Endothelial cells act as gatekeepers for LTßR-dependent thymocyte emigration.

The emigration of mature thymocytes from the thymus is critical for establishing peripheral T cell compartments. However, the pathways controlling this process and the timing of egress in relation to postselection developmental stages are poorly defined. Here, we reexamine thymocyte egress and test current and opposing models in relation to the requirement for LTßR, a regulator of thymic microenvironments and thymocyte emigration. Using cell-specific gene targeting, we show that the requirement for LTßR in thymocyte egress is distinct from its control of thymic epithelium and instead maps to expression by endothelial cells. By separating emigration into sequential phases of perivascular space (PVS) entry and transendothelial migration, we reveal a developmentally ordered program of egress where LTßR operates to rate limit access to the PVS. Collectively, we show the process of thymic emigration ensures only the most mature thymocytes leave the thymus and demonstrate a role for LTßR in the initiation of thymus emigration that segregates from its control of medulla organization.

α-Galactosylceramide and peptide-based nano-vaccine synergistically induced a strong tumor suppressive effect in melanoma.

α-Galactosylceramide (GalCer) is a glycolipid widely known as an activator of Natural killer T (NKT) cells, constituting a promising adjuvant against cancer, including melanoma. However, limited clinical outcomes have been obtained so far. This study evaluated the synergy between GalCer and major histocompatibility complex (MHC) class I and MHC class II melanoma-associated peptide antigens and the Toll-Like Receptor (TLR) ligands CpG and monophosphoryl lipid A (MPLA), which we intended to maximize following their co-delivery by a nanoparticle (NP). This is expected to improve GalCer capture by dendritic cells (DCs) and subsequent presentation to NKT cells, simultaneously inducing an anti-tumor specific T-cell mediated immunity. The combination of GalCer with melanoma peptides and TLR ligands successfully restrained tumor growth. The tumor volume in these animals was 5-fold lower than the ones presented by mice immunized with NPs not containing GalCer. However, tumor growth was controlled at similar levels by GalCer entrapped or in its soluble form, when mixed with antigens and TLR ligands. Those two groups showed an improved infiltration of T lymphocytes into the tumor, but only GalCer-loaded nano-vaccine induced a prominent and enhanced infiltration of NKT and NK cells. In addition, splenocytes of these animals secreted levels of IFN-γ and IL-4 at least 1.5-fold and 2-fold higher, respectively, than those treated with the mixture of antigens and adjuvants in solution. Overall, the combined delivery of the NKT agonist with TLR ligands and melanoma antigens via this multivalent nano-vaccine displayed a synergistic anti-tumor immune-mediated efficacy in B16F10 melanoma mouse model. STATEMENT OF SIGNIFICANCE: Combination of α-galactosylceramide (GalCer), a Natural Killer T (NKT) cell agonist, with melanoma-associated antigens presented by MHC class I (Melan-A:26) and MHC class II (gp100:44) molecules, and Toll-like Receptor (TLR) ligands (MPLA and CpG), within nanoparticle matrix induced a prominent anti-tumor immune response able to restrict melanoma growth. An enhanced infiltration of NKT and NK cells into tumor site was only achieved when the combination GalCer, antigens and TLR ligands were co-delivered by the nanovaccine.

Redefining thymus medulla specialization for central tolerance.

During αßT cell development, the thymus medulla represents an essential microenvironment for T cell tolerance. This functional specialization is attributed to its typical organized topology consisting of a branching structure that contains medullary thymic epithelial cell (mTEC) networks to support negative selection and Foxp3+ T-regulatory cell (T-reg) development. Here, by performing TEC-specific deletion of the thymus medulla regulator lymphotoxin ß receptor (LTßR), we show that thymic tolerance mechanisms operate independently of LTßR-mediated mTEC development and organization. Consistent with this, mTECs continue to express Fezf2 and Aire, regulators of intrathymic self-antigens, and support T-reg development despite loss of LTßR-mediated medulla organogenesis. Moreover, we demonstrate that LTßR controls thymic tolerance by regulating the frequency and makeup of intrathymic dendritic cells (DCs) required for effective thymocyte negative selection. In all, our study demonstrates that thymus medulla specialization for thymic tolerance segregates from medulla organogenesis and instead involves LTßR-mediated regulation of the thymic DC pool.

Inflammation drives thrombosis after Salmonella infection via CLEC-2 on platelets.

Thrombosis is a common, life-threatening consequence of systemic infection; however, the underlying mechanisms that drive the formation of infection-associated thrombi are poorly understood. Here, using a mouse model of systemic Salmonella Typhimurium infection, we determined that inflammation in tissues triggers thrombosis within vessels via ligation of C-type lectin-like receptor-2 (CLEC-2) on platelets by podoplanin exposed to the vasculature following breaching of the vessel wall. During infection, mice developed thrombi that persisted for weeks within the liver. Bacteria triggered but did not maintain this process, as thrombosis peaked at times when bacteremia was absent and bacteria in tissues were reduced by more than 90% from their peak levels. Thrombus development was triggered by an innate, TLR4-dependent inflammatory cascade that was independent of classical glycoprotein VI-mediated (GPVI-mediated) platelet activation. After infection, IFN-γ release enhanced the number of podoplanin-expressing monocytes and Kupffer cells in the hepatic parenchyma and perivascular sites and absence of TLR4, IFN-γ, or depletion of monocytic-lineage cells or CLEC-2 on platelets markedly inhibited the process. Together, our data indicate that infection-driven thrombosis follows local inflammation and upregulation of podoplanin and platelet activation. The identification of this pathway offers potential therapeutic opportunities to control the devastating consequences of infection-driven thrombosis without increasing the risk of bleeding.

Three-dimensional spheroid cell culture of umbilical cord tissue-derived mesenchymal stromal cells leads to enhanced paracrine induction of wound healing.

INTRODUCTION: The secretion of trophic factors by mesenchymal stromal cells has gained increased interest given the benefits it may bring to the treatment of a variety of traumatic injuries such as skin wounds. Herein, we report on a three-dimensional culture-based method to improve the paracrine activity of a specific population of umbilical cord tissue-derived mesenchymal stromal cells (UCX®) towards the application of conditioned medium for the treatment of cutaneous wounds. METHODS: A UCX® three-dimensional culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX® spheroids of extracellular matrix proteins and trophic factors involved in the wound-healing process was analysed. The skin regenerative potential of UCX® three-dimensional culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX® two-dimensional culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively. RESULTS: UCX® spheroids kept in our three-dimensional system remained viable and multipotent and secreted considerable amounts of vascular endothelial growth factor A, which was undetected in two-dimensional cultures, and higher amounts of matrix metalloproteinase-2, matrix metalloproteinase-9, hepatocyte growth factor, transforming growth factor ß1, granulocyte-colony stimulating factor, fibroblast growth factor 2 and interleukin-6, when compared to CM2D. Furthermore, CM3D significantly enhanced elastin production and migration of keratinocytes and fibroblasts in vitro. In turn, tubulogenesis assays revealed increased capillary maturation in the presence of CM3D, as seen by a significant increase in capillary thickness and length when compared to CM2D, and increased branching points and capillary number when compared to basal medium. Finally, CM3D-treated wounds presented signs of faster and better resolution when compared to untreated and CM2D-treated wounds in vivo. Although CM2D proved to be beneficial, CM3D-treated wounds revealed a completely regenerated tissue by day 14 after excisions, with a more mature vascular system already showing glands and hair follicles. CONCLUSIONS: This work unravels an important alternative to the use of cells in the final formulation of advanced therapy medicinal products by providing a proof of concept that a reproducible system for the production of UCX®-conditioned medium can be used to prime a secretome for eventual clinical applications.

In vivo delivery of peptides and Toll-like receptor ligands by mannose-functionalized polymeric nanoparticles induces prophylactic and therapeutic anti-tumor immune responses in a melanoma model.

We hypothesized that the co-entrapment of melanoma-associated antigens and the Toll-like receptor (TLR) ligands Poly(I:C) and CpG, known to be Th1-immunopotentiators, in mannose-functionalized aliphatic polyester-based nanoparticles (NPs) could be targeted to mannose receptors on antigen-presenting cells and induce anti-tumor immune responses. High entrapment efficiencies of antigens and immunopotentiators in 150nm NPs were obtained. The co-entrapment of the model antigen ovalbumin and the TLR ligands was crucial to induce high IgG2c/IgG1 ratios and high levels of IFN-γ and IL-2. Mannose-functionalization of NPs potentiated the Th1 immune response. The nanoparticulate vaccines decreased the growth rate of murine B16F10 melanoma tumors in therapeutic and prophylatic settings. The combination of mannose-functionalized NPs containing MHC class I- or class II-restricted melanoma antigens and the TLR ligands induced the highest tumor growth delay. Overall, we demonstrate that the multifunctional properties of NPs in terms of targeting and antigen/adjuvant delivery have high cancer immunotherapeutic potential.

IP-10/CXCL10 induction in human pancreatic cancer stroma influences lymphocytes recruitment and correlates with poor survival.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant desmoplastic reaction driven by pancreatic stellate cells (PSCs) that contributes to tumor progression. Here we sought to characterize the interactions between pancreatic cancer cells (PCCs) and PSCs that affect the inflammatory and immune response in pancreatic tumors. Conditioned media from mono- and cocultures of PSCs and PCCs were assayed for expression of cytokines and growth factors. IP-10/CXCL10 was the most highly induced chemokine in coculture of PSCs and PCCs. Its expression was induced in the PSCs by PCCs. IP-10 was elevated in human PDAC specimens, and positively correlated with high stroma content. Furthermore, gene expression of IP-10 and its receptor CXCR3 were significantly associated with the intratumoral presence of regulatory T cells (Tregs). In an independent cohort of 48 patients with resectable pancreatic ductal adenocarcinoma, high IP-10 expression levels correlated with decreased median overall survival. Finally, IP-10 stimulated the ex vivo recruitment of CXCR3+ effector T cells as well as CXCR3+ Tregs derived from patients with PDAC. Our findings suggest that, in pancreatic cancer, CXCR3+ Tregs can be recruited by IP-10 expressed by PSCs in the tumor stroma, leading to immunosuppressive and tumor-promoting effects.

Resolving Salmonella infection reveals dynamic and persisting changes in murine bone marrow progenitor cell phenotype and function.

The generation of immune cells from BM precursors is a carefully regulated process. This is essential to limit the potential for oncogenesis and autoimmunity yet protect against infection. How infection modulates this is unclear. Salmonella can colonize systemic sites including the BM and spleen. This resolving infection has multiple IFN-γ-mediated acute and chronic effects on BM progenitors, and during the first week of infection IFN-γ is produced by myeloid, NK, NKT, CD4(+) T cells, and some lineage-negative cells. After infection, the phenotype of BM progenitors rapidly but reversibly alters, with a peak ∼ 30-fold increase in Sca-1(hi) progenitors and a corresponding loss of Sca-1(lo/int) subsets. Most strikingly, the capacity of donor Sca-1(hi) cells to reconstitute an irradiated host is reduced; the longer donor mice are exposed to infection, and Sca-1(hi) c-kit(int) cells have an increased potential to generate B1a-like cells. Thus, Salmonella can have a prolonged influence on BM progenitor functionality not directly related to bacterial persistence. These results reflect changes observed in leucopoiesis during aging and suggest that BM functionality can be modulated by life-long, periodic exposure to infection. Better understanding of this process could offer novel therapeutic opportunities to modulate BM functionality and promote healthy aging.

The role of human umbilical cord tissue-derived mesenchymal stromal cells (UCX®) in the treatment of inflammatory arthritis.

BACKGROUND: ECBio has developed proprietary technology to consistently isolate, expand and cryopreserve a well-characterized population of stromal cells from human umbilical cord tissue (UCX® cells). The technology has recently been optimized in order to become compliant with Advanced Medicine Therapeutic Products. In this work we report the immunosuppressive capacity of UCX® cells for treating induced autoimmune inflammatory arthritis. METHODS: UCX® cells were isolated using a proprietary method (PCT/IB2008/054067) that yields a well-defined number of cells using a precise proportion between tissue digestion enzyme activity units, tissue mass, digestion solution volume and void volume. The procedure includes three recovery steps to avoid non-conformities related to cell recovery. UCX® surface markers were characterized by flow cytometry and UCX® capacity to expand in vitro and to differentiate into adipocyte, chondrocyte and osteoblast-like cells was evaluated. Mixed Lymphocyte Reaction (MLR) assays were performed to evaluate the effect of UCX® cells on T-cell activation and Treg conversion assays were also performed in vitro. Furthermore, UCX® cells were administered in vivo in both a rat acute carrageenan-induced arthritis model and rat chronic adjuvant induced arthritis model for arthritic inflammation. UCX® anti-inflammatory activity was then monitored over time. RESULTS: UCX® cells stained positive for CD44, CD73, CD90 and CD105; and negative for CD14, CD19 CD31, CD34, CD45 and HLA-DR; and were capable to differentiate into adipocyte, chondrocyte and osteoblast-like cells. UCX® cells were shown to repress T-cell activation and promote the expansion of Tregs better than bone marrow mesenchymal stem cells (BM-MSCs). Accordingly, xenogeneic UCX® administration in an acute carrageenan-induced arthritis model showed that human UCX® cells can reduce paw edema in vivo more efficiently than BM-MSCs. Finally, in a chronic adjuvant induced arthritis model, animals treated with intra-articular (i.a.) and intra-peritoneal (i.p.) infusions of UCX® cells showed faster remission of local and systemic arthritic manifestations. CONCLUSION: The results suggest that UCX® cells may be an effective and promising new approach for treating both local and systemic manifestations of inflammatory arthritis.

Immunoregulatory function of IL-27 and TGF-ß1 in cardiac allograft transplantation.

BACKGROUND: Deciphering the mechanisms of tolerance represents a crucial aim of research in transplantation. We previously identified by DNA chip interleukin (IL)-27 p28 and transforming growth factor (TGF)-ß1 as overexpressed in a model of rat cardiac allograft tolerance mediated by regulatory CD4CD25 T cells. The role of these two molecules on the control of the inflammatory response remains controversial. However, both are involved in the regulation of the T helper 17/Treg axis, suggesting their involvement in tolerance. METHODS: We analyzed regulation of IL-27 and TGF-ß1 expression in allograft response and their role in tolerance by using blocking anti-TGF-ß antibody and by generating an adeno-associated virus encoding IL-27. RESULTS: Here, we confirmed the overexpression of IL-27 and TGF-ß1 in tolerated cardiac allografts in two different rodent models. We observed that their expression correlates with inhibition of T helper 17 differentiation and with expansion of regulatory CD4CD25 T cells. We showed in a rat model that anti-TGF-ß treatment abrogates infectious tolerance mediated by the transfer of regulatory CD4CD25 T cells. Moreover, overexpression of IL-27 by adeno-associated virus administration in combination with a short-term immunosuppression allows prolongation of cardiac allograft survival and one tolerant recipient. We found that IL-27 overexpression did not induce Foxp3CD4CD25 T-cell expansion but rather IL-10-expressing CD4 T cells in the tolerant recipient. CONCLUSIONS: Taken together, these data suggest that both TGF-ß1 and IL-27 play a role in the mechanisms of tolerance. However, in contrast to TGF-ß1, IL-27 seems not to be involved in regulatory CD4CD25 T-cell expansion but rather in their mode of action.

Th17 cells in alemtuzumab-treated patients: the effect of long-term maintenance immunosuppressive therapy.

BACKGROUND: Leukocyte depletion at the time of transplantation with alemtuzumab (Campath-1H) has been demonstrated to be a potential strategy for reducing long-term exposure to immunosuppressive drugs. Although the impact of alemtuzumab treatment on the immune system has been explored, the effects of long-term immunosuppressive therapy in alemtuzumab-treated patients still need to be elucidated. METHODS: T-regulatory cells and Th1/Th17 responses were assessed by flow cytometry and real-time polymerase chain reaction more than 4 years after transplantation in 10 kidney recipients treated with alemtuzumab induction. Seven patients were converted to sirolimus monotherapy at 12 months posttransplant, whereas the remaining three patients with history of graft rejection were treated with sirolimus and mycophenolate mofetil. In addition, we sorted and expanded interleukin (IL)-17A-producing CCR6CD4 T cells and assessed their susceptibility to suppression by regulatory T (Treg) cells in in vitro suppression tests. RESULTS: Three years of mammalian target of rapamycin inhibitor monotherapy correlates with an increase in the number of IL-17A producing cells, compared with patients treated with sirolimus and mycophenolate mofetil. In these patients, IL-17A expression was compensated for by an increase in Treg cell frequency and number. In addition, we demonstrated that both proliferation and cytokine production by Th17 cells can be effectively regulated by Treg cells. CONCLUSIONS: Our results demonstrate that history of rejection and long-term maintenance immunosuppression has an impact on the number of circulating Treg and Th17 cells. However, more importantly, we have shown that Treg cells can effectively regulate Th17cells both in vitro and in vivo.

Regulatory T cells can prevent memory CD8+ T-cell-mediated rejection following polymorphonuclear cell depletion.

Accumulating evidence suggests that alloreactive memory T cells (Tm) may form a barrier to tolerance induction in large animals and humans due in part to a resistance to suppression by Treg. However, why Tm are resistant to regulation and how the Tm response to an allograft differs from that of naïve T cells, which are amenable to suppression by Treg, remains unknown. Here, we show that accelerated graft rejection mediated by CD8(+) Tm was due to the enhanced recruitment of PMN to allografts in a mouse skin allograft model. Importantly, depletion of PMN slowed the kinetics of (but did not prevent) rejection mediated by Tm and created a window of opportunity that allowed subsequent suppression of rejection by Treg. Taken together, we conclude that CD8(+) Tm are not intrinsically resistant to suppression by Treg but may rapidly inflict substantial graft damage before the establishment of regulatory mechanisms. These data suggest that if Tm responses can be attenuated transiently following transplantation, Treg may be able to maintain tolerance through the suppression of both memory and naïve alloreactive T-cell responses in the long term.

Immunosenescence increases the rate of acceptance of kidney allotransplants in elderly recipients through exhaustion of CD4+ T-cells.

Compromised immunity is the hallmark of ageing. Paradoxically, it may be "an ally" in facilitating acceptance of allogeneic grafts in the elderly. In this retrospective study we looked for biomarkers of immunosenescence that distinguish elderly recipients less prone to reject kidney allografts. Recruited kidney recipients aged > or = 60 or < 60 were designated 'elderly' and 'young', respectively. Both age-groups were divided according to the history of acute rejection. The phenotype, length of telomeres, expression of FoxP3 and proliferative responses were assessed in CD4(+) and CD8(+) T-cell subsets. In addition, IL6, IL10 and TGFbeta were measured on the level of mRNA and serum protein. Acute-rejection-free history in elderly transplant recipients was associated with short telomeres, a decreased proportion of CD28(+) T-cells associated with CMV-seropositivity and low proliferation of CD4(+) T-cells. In contrast, elderly recipients who experienced acute rejection kept preserved telomere length, had a higher number of functional CD4(+)CD28(+) cells and exhibited vigorous proliferation in vitro. These differences were not found in the young group. The major conclusion of this study is that the impaired condition of CD4(+) T-cells, so-called immunosenescence, renders transplant recipients less responsive to an allogeneic kidney graft, an effect that was limited to transplant recipients of > 60 years of age.

Location and time-dependent control of rejection by regulatory T cells culminates in a failure to generate memory T cells.

Adaptive CD25(+)CD4(+) regulatory T cells (Treg) can be induced following exposure to alloantigen and may function alongside naturally occurring Treg to suppress allograft rejection when present in sufficient numbers. However, the location of the Treg as they function in vivo and the mechanisms used to control donor-reactive T cells remains ill-defined. In this study, we used a CD8(+) TCR transgenic model of skin allograft rejection to characterize in vivo activity of donor-reactive Treg cells during induction of transplantation tolerance. We demonstrate that, initially after skin transplantation, Treg attenuate the priming of donor-reactive naive CD8(+) T cells in the lymphoid tissue draining the graft site. However, with time, peripheral suppression is overcome despite the continued presence of Treg, resulting in the priming of donor-reactive CD8(+) T cells and graft infiltration by the resultant effector T cells and induction of a "Tc1-like" intragraft gene expression profile. These intragraft effector CD8(+) T cells are then prevented from eliciting rejection by Treg that simultaneously infiltrate the skin allografts, resulting in a failure to generate donor-reactive memory CD8(+) T cells. Overall, these data demonstrate for the first time that donor-reactive Treg can suppress allograft rejection using distinct mechanisms at different sites in vivo with the overall outcome of preventing the generation of donor-reactive memory T cells.

Allograft rejection mediated by memory T cells is resistant to regulation.

Alloreactive memory T cells may be refractory to many of the tolerance-inducing strategies that are effective against naive T cells and thus present a significant barrier to long-term allograft survival. Because CD4(+)CD25(+) regulatory T cells (Tregs) are critical elements of many approaches to successful induction/maintenance of transplantation tolerance, we used MHC class I and II alloreactive TCR-transgenic models to explore the ability of antigen-specific Tregs to control antigen-specific memory T cell responses. Upon coadoptive transfer into RAG-1(-/-) mice, we found that Tregs effectively suppressed the ability of naive T cells to reject skin grafts, but neither antigen-unprimed nor antigen-primed Tregs suppressed rejection by memory T cells. Interestingly, different mechanisms appeared to be active in the ability of Tregs to control naive T cell-mediated graft rejection in the class II versus class I alloreactive models. In the former case, we observed decreased early expansion of effector cells in lymphoid tissue. In contrast, in the class I model, an effect of Tregs on early proliferation and expansion was not observed. However, at a late time point, significant differences in cell numbers were seen, suggesting effects on responding T cell survival. Overall, these data indicate that the relative resistance of both CD4(+) and CD8(+) alloreactive memory T cells to regulation may mediate resistance to tolerance induction seen in hosts with preexisting alloantigen-specific immunity and further indicate the multiplicity of mechanisms by which Tregs may control alloimmune responses in vivo.

Effector and memory CD8+ T cells can be generated in response to alloantigen independently of CD4+ T cell help.

There is now considerable evidence suggesting that CD8(+) T cells are able to generate effector but not functional memory T cells following pathogenic infections in the absence of CD4(+) T cells. We show that following transplantation of allogeneic skin, in the absence of CD4(+) T cells, CD8(+) T cells become activated, proliferate, and expand exclusively in the draining lymph nodes and are able to infiltrate and reject skin allografts. CD44(+)CD8(+) T cells isolated 100 days after transplantation rapidly produce IFN-gamma following restimulation with alloantigen in vitro. In vivo CD44(+)CD8(+) T cells rejected donor-type skin allografts more rapidly than naive CD8(+) T cells demonstrating the ability of these putative memory T cells to mount an effective recall response in vivo. These data form the first direct demonstration that CD8(+) T cells are able to generate memory as well as effector cells in response to alloantigen during rejection in the complete absence of CD4(+) T cells. These data have important implications for the design of therapies to combat rejection and serve to reinforce the view that CD8(+) T cell responses to allografts require manipulation in addition to CD4(+) T cell responses to completely prevent the rejection of foreign organ transplants.

Chemokine gene expression during allograft rejection: comparison of two quantitative PCR techniques.

The ability to analyse expression of genes rapidly in small samples of tissue is essential for the clinical assessment of many conditions, including the onset of rejection after transplantation. Chemokines have been shown to play a critical role in leukocyte recruitment to transplanted organs and in leukocyte localisation within tissues and antagonism of certain chemokines or chemokine receptors, identified as being up-regulated during allograft rejection, it has been shown to delay leukocyte infiltration into the graft and to prolong graft survival. The analysis of chemokine and chemokine receptor expression in allografts after transplantation may therefore be a useful early indicator of the onset of rejection. RT-PCR techniques are the most sensitive for the detection of low abundance mRNA when the amount of tissue sample is limited. Here we compared competitive-quantitative RT-PCR (CQ-PCR) with real-time PCR for the sequential quantification of chemokine transcripts after transplantation of a fully MHC mismatched mouse cardiac allograft. Although CQ-PCR was found to be an accurate and sensitive technique, real-time PCR was more sensitive and reproducible. Despite the reproducibility, differences in sensitivity between the two techniques were high. Real-time PCR avoids hazardous post-PCR manipulations thereby decreasing the potential risk of sample contamination, and offers the advantage that several genes can be analysed from small tissue samples in a shorter period of time, a key parameter for graft biopsy samples. Real-time PCR was therefore used to extend the analysis of intragraft mRNA chemokine expression levels. Expression of CXCL5 and CCL2 was found to be independent of T cell infiltration while intragraft expression of CCL3, CCL4, CCL5, CXCL9, CXCL10, XCL1 and CCL1 was clearly T cell dependent and increased significantly with time after transplantation. Overall, real-time PCR analysis showed that chemokine gene expression during rejection is clearly distinct from that in non-rejecting syngeneic grafts and is altered by the onset of infiltration of alloantigen-reactive T cells into the graft.