Tertiary Lymphoid Tissues Generate Effector and Memory T Cells That Lead to Allograft Rejection

American Journal of Transplantation 2007; 7: 1071 1079 Blackwell Munksgaard C 2007 The Authors Journal compilation C 2007 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/j.1600-6143.2007.01756.x Tertiary Lymphoid Tissues Generate Effector and Memory T Cells That Lead to Allograft Rejection I. W. Nasr a, M. Reel b, M. H. Oberbarnscheidt b, R. H. Mounzer c, F. K. Baddoura d, N. H. Ruddle c,e and F. G. Lakkis a,, a Thomas E. Starzl Transplantation Institute, Departments of Surgery and Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA b Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT c Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT d Pathology and Laboratory Medicine, Veterans Affairs Medical Center & State University of New York, Buffalo, NY e Section of Immunobiology, Yale University School of Medicine, New Haven, CT Co-senior authors Corresponding author: Fadi G. Lakkis, lakkisf@upmc.edu Tertiary lymphoid tissues are lymph node-like cell aggregates that arise at sites of chronic inflammation. They have been observed in transplanted organs undergoing chronic rejection, but it is not known whether they contribute to the rejection process by supporting local activation of naïve lymphocytes. To answer this question, we established a murine transplantation model in which the donor skin contains tertiary lymphoid tissues due to transgenic expression of lymphotoxin-a (RIP-LT a ), whereas the recipient lacks all secondary lymphoid organs and does not mount primary alloimmune responses. We demonstrate in this model that RIP-LT a allografts that harbor tertiary lymphoid tissues are rejected, while wild-type allografts that lack tertiary lymphoid tissues are accepted. Wildtype allografts transplanted at the same time as RIP- LT a skin or 60 days later were also rejected, suggesting that tertiary lymphoid tissues, similar to secondary lymphoid organs, generate both effector and memory immune responses. Consistent with this observation, naive T cells transferred to RIP-LTa skin allograft but not syngeneic graft recipients proliferated and differentiated into effector and memory T cells. These findings provide direct evidence that tertiary lymphoid structures perpetuate the rejection process by supporting naïve T-cell activation. Key words: Immunologic memory, lymphoid neogenesis, lymphotoxins, rejection, T cells, transplantation Received 22 November 2006, revised 10 January 2007 and accepted for publication 16 January 2007 Introduction Tertiary lymphoid tissues are ectopic accumulations of lymphoid cells formed in sites that are normally devoid of canonical lymphoid organs. They arise in states of chronic inflammation through a process called lymphoid neogenesis (1). They have been observed in autoimmunity (Hashimoto s thyroiditis, rheumatoid arthritis, myasthenia gravis, Sjogren s syndrome and multiple sclerosis), chronic microbial infection (hepatitis C, Helicobacter pylori, and Lyme disease) and chronic allograft rejection (2,3). Tertiary lymphoid tissues share several key morphological characteristics with secondary lymphoid organs such as peripheral lymph nodes. These include the presence of high endothelial venules (HEV), discrete T- and B-cell accumulations (often accompanied by germinal centers), CD11c + dendritic cells (DC), and follicular dendritic cell (FDC) networks (3). HEV in tertiary lymphoid tissues express peripheral node addressin (PNAd) or mucosal addressin cell adhesion molecule (MAdCAM-1) (1,4), glycoproteins that mediate the extravasation of naïve T cells (5). Chemokines that recruit naïve T cells (CCL19, CCL21), B cells (CXCL13) and DC (CCL21) have also been detected (4,6 8). The biochemical pathways responsible for the genesis of secondary lymphoid tissues during ontogeny are responsible for lymphoid neogenesis in adult animals (1,4,9). Central to these pathways are the lymphotoxin cytokine family, which includes lymphotoxin (LT)-a 1 b 2, and the LT-b receptor (LTbR) (10). The morphological similarities between tertiary lymphoid tissues and peripheral lymph nodes, and their proximity to the source of antigen, have led to the hypothesis that tertiary lymphoid tissues generate adaptive immune responses that contribute to disease pathogenesis (2,3,11). This hypothesis is supported primarily by studies of B-cell differentiation. Oligoclonal B-cell expansion, somatic hypermutation of immunoglobulin variable genes and differentiation of naïve B cells into memory and plasma cells have been observed within tertiary lymphoid tissue germinal centers in patients with rheumatoid arthritis (12), Sjogren syndrome (13), autoimmune thyroiditis (14), myasthenia gravis (15) and multiple sclerosis (16). Likewise, immunization with heterologous antigen generates plasma cells and immunoglobulin isotype switching in tertiary lymphoid tissues induced in mice by transgenic overexpression of LTa (1). Evidence that naïve T cells are activated in tertiary lymphoid tissues derives from murine 1071

Nasr et al. studies in which the over-production of either LTa or LIGHT by tumor cells or pancreatic islets leads to the formation of local lymph node like structures and enhances tumor rejection or the development of diabetes, respectively (17 19). These findings suggest that tertiary lymphoid tissues may generate primary immune responses. In addition to autoimmunity and infection, tertiary lymphoid tissues arise in the setting of organ transplantation. PNAd + HEV, lymphocyte clusters and lymphatic channels reminiscent of lymph node architecture have been described in human and mouse heart or kidney allografts in association with chronic rejection (20 23). PNAd + blood vessels, in the absence of lymphocyte clusters, have also been reported in allografts undergoing acute rejection (24,25). Despite these histopathological associations, it is unclear whether tertiary lymphoid tissues contribute to the rejection process by providing local sites for the activation of recipient naï ve lymphocytes or are simply an epiphenomenon of the inflammatory process that accompanies rejection. To address this question, we set up a murine skin transplantation model in which the transplanted donor skin contains tertiary lymphoid tissues due to the transgenic overproduction of LTa(the RIP-LT a mouse) (26), whereas the recipient lacks all secondary lymphoid tissues due to a defect in the LTbR signaling pathway (aly/aly mice from whom the spleen was surgically removed) (27,28). In this model, the aly/aly host is severely compromised in its ability to mount primary immune responses (29,30), including those that lead to allograft rejection (31), and the only lymph node like tissue present is confined to the RIP-LT a graft. Using this model, we demonstrate that intra-graft tertiary lymphoid tissues, like secondary lymphoid organs, generate both effector and memory T-cell responses that lead to graft rejection. These findings provide evidence that tertiary lymphoid structures perpetuate the rejection process by supporting naï ve T-cell activation within the transplanted organ itself. Materials and Methods Mice Wildtype (wt) C57BL/6 (B6, Thy1.2, H-2 b ), B6.PL-Thy1a/Cy (B6.1.1, Thy1.1, H-2 b ), and CB6F1/J (H-2 d/b ) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Alymphoplastic (aly) mice (Map3k14 /, Thy1.2, H-2 b ), which lack lymph nodes and Peyer s patches due to a spontaneous point mutation in NFjB-inducing kinase (NIK) (27,28), were purchased from CLEA Japan (Osaka, Japan) and bred under specific pathogen-free conditions. Homozygous aly/aly mice underwent splenectomy to create mice that lack all secondary lymphoid tissues (aly/aly-spleen). RIP-LT a mice (B6, Thy1.2, H-2 b ) (26) were maintained in our colony and bred with wt (B6, Thy1.2) mice at the Yale Animal Resource Center. RIP-LT a(b6, Thy1.2, H-2 b ) mice were crossed with BALB/cJ mice to generate (RIP-LT a BALB/c)F1 (H-2 d/b ) donors for allogeneic skin transplantation. Surgical procedures Splenectomy was performed according to established techniques via a subcostal incision in the mid-axillary line (32). Completeness of splenectomy was verified postmortem in each mouse. Full thickness skin transplantation was performed as follows. Abdominal skin from donor Balb/c or C3H mice was harvested and subcutaneous layers reduced by gentle scraping. A 1.5- cm 2 graft bed was prepared on the recipient s dorsolateral aspect. Donor skin was cut to fit the prepared bed and secured with sutures and bandage for 7 days, after which the bandage was removed and the graft was monitored every other day. Rejection was defined as >90% graft necrosis. Isolation and adoptive transfer of naïve T cells Spleen and lymph node cells were harvested from Thy1.1 + B6 mice and enriched for T cells using Nylon Wool Fiber Columns (Polysciences, Inc., Warrington, PA). Nylon wool-purified T cells were then labeled with fluorochrome-tagged antibodies against CD4, CD8 and CD44 and sorted for naive CD4 + CD44 lo and CD8 + CD44 lo populations (>98% purity) using a FACS Aria high-speed cell sorter (BD Biosciences, CA). Sorted CD4 and CD8 naive T cells (Thy1.1 + ) were labeled with CFSE (Molecular Probes, Eugene, OR) for 10 min at 37 C, washed with PBS and adoptively transferred i.v. to aly/aly-spleen recipients (Thy1.2 + ) that had received either allogeneic or syngeneic skin grafts. 5 10 6 naïve CD4 or CD8 T cells were transferred to each mouse. Cells were analyzed prior to adoptive transfer to establish their baseline phenotype. Cell isolation after adoptive transfer Cells were harvested 15 or 80 days after adoptive transfer from the blood, liver, lungs, bone marrow and skin graft. Recipient mice were perfused with heparin (100 U/mL) at the time of sacrifice. Blood was removed by cardiac puncture and RBCs were lysed. The liver was perfused via the portal vein with 50 U/mL collagenase IV solution (Worthington Biochemical, Lakewood, NJ), removed and passed through a 70-lm strainer. The tissue suspension was incubated at 37 C with 50 U/mL collagenase IV in RPMI 1640 plus 5% FCS for 30 min. Lymphocytes were then separated on a 25% Optiprep gradient (Accurate Chemical and Scientific Corp., Westbury, NY). Lungs were perfused with PBS via right ventricular cannulation, removed, and incubated at 37 C in 50 U/mL collagenase IV solution for 60 min. The digested tissue was passed through a 100-lm strainer followed by centrifugation and RBC lysis. To obtain bone marrow cells, the femur and tibia were flushed with PBS and the cell suspension was centrifuged followed by RBC lysis. The skin graft was minced and digested at 37 C for 2 hrs in a solution containing 2.7 mg/ml collagenase, 1 mg/ml DNAse, and 0.25mg/ml hyaluronidase. The digested tissue was passed through a 100 lm strainer. Isolated cells were stained and analyzed by flow cytometry after gating on the Thy1.1 + population. Flow cytometry Fluorochrome-conjugated antibodies were purchased from BD Pharmingen (San Diego, CA), ebioscience (San Diego, CA) or SouthernBiotech (Birmingham, AL). Antibodies were against CD4 (RM4-5), CD8a (53-6.7), CD44 (Pgp- 1), CD62L (MEL-14) and CD90.1 (OX-7). Flow cytometry was performed on FACS Aria or LSRII analyzers (BD Biosciences, San Diego, CA). Data were analyzed using Flowjo software (Treestar Corp., CA). Histologic analyses Native skin from age-matched wt and RIP-LTa B6 mice housed under SPF conditions was harvested, fixed in Zinc-formalin and embedded in paraffin. Routine Hematoxylin & Eosin (H&E) staining and specialized immunohistochemical analysis of endothelial PNAd expression using MECA-79 antibodies (BD Biosciences) were performed on deparaffinized sections as previously described (21). Frozen sections of skin grafts were stained with hematoxylin or fluorescein-conjugated anti-cd4 and B220 antibodies. Sections were interpreted by the pathologist (FKB) in a blinded fashion. 1072 American Journal of Transplantation 2007; 7: 1071 1079

Tertiary Lymphoid Tissues and Rejection Results Skin from RIP-LTa mice contains morphological features of tertiary lymphoid tissues The RIP-LT a mouse expresses LTa under the control of the rat insulin promoter (RIP) (26). Because of the lack of high tissue specificity of this promoter, LTa is overexpressed in the pancreatic islets, kidneys and skin (26). The pancreata and kidneys of adult RIP-LT a mice exhibit well-defined tertiary lymphoid tissues that contain PNAd + HEV, T and B lymphocyte clusters, and in some cases germinal centers (1,26). Lymphoid neogenesis in these mice is consistent with the roles of LTa alone and in combination with LTb to form LTa 1 b 2 in the development of tertiary lymphoid tissues (4). In the current study, we opted to graft skin from RIP-LT a donors onto hosts that lack all secondary lymphoid tissues to test whether intra-graft tertiary lymphoid tissues initiate the rejection process. The skin grafting model is preferable to transplanting either pancreatic islets or kidneys because only a small number of islets can be harvested from RIP-LT a donors (personal observations), and kidney transplantation is suboptimal for studying rejection as renal allografts are often accepted spontaneously in mice (33). Therefore, as a first step towards testing our hypothesis, we compared skin from RIP-LT a and wt C57BL/6 mice for the presence of morphological characteristics of tertiary lymphoid tissues. We found that native RIP-LT a skin harbors PNAd + vessels, with morphological characteristics of HEVs, surrounded by mononuclear cell clusters. These structures were predominantly located in the deep dermis and subcutaneous tissues (Figure 1). PNAd in the affected vessels was restricted to blood vessel endothelial cells (Figure 1). In contrast, skin from age-matched, wt control mice contained neither PNAd + vessels nor mononuclear cell clusters (Figure 1). RIP-LTa but not wt skin allografts are rejected by mice that lack secondary lymphoid organs aly/aly-spleen mice, which lack lymph nodes, Peyer s patches and spleen, are severely compromised in their ability to mount primary immune responses against viruses (29), allogeneic tumors (30), and allografts (31). To test whether tertiary lymphoid tissues support primary immune activation, we asked whether the presence of tertiary lymphoid structures in an allogeneic RIP-LT a skin graft restores the rejection response in aly/aly-spleen recipients. As shown in Figure 2A, (C57BL6 x BALB/c)F1 (H-2 b,d ) wt skin transplanted to aly/aly-spleen (H-2 b ) mice was accepted spontaneously (MST > 200 days, n = 6). In contrast, (RIP-LT a x BALB/c)F1 (H-2 b,d ) skin grafts, which contain tertiary lymphoid tissues, were acutely rejected by a separate group of aly/aly-spleen mice (MST = 18 days, n = 12). Graft rejection was evident by gross morphology and was confirmed histologically by presence of Figure 1: RIP-LT a skin contains morphological features of tertiary lymphoid tissues. Native skin was harvested from age-matched RIP-LT a and wt B6 mice. Micrographs demonstrate PNAd + vascular endothelial cells (brown) and peri-vascular mononuclear cell clusters (blue) in RIP-LT a skin only. American Journal of Transplantation 2007; 7: 1071 1079 1073

Nasr et al. disrupted tissue architecture, scarring of the epidermis and a dense mononuclear cell infiltrate consisting of neutrophils and lymphocytes (Figure 2B). Finally, syngeneic (H- 2 b ) RIP-LT a skin grafts were accepted indefinitely (> 200 days, n = 12) by aly/aly-spleen recipients, indicating that the acute loss of RIP-LT a skin allografts was not due to altered graft healing that could have resulted from excessive, local LTa production. RIP-LTa skin grafts precipitate the rejection of wt skin allografts in mice that lack secondary lymphoid organs The rejection of RIP-LT a skin allografts by aly/aly-spleen mice in the preceding experiment (Figure 2) suggests that intra-graft tertiary lymphoid tissues are sufficient for mounting primary alloimmune responses. This finding, however, does not convincingly demonstrate that tertiary lymphoid tissues function as secondary lymphoid organs, since foreign antigen, lymphocytes and target tissue are artificially clustered together in the RIP-LT a skin allograft model. Lymph nodes capture passing antigen or antigenloaded DC and generate effector lymphocytes that then encounter the antigen at a remote nonlymphoid site (34). To address this discrepancy, we transplanted syngeneic B6 (H-2 b ) RIP-LT a skin onto aly/aly-spleen mice, allowed the grafts sufficient time to heal (>21 days), and then transplanted allogeneic (C57BL6 x BALB/c)F1 (H-2 b,d ) wt skin to the same mice at least 1 cm apart from the first graft. In this model, the syngeneic RIP-LT a graft provided the tertiary lymphoid tissues, whereas the wt graft served as both the source of antigen and the remote target tissue. As shown in Figure 2A, wt allogeneic skin grafts transplanted alone to aly/aly-spleen mice were not rejected. In contrast, the same wt grafts transplanted to aly/aly-spleen mice that had received syngeneic RIP-LT a skin grafts were rejected acutely (MST = 31 days, n = 6) (Figure 3a), suggesting that tertiary lymphoid tissues, like lymph nodes, mount primary immune responses against antigens that originate at a remote site. To further confirm that tertiary lymphoid tissues generate an effector response that causes rejection irrespective of the location of the target graft, allogeneic (RIP-LT a BALB/c)F1 ((H-2 b,d ) and (C57BL6 x BALB/c)F1 (H-2 b,d ) wt skin were simultaneously transplanted to aly/aly-spleen mice. The grafts were placed on opposite sides of the recipient, at least 1 cm apart. As shown in Figure 3B, both allografts were rejected at the same tempo (MST = 52 days, n = 6/group), while wt skin allografts transplanted alone to aly/aly-spleen mice were not rejected (Figure 2A). The unusually delayed rejection time of 52 days may have been due to the increased size of tissue targeted by rejection as the mice received two skin grafts each (35). Therefore, these results indicate that tertiary lymphoid tissues, like secondary lymphoid organs, generate an effector immune response that clears antigen at a disparate nonlymphoid site. Figure 2: RIP-LT a skin allografts are rejected by mice that lack secondary lymphoid organs. (A) Survival of allogeneic (F1) RIP- LT a (n = 12), allogeneic (F1) wt (n = 6), and control syngeneic (B6) RIP-LT a skin grafts (n = 12) transplanted to aly/aly-spleen hosts. Accepted grafts were monitored >200 days. (B) Top panels: Gross appearance of skin grafts >200 days after transplantation (F1 wt) or at time of rejection (F1 RIP-LT a). Lower panels: Microscopic appearance of F1 wt and F1 RIP-LT a skin grafts 25 days after transplantation to aly/aly-spleen hosts (H&E stain). RIP-LTa skin allografts generate immunologic memory in mice that lack secondary lymphoid organs A hallmark of adaptive immune responses is the generation of immunologic memory (36). The preceding experiments (Figure 3) provided evidence that tertiary lymphoid tissues mount an effector alloimmune response. To investigate whether tertiary lymphoid tissues generate immunologic memory, we transplanted allogeneic (RIP-LT a x BALB/c)F1 (H-2 b,d ) skin to aly/aly-spleen mice, waited until the skin grafts were acutely rejected, and 60 days later 1074 American Journal of Transplantation 2007; 7: 1071 1079

A B 100 80 60 40 20 100 B6 RIP-LTα F1 WT 0 0 20 40 60 80 100 80 Time (days) 100 80 60 40 20 Tertiary Lymphoid Tissues and Rejection 0 0 20 40 60 80 100 Time (days) F1 RIP-LTα F1 WT Figure 4: RIP-LT a skin grafts generate immunologic memory in mice that lack secondary lymphoid organs. Allogeneic (F1) RIP-LT a skin grafts transplanted to aly/aly-spleen hosts were rejected within 21 days. Allogeneic (F1) wt skin grafts transplanted to the same mice 60 days after rejection of the RIP-LT a allografts were also rejected acutely (n = 6). 60 40 20 F1 RIP-LTα F1 WT 0 0 20 40 60 80 100 Time (days) Figure 3: RIP-LT a skin grafts generate an effector immune response in mice that lack secondary lymphoid organs. (A) Survival of syngeneic (B6) RIP-LT a and allogeneic (F1) wt skin grafts transplanted to the same aly/aly-spleen recipients (n = 6/group). Syngeneic RIP-LT a grafts were allowed to heal for at least 21 days before transplanting the allogeneic wt skin grafts. (B) Survival of allogeneic (F1) RIP-LT a and (F1) wt skin grafts transplanted simultaneously to aly/aly-spleen mice (n = 6/group). In both experiments, RIP-LT a skin grafts precipitated the rejection of allogeneic wt grafts. grafted allogeneic (C57BL6 x BALB/c)F1 (H-2 b,d ) wt skin to the same recipients. As shown in Figure 4, wt allografts were promptly rejected (MST = 16 days, n = 6). Rejection of wt allografts occurred in the absence of host secondary lymphoid organs, long after the RIP-LT a skin allograft that contains tertiary lymphoid structures had been eliminated. This rejection occurred at a more rapid rate than first set rejection of wt skin allografts shown in Figure 3A (MST = 16 vs. 31 days). These findings indicate that tertiary lymphoid tissues generate immunologic memory. RIP-LTa skin allografts generate effector and memory T cells Allograft rejection is a T-cell-dependent process (37). Therefore, graft rejection observed in the preceding experiments (Figures 3 and 4) suggests that tertiary lymphoid tissues induce the differentiation of naïve T cells into effector and memory cells. To test this possibility, we transferred congenic (Thy1.1 + ), CFSE-labeled, naïve T cells (CD44 lo CD4 + or CD8 + cells sorted from naïve mice) to aly/aly-spleen hosts (Thy1.2 + ) that had received either an allogeneic or syngeneic RIP-LT a skin graft 2 days earlier. aly/aly-spleen hosts were sacrificed 15 days after cell transfer and the liver, lungs, bone marrow, blood and skin graft were analyzed for the presence of effector phenotype Thy1.1 + cells. Alternatively, aly/aly-spleen hosts were sacrificed 80 days after cell transfer and the same tissues, except for allogeneic skin grafts that had been rejected approximately 60 days earlier, were analyzed for the presence of memory phenotype Thy1.1 + cells. Fifteen days after cell transfer, Thy1.1 + CD8 + cells were detected in all tissues studied but were most abundant in the liver and lungs. Eighty days after cell transfer, Thy1.1 + CD8 + cells were again present in the liver and lungs, but could not be detected in the blood or bone marrow. The preferential migration of CD8 + effector and memory T cells to the liver and lungs is consistent with the nonlymphoid tissue tropism of effector and memory T cells in either wt or aly/aly-spleen mice (38,39). Transferred Thy1.1 + CD4 + T cells could not be retrieved in sufficient numbers at either time point thus, restricting phenotypic analysis to the CD8 + population. Analysis of retrieved cells revealed that naïve Thy1.1 + CD8 + T cells transferred 15 days earlier to recipients of allogeneic RIP-LT a skin had proliferated (as measured by CFSE dilution) and American Journal of Transplantation 2007; 7: 1071 1079 1075

Nasr et al. Figure 5: RIP-LT a skin allografts induce the differentiation of adoptively transferred naïve CD8 T cells to effector and memory T cells. 5 10 6 CD8 + CD44 low T cells were sorted from naï ve congenic (Thy1.1 + ) B6 mice, CFSE labeled and transferred to aly/aly-spleen mice (Thy1.2 + ) that had received either an allogeneic (2nd and 3rd columns) or syngeneic (ctrl 1) RIP-LT a, or an allogeneic wildtype (ctrl 2), skin graft 2 days earlier. Mice were sacrificed either 15 or 80 days later and CD8 + Thy1.1 + T cells isolated from the liver were gated and analyzed by flow cytometry. Purity and phenotype of sorted CD8 + Thy1.1 + T cells prior to adoptive transfer are shown in the lefthand column. acquired a CD62L lo CD44 hi phenotype, indicating naï ve to effector T-cell differentiation (Figure 5). In contrast, naï ve CD8 + Thy1.1 + transferred to control mice that received syngeneic RIP-LT a skin grafts, or allogeneic WT skin grafts, did not divide and retained their naïve phenotype (Figure 5). Thy1.1 + CD8 + cells retrieved 80 days after transfer (approximately 60 days after rejection of the allogeneic RIP-LT a graft) had also divided and acquired a CD44 hi phenotype, consistent with naï ve to memory T-cell differentiation (Figure 5). These cells were predominantly CD62L hi, suggesting a central memory phenotype. Discussion Tertiary lymphoid tissues have been observed in mouse and human organ transplants, particularly in the setting of chronic rejection (20 23). However, a causal relationship between intra-graft tertiary lymphoid tissues and the 1076 American Journal of Transplantation 2007; 7: 1071 1079

Tertiary Lymphoid Tissues and Rejection rejection process had not been established. Here, we demonstrated that skin grafts containing tertiary lymphoid structures restore the ability of hosts lacking secondary lymphoid organs to mount a primary rejection response and to generate effector and memory cells from naï ve T cells. Prior studies testing whether naï ve T cells could be primed within the tertiary lymphoid microenvironment relied on wild-type mouse models in which tertiary lymphoid tissues are induced in the periphery and the immune response to foreign antigen is then analyzed (17,18). A principal challenge to interpreting these experiments is distinguishing between T-cell priming in secondary lymphoid organs and priming in the tertiary lymphoid microenvironment. More recently, Moyron-Quiroz et al. utilized mice that lack canonical secondary lymphoid organs to investigate whether inducible bronchus associated lymphoid tissues (ibalt) are sufficient to generate protective immunity (40). ibalt are occasionally found in the airways of humans and mice and are induced or increased in number and size following respiratory infection or inflammation (41). By infecting splenectomized LTa knockout mice with influenza virus, these authors provided evidence that ibalt support lymphocyte proliferation and provide acute protective immunity against the virus (40). Tertiary lymphoid tissues also participate in autoimmunity. Fu and colleagues showed that tertiary lymphoid structures induced by transgenic expression of the LTbR ligand LIGHT in the pancreatic islets of NOD mice accelerate the progression to diabetes (19). Diabetes was observed in transgenic NOD mice even when the pancreatic draining lymph nodes were removed, indicating that activation of autoreactive T cells took place within tertiary lymphoid tissues. In our study, we demonstrate that tertiary lymphoid tissues present in a transplanted organ not only support productive primary immune responses (acute rejection) but also generate longlasting immunologic memory. The generation of memory T cells, a hallmark of adaptive immune responses initiated in secondary lymphoid organs, provides additional proof that tertiary lymphoid tissues are immunologically competent. Under conditions of disrupted lymphocyte entry into the lymph nodes and spleen, the bone marrow may serve as an alternative site of lymphocyte priming for systemic antigens administered at high doses (42, 43). Similarly, a recent study suggested that the liver may prime naï ve T cells if antigen presentation is restricted to liver cells (44). Neither possibility is likely to explain the RIP-LT a skin graft restoration of acute rejection and antigen-driven T-cell differentiation in aly/aly-spleen mice (this study). First, acute allograft rejection was observed only when skin grafts containing tertiary lymphoid structures were transplanted. Transplanting wt skin allografts did not elicit a rejection process despite the presence of functional bone marrow and liver microenvironments in aly/aly-spleen recipients. Second, the fact that the majority of effector and memory T cells were detected in the liver after transferring naï ve congenic T cells to transplanted mice (Figure 5) does not necessarily imply that T-cell activation occurred in the liver. Effector T cells and either central or effector memory T cells generated in secondary lymphoid organs are known to accumulate in the liver of both wt and aly/aly-spleen mice irrespective of the original site of antigen entry (38,39,45). The ultimate proof that naïve T-cell activation occurs within tertiary lymphoid tissues awaits intra-vital visualization of DC-T-cell interactions within the tertiary lymphoid microenvironment of transplanted or native organs subjected to chronic inflammation. Chronic rejection is an inflammatory process that causes slow but intractable failure of vascularized organ allografts (46). It is characterized by lymphocyte infiltration, narrowing of vessel lumina and progressive graft fibrosis. The realization that lymphocyte infiltrates in chronic rejection often take the form of tertiary lymphoid tissues raises the possibility that these organs contribute to the pathogenesis of chronic rejection by perpetuating alloimmune responses in the local microenvironment (20 23). T- and B-cell activation within the microenvironment of a transplanted organ may be particularly difficult to switch off because of the local abundance of donor antigen, increased likelihood of epitope spreading, and potential absence of regulatory mechanisms that normally operate within secondary lymphoid organs (11). Therefore, understanding the contribution of tertiary lymphoid tissues to allograft pathology is essential for developing novel therapies to interrupt chronic rejection. Our study provides the first step toward achieving this goal as it demonstrates that intra-graft tertiary lymphoid tissues are immunologically functional they support the differentiation of naï ve T cells into effector and memory T cells that mediate rejection. Acknowledgment This work was supported by the Roche Organ Transplantation Research Foundation (FGL) and by NIH grants AI 49466 (FGL), AI 44644 (FGL), DK 57731 (NHR), and CA 16885 (NHR). 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