric analysis demonstrated that approximately one half of the T cells isolated from the MLNs and slightly more of the cells from the PPs of transplanted intestines were of recipient origin at 24 h (Figure 1C). Analysis of cells stained with antibodies specific for CD4 and CD8 revealed that both of these T cell subsets displayed a similar ratio of donor:recipient T cells with approximately half of the CD4 + and CD8 + T cells isolated from the MLN and PPs being of recipient origin (data not shown). Thus, the infiltration of donor secondary lymphoid organs by recipient lymphocytes occurs several days before histologic evidence of rejection, as indicated by epithelial injury, is evident. Recipient T cells that have migrated to the MLNs of intestinal allografts display an activated phenotype, proliferate vigorously and produce IFNc The observation that recipient T cells migrated rapidly and in significant numbers to the lymphoid organs of both allogeneic and syngeneic intestinal grafts raised the possibility that this migration pattern could represent the physiologic trafficking of T cells to the secondary lymphoid organs of the transplanted intestine. We reasoned that if the recipient T cells infiltrating the lymphoid organs of allografts contributed significantly to the recipient alloimmune response, these cells should display different phenotypic and functional properties than T cells infiltrating syngeneic intestinal grafts. In order to test this hypothesis intestines from CD45.2 C3H or C57BL/6 donors were transplanted into CD45.1 C57BL/6 recipients. Consistent with the prediction that recipient T cells were activated in the secondary lymphoid organs of allografts but not isografts, significantly more recipient (CD45.1) CD8 + and CD4 + T cells infiltrating the MLNs of intestinal allografts had up-regulated their expression of CD25 and CD69 and down-regulated their expression of CD62L relative to recipient T cells infiltrating the MLNs of syngeneic intestinal grafts (Figure 2). Similar results were obtained when recipient T cells migrating to the PPs of intestinal grafts were examined (data not shown). In addition to these phenotypic differences, we examined the recipient T cells infiltrating the MLNs of transplanted intestinal iso- and allografts for functional differences. For these experiments CFSE-labeled, purified T cells from naïve CD45.1 C57BL/6 mice were transferred American Journal of Transplantation 2006; 6:

4 Wang et al. intravenously into congenic C57BL/6 (CD45.2) recipients of C3H or C57BL/6 (both CD45.2) intestinal grafts. Four days after transplantation the proliferation of the labeled CD45.1 T cells infiltrating the graft MLN as well as various compartments within the recipient was assessed using flow cytometry to quantify CFSE dilution. Consistent with the activated phenotype of recipient T cells infiltrating the MLNs of allografts and the quiescent phenotype of recipient T cells infiltrating the MLNs of isografts, adoptively transferred, CFSE-labeled recipient strain T cells isolated from the MLNs of intestinal allografts had undergone extensive proliferation with >68% of the CD8 + T cells demonstrating evidence of having divided at least once compared to <7% of the recipient strain T cells infiltrating the MLNs of intestinal isografts (Figure 3D). Furthermore, not only was the percentage of cells proliferating in the MLNs of allografts much greater than that in isografts, but the number of cell divisions for those cells that did proliferate was much greater (Figure 3A C). Similar results were observed for CD4 + T cells (data not shown). In order to compare the function of T cells within these various lymphoid compartments following intestinal transplantation T cells were isolated from the recipient spleen, recipient peripheral LNs and graft MLNs. These purified T cells were cultured for 72 h with donor strain allogeneic or syngeneic stimulator cells and the number of IFNc - producing cells quantified by ELISPOT. As shown in Figure 4A,B, the frequency of T cells within the MLNs of allografts that produced IFNc when stimulated with donor antigen was significantly greater than that of T cells isolated from the MLNs of intestinal isografts (Figure 4A,B). This finding indicates that following activation, T cells infiltrating allograft secondary lymphoid organs differentiate into effector cells. These data demonstrate that recipient T cells rapidly migrate to the secondary lymphoid organs of transplanted intestinal allografts where they display evidence of activation and effector function but do not address the relative Figure 1: Rapid infiltration of allograft lymphoid organs by recipient T cells. (A) Flow cytometric analysis on days 3, 4 and 5ofCD8 + T cells isolated from the MLNs of C3H intestines transplanted into splenectomized LTa / recipients. Cells of donor origin are H2K k+ while those of recipient origin are H2K k. (B) Confocal imaging on day 1 of recipient lymphocytes infiltrating the MLNs and PPs of C3H (H2 k ) and C57BL/6 (CD45.1) intestines transplanted into wild type C57BL/6 (H2 b, CD45.2) recipients. The T cell and B cell areas are identified by staining with Thy1.2 and B220 respectively. Recipient T cells infiltrating allografts are identified by positive staining for H2K b while recipient T cells infiltrating isografts are identified by positive staining for CD45.2. (C) Summary of data generated by flow cytometric analysis on day 1 of the MLNs and PPs of intestinal isografts (C57BL/6 CD45.2) and allografts (C3H CD45.2) transplanted into C57BL/6 CD45.1 recipients (N = 4 mice per group). Cells isolated from the MLNs and PPs of intestinal grafts were stained with antibodies specific for CD45.1, CD45.2, CD4, and CD8. Data depict the mean percentage of donor (CD45.2) and recipient (CD45.1) origin cells ± the standard deviation for the total number of cells staining positive for CD4 or CD American Journal of Transplantation 2006; 6:

5 Donor Lymphoid Organs Figure 2: Recipient T cells infiltrating allograft lymphoid organs display an activated phenotype. Intestines from CD45.2 C3H or C57BL/6 donors were transplanted into CD45.1 C57BL/6 recipients. One day following transplantation cells isolated from the MLNs were analyzed by gating on the CD45.1 (recipient) cells stained with mab specific for CD4 or CD8 and CD25, CD69, and CD62L. Data are depicted as the mean percentage of the cells expressing each activation marker ± the standard deviation (N = 4 mice per group, denotes p < 0.05). contributions of the donor and recipient lymphoid organs in the immune response to intestinal allografts. Quantification of the proliferative response of recipient T cells isolated from various recipient and donor compartments demonstrated that the cells isolated from the allograft MLNs had proliferated to a much greater extent that those isolated from the recipient blood, peripheral LN, MLNs or spleen (Figure 3A D). Similarly, the frequency of T cells producing IFNc in response to donor allogeneic cells was greater in the allograft (donor) MLNs than in either the recipient spleen or peripheral LN (Figure 4A,B). Based on these observations donor secondary lymphoid organs appear to be a primary site at which immune responses are initiated/occur at early time points following intestinal transplantation. rejected by C57BL/6 recipients in a manner indistinguishable from C3H intestines (18), B6C3F2 intestines that expressed H-2K k and were homozyogous for the wild-type NIK gene and therefore had normal MLNs and PPs were promptly rejected (Figure 5A C). However, intestinal allografts from littermates homozyogous for the aly mutation and therefore lacking secondary lymphoid organs developed only very mild rejection (as indicated by a slight increase in the number of apoptotic crypt cells) at a time point by which all intestinal allografts from the control wild-type mice had developed severe rejection. These experiments demonstrate that donor secondary lymphoid organs are important contributors to the recipient immune response to intestinal allografts. MLNs within the donor intestinal allograft are sufficient to mediate rejection While our results supported the hypothesis that donor secondary lymphoid organs included with the transplanted intestine contributed to the recipient anti-donor response, they did not address whether donor lymphoid organs were sufficient to support allograft rejection. Given the intestine s specialized role in immune surveillance, we considered the alternative possibility that enterocytes and/or dendritic cells residing within the transplanted intestine might be capable of initiating an anti-donor response by recipient T cells in the complete absence of recipient and donor secondary lymphoid organs. In order to further investigate the role of allograft lymphoid organs in the rejection process, we generated mice that expressed allogeneic MHC molecules but lacked lymph nodes and PPs. B6C3F2 mice homozygous for the mutated NIK gene and therefore lacking MLNs and PPs (designated B6C3F2 aly/aly) and their littermates homozygous for the wild-type NIK gene (designated B6C3F2 WT/WT) that expressed at least one K k allele were used as intestine donors. Consistent with the rapid rejection of wild-type C3H intestines transplanted into splenectomized LTa / recipients and our previous demonstration that B6C3F1 intestines are promptly Discussion Previous studies have demonstrated the pivotal role of recipient lymphoid organs during the initiation of immune responses to alloantigens. Donor-reactive T cells that had differentiated into cytokine producing effectors in response to skin allografts were shown to preferentially populate the draining lymph nodes of wild-type recipient mice at early time points and to subsequently accumulate in the spleen at later time points (19). Using the ELISPOT assay, this pattern was shown to apply to T cells primed via either the direct or the indirect pathways of antigen presentation. More recently the central role of recipient lymph nodes during the immune response to heart allografts was confirmed using a TCR transgenic model in which T cells transgenic for a TCR that is specific for a single MHC class I antigen (K d ) were transferred into wild-type recipients transplanted with a heart that was syngeneic except for the expression of the MHC class I transgene K d (20). The transferred transgenic T cells rapidly disappeared from the blood and accumulated in the peripheral LNs where they proliferated. After several days the primed cells migrated to the heart allograft where they mediated rejection but did not American Journal of Transplantation 2006; 6:

6 Wang et al. Figure 3: Continued. proliferate further. These observations together with the previously discussed reports which demonstrated prolonged survival of heart allografts in splenectomized mice that lack peripheral LNs due to genetic mutations (9,10) provide conclusive evidence that the alloimmune response to at least some organs is critically dependent upon the environment provided by recipient secondary lymphoid organs. Figure 3: Allograft mesenteric lymph nodes are the primary site of T cell proliferation at early time points following intestinal transplantation. Anin vivo proliferation assay was used to assess the proliferation of adoptively transferred CFSE-labeled C57BL/6 CD45.1 congenic T cells isolated from various compartments of C57BL/6 CD45.2 recipients of intestinal grafts (all CD45.2) 96 h after transplantation and cell transfer. (A) Representative dot plots and histograms depicting the proliferation of CD45.1 congenic T cells in the recipient peripheral LNs, (B) the recipient spleen and (C) the donor MLNs. (D) Summary of the proliferation of adoptively transferred CD45.1 T cells in different recipient and donor compartments (N = 3 mice for both syngeneic and allogeneic transplanted intestines, denotes p = 0.01 for donor MLN vs. recipient lymphoid organs). Data are depicted as the mean ± the standard deviation. While the contribution of recipient secondary lymphoid organs to the process of rejection is well recognized, the role of the graft lymphoid structures has been less fully explored. The very early migration of recipient lymphocytes to the lymphoid organs of both allogeneic and syngeneic intestinal grafts suggests that this process may initially be alloantigen independent. However, the observation that recipient T cells acquire an activated phenotype, proliferate and produce cytokines such as IFNc within the lymphoid structures of allografts but not isografts indicates that maturation of the immune response is alloantigenspecific. Experiments conducted using lymphoid organ deficient donors or recipients demonstrated that either recipient or donor secondary lymphoid organs were sufficient to promote rejection. However, in the absence of both donor and recipient lymphoid structures, rejection was significantly inhibited. We interpret this finding to indicate the important role of both donor and recipient lymphoid organs in the immune response to intestinal allografts. Alternatively, while we are not aware of data that demonstrate reduced numbers of T cells in LTa / or aly/aly mice, it may be hypothesized that rejection is impaired in mice lacking lymphoid organs due to a concomitant reduction in the number of lymphocytes. However, we do not favor this hypothesis for several reasons. First, splenectomized LTa / recipients, that by this hypothesis would have reduced numbers of lymphocytes, reject intestinal allografts that contain lymphoid structures with normal kinetics. This finding implies that any reduction in recipient lymphocyte numbers that may exist in LTa / mice is not sufficient to impair rejection. Furthermore, in a heart transplant model the transfer of even relatively low numbers of K d -specific 2568 American Journal of Transplantation 2006; 6:

7 Donor Lymphoid Organs Figure 4: Increased frequency of primed, donor-reactive T cells in the mesenteric lymph nodes of intestinal allografts. T cells isolated from the peripheral LN and spleen of C57BL/6 recipients and the MLNs of either C3H or C57BL/6 donors 72 h after intestinal transplantation were cultured with donor or self stimulator cells for 18 h in an ELISPOT assay. (A) Photographs of representative wells depicting the frequency of IFNc -producing T cells isolated from the graft MLNs, recipient peripheral LN, and recipient spleen following culture with C3H stimulators. (B) Summary data indicating the frequency of IFNc -producing T cells isolated from different donor and recipient lymphoid compartments. Data are depicted as the mean ± the standard deviation (N = 3 mice per group, denotes p < 0.03 for donor MLN vs. recipient lymphoid organs). For each group the frequency of IFNc -producing cells shown was calculated by subtracting the frequency of cells producing IFNc following stimulation with C57BL/6 cells (background) from the frequency of cells producing IFNc following stimulation with C3H cells. TCR transgenic T cells into immunocompetent mice bearing allografts from K d transgenic donors has been shown to be sufficient to produce prompt rejection (20). The potential contribution of a reduction in the number of donor origin lymphocytes within intestinal allografts that lack lymphoid structures also seems unlikely as lymphoid organ-deficient allografts are rejected promptly by wild-type recipients (see below). Thus, while we have not formally excluded the possibility that lymphocyte numbers are reduced in mice lacking lymphoid organs to the point that rejection is significantly impaired, we believe the data more strongly support the absence of the lymphoid structures themselves as the factor responsible for the prolonged survival of intestinal allografts in our studies. Consistent with the important role of recipient lymphoid organs in alloimmunity, in experiments not shown we have found that intestinal allografts from lymphotoxin b receptor knockout donors that lack MLNs and PPs were rejected promptly by wild-type recipients. Thus, while donor secondary lymphoid organs are sufficient to mediate rejection of intestinal allografts, they are not necessary. Based on the tempo and severity of rejection, it is not possible to compare the relative contributions of recipient and donor secondary lymphoid organs to the rejection process. However, the extensive proliferation of recipient T cells that occurs primarily within the MLNs of intestinal allografts together with the significantly increased frequency of IFNc - producing donor-reactive T cells in the allograft MLNs relative to the recipient LNs and spleen suggests that the secondary lymphoid organs of the allograft play a primary role in initiating the recipient anti-donor immune response. We believe these findings may be of relevance to clinical transplantation and may help explain the greater immunogenicity of organs such as the intestine or lung that contain significant amounts of lymphoid tissues relative to organs such as the heart, kidney and liver that contain relatively little lymphoid tissue. The histologic evidence of very mild rejection of intestinal allografts at day 14 in recipients lacking both donor and recipient secondary lymphoid organs is an important American Journal of Transplantation 2006; 6:

8 Wang et al. Figure 5: Rejection of intestinal allografts is impaired when both the recipient and donor lack secondary lymphoid organs. Intestines from H- 2K k expressing, lymph node-deficient donors (designated B6C3F2 aly/aly) were transplanted into splenectomized C57BL/6, lymph node-deficient recipients (LTa / ). For the control group intestines from H-2K k expressing, littermates that were homozygous for wild type NIK (designated B6C3F2 wt/wt) were transplanted into splenectomized C57BL/6, lymph node-deficient recipients (LTa / ). (A) The gross appearance of representative B6C3F2 intestines expressing wild type NIK (and hence containing normal lymphoid organs) or the aly/aly mutation of NIK (thus lacking all secondary lymphoid organs) 14 days after transplantation into splenectomized C57BL/6 LTa / recipients. (B) The histologic appearance on day 14 of representative H-2K k expressing B6C3F2 intestines expressing wild type NIK or the aly/aly mutation transplanted into splenectomized C57BL/6 LTa / recipients. (C) Mean rejection scores on day 14 of H-2K k expressing B6C3F2 wt/wt and aly/aly intestines transplanted into splenectomized C57BL/6 LTa / recipients. Each point represents an individual recipient (p < 0.03 wild type vs. aly/aly donors). observation. Heart allografts from wild-type donors were observed to be rejected in a delayed manner by lymphotoxin a and lymphotoxin b receptor knockout recipients lacking secondary lymphoid organs due to the eventual formation of tertiary lymphoid organs (10). However, in our experiments the mild rejection observed when both the donor and recipient lack secondary lymphoid organs cannot be due to lymphoid organ neogenesis as genetic mutations of both the donor and recipient preclude the formation of tertiary lymphoid organs. Thus, while donor secondary lymphoid organs contribute significantly to the rejection of intestinal allografts and appear to play a greater role in the early immune response following intestinal transplantation than do recipient secondary lymphoid organs, our data suggest that components of the transplanted intestine other than the secondary lymphoid organs may also be capable of promoting rejection, albeit in a much delayed fashion. A recent report that naïve recipient mice lacking secondary lymphoid organs reject xenogeneic skin grafts demonstrates that in some settings rejection can occur in the complete absence of secondary lymphoid organs (21). One possible explanation for our observations is that the unique immunologic and structural environment of the intestine facilitates direct T-cell priming by the large 2570 American Journal of Transplantation 2006; 6:

9 Donor Lymphoid Organs number of donor dendritic cells residing within the transplanted intestine. Alternatively, the finding that under appropriate conditions intestinal epithelial cells can function as antigen-presenting cells in vitro is intriguing and suggests that the graft epithelial cells may directly contribute to the priming of recipient T cells after transplantation (22). Future experiments will more fully investigate the mechanisms of intestinal allograft rejection in the complete absence of secondary lymphoid organs. In summary, the implication of our data is that the greater immunogenicity of intestinal allografts may be at least partially explained by the large amount of organized lymphoid tissue included with the transplanted intestine. Thus interfering with the early migration of recipient T cells to the secondary lymphoid organs of intestinal allografts may diminish their enhanced immunogenicity. Because the removal of donor lymphoid tissues from the intestinal allograft either surgically or functionally using irradiation is not technically possible and/or results in significant damage to the graft, the therapeutic exploitation of this finding will require the identification of the molecular regulators of lymphocyte trafficking to the lymphoid organs of inflamed intestines and the development of agents capable of inhibiting this migration. Acknowledgment This work was supported by Grants AI and AI51224 (to K.A.N), DK64730 (to I.R.W.) and DK64399 (the Emory Endothelial Pathobiology Research Development Center). References 1. Zhang Z, Zhu L, Quan D et al. Pattern of liver, kidney, heart, and intestine allograft rejection in different mouse strain combinations. Transplantation 1996; 62: Grant D, Abu-Elmagd K, Reyes J et al report of the intestine transplant registry: A new era has dawned. 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