Cell-mediated Allograft Rejection Research Articles

A Novel Pathway for CD8+ T Cell Activation by Donor Derived Non-Haematopoietic Cells Leading to Acute Allograft Rejection

Introduction: Although seminal studies have shown that CD8+ T cells can reject cardiac allografts that lack donor haematopoietic cells (HPCs) through a direct cognate interaction with graft parenchymal cells, the observation from other studies that secondary lymphoid tissue (SLT) is essential for graft rejection creates a paradox yet to be resolved. Here we examine the hypothesis that CD8+ T cell activation by graft parenchymal cells requires acquisition and re-presentation of intact MHC class I alloantigen by recipient antigen presenting cells (APCs) within SLT. If proven, this hypothesis would not only resolve this paradox, but would also represent the first definitive demonstration of the contribution of the semi-direct pathway to graft rejection. Methods: CD8+ T cell-mediated allograft rejection following activation by donor parenchymal cells was examined using a transgenic heterotopic murine heart transplant model. Balb/c cardiac donors were lethally irradiated and treated with depleting antibodies ensuring complete eradication of HPCs, such that parenchymal cells in these grafts were the only source of alloantigen presentation. These HPC-depleted Balb/c (Balb/cHPC-) cardiac allografts were transplanted into: 1) 2C transgenic mice (monoclonal population of CD8+ T cells against Ld MHC class I); 2) Splenectomised aly/aly (aly/alyspl) mice (complete absence of SLT) with adoptive transfer of 2C CD8+ T cells, to investigate the importance of SLT in this pathway; 3) CD11c-DTR transgenic mice, in which host dendritic cells (DCs) are selectively depleted by diphtheria toxin, allowing delineation of the role of these cells in parenchymal alloantigen presentation to CD8+ T cells. Results: 2C transgenic mice rejected Balb/cHPC- grafts as rapidly as non-depleted Balb/c grafts (MST = 5d vs. 4d respectively; p=0.4) suggesting an effective mechanism for parenchymal cell driven CD8+ T cell mediated rejection. Balb/cHPC- allografts showed prolonged survival (>50d) in aly/alyspl mice given 2C CD8+ T cells, whereas in non-splenectomised controls all grafts rejected (MST = 17d; p=0.01) suggesting an essential role for SLT in this pathway. In addition, when aly/alyspl mice were given activated 2C CD8+ T cells (from a 2C recipient of a Balb/c cardiac graft) they rapidly rejected Balb/cHPC- allografts (MST = 7d; p=0.01) indicating that SLT plays a fundamental role in the activation of CD8+ T cells. To examine, therefore, whether recipient DCs were required to present to CD8+ T cells within SLT, CD4+-depleted CD11c-DTR mice given 2C CD8+ T cells were transplanted with Balb/cHPC- allografts. When these mice were also treated with diphtheria toxin to deplete host DCs they demonstrated significantly delayed rejection kinetics when compared with untreated controls (MST = 26d vs. 16d; p=0.02) highlighting a key role for these cells. Conclusion: These results indicate that allorecognition of graft parenchymal cells represents an important mechanism of CD8+ T cell mediated rejection. They support the role of a novel pathway in which recipient DCs acquire intact MHC class I from donor parenchymal cells and present to CD8+ T cells in SLT leading to their activation.

Vav1 GEF activity is required for T cell mediated allograft rejection

The GDP exchange factor (GEF) Vav1 is a central signal transducer downstream of the T cell receptor and has been identified as a key factor for T cell activation in the context of allograft rejection. Vav1 has been shown to transduce signals both dependent and independent of its GEF function. The most promising approach to disrupt Vav1 activity by pharmacological inhibition would be to target its GEF function. However, the contribution of Vav1 GEF activity for allogeneic T cell activation has not been clarified yet. To address this question, we used knock-in mice bearing a mutated Vav1 with disrupted GEF activity but intact GEF-independent functions. T cells from these mice showed strongly reduced proliferation and activation in response to allogeneic stimulation. Furthermore, lack of Vav1 GEF activity strongly abrogated the in vivo expansion of T cells in a systemic graft-versus-host model. In a cardiac transplantation model, mice with disrupted Vav1 GEF activity show prolonged allograft survival. These findings demonstrate a strong requirement for Vav1 GEF activity for allogeneic T cell activation and graft rejection suggesting that disruption of Vav1 GEF activity alone is sufficient to induce significant immunosuppression.

Serum hematopoietic growth factors as diagnostic and prognostic markers of acute renal allograft rejection: A potential role for serum stem cell factor

ObjectivesHematopoietic growth factors (HGFs) are proliferative and chemotactic agents for hematopoietic stem cells, although their functions on renal transplantation are rarely reported. This study aimed to investigate the association of HGFs with acute T cell-mediated renal rejection. Experimental designA cytokine bead array was used to analyze 10 HGFs in the sera of 32 patients with acute T cell-mediated renal allograft rejection before and during rejection and after rejection reversal. Stable renal allograft recipients (n=38) were also investigated. ResultsSerum levels of stem cell factor (SCF), IL-3, flt3 ligand, G-CSF, M-CSF and GM-CSF were elevated during episodes of rejection compared with before and after rejection. Serum concentrations of SCF, G-CSF, flt3 ligand and IL-3 were significantly higher than in the absence of rejection. Moreover, serum SCF levels were significantly higher in patients before rejection versus stable controls. ConclusionsHGFs are reported to be involved in acute T cell-mediated renal allograft rejection. A direct correlation was observed between SCF levels and the occurrence of acute renal allograft rejection which may represent an effective diagnostic and predictive biomarker for acute cellular renal allograft rejection.

Anti-Complement Component C5 mAb Synergizes with CTLA4Ig to Inhibit Alloreactive T cells and Prolong Cardiac Allograft Survival in Mice

While activation of serum complement mediates antibody-initiated vascular allograft injury, increasing evidence indicates that complement also functions as a modulator of alloreactive T cells. We tested whether blockade of complement activation at the C5 convertase step affects T cell-mediated cardiac allograft rejection in mice. The anti-C5 mAb BB5.1, which prevents the formation of C5a and C5b, synergized with subtherapeutic doses of CTLA4Ig to significantly prolong the survival of C57BL/6 heart grafts that were transplanted into naive BALB/c recipients. Anti-C5 mAb treatment limited the induction of donor-specific IFNγ-producing T cell alloimmunity without inducing Th2 or Th17 immunity in vivo and inhibited primed T cells from responding to donor antigens in secondary mixed lymphocyte responses. Additional administration of anti-C5 mAb to the donor prior to graft recovery further prolonged graft survival and concomitantly reduced both the in vivo trafficking of primed T cells into the transplanted allograft and decreased expression of T cell chemoattractant chemokines within the graft. Together these results support the novel concept that C5 blockade can inhibit T cell-mediated allograft rejection through multiple mechanisms, and suggest that C5 blockade may constitute a viable strategy to prevent and/or treat T cell-mediated allograft rejection in humans.

Acquisition of Humoral Transplantation Tolerance upon De Novo Emergence of B Lymphocytes

A major obstacle to transplantation tolerance is humoral immunity. In this paper, we demonstrate that the intrinsic developmental propensity of the B lymphocyte compartment for acquisition of self-tolerance can be harnessed to induce humoral unresponsiveness to transplanted alloantigens. In the current study, when transitional B cells developed in the presence of donor lymphoid cells, the mature B lymphocyte compartment failed to mount a donor-specific alloantibody response to an organ transplant--despite unrestrained acute T cell-mediated allograft rejection. Specifically, we generated an experimental system wherein a B6 strain B cell compartment developed de novo in the presence of F1 (B6xBALB/c) lymphoid cells and in a T cell-deficient setting. Following establishment of a steady-state B cell compartment, these B6 mice were transplanted with heterotopic cardiac allografts from allogeneic BALB/c donors. The mice were then inoculated with purified syngeneic B6 T cells. As expected, all cardiac allografts were acutely rejected. However, the B lymphocyte compartment of these mice was completely inert in its capacity to form a BALB/c-specific alloantibody response. Using an alloantigen-specific Ig transgenic system, we demonstrated that this profound degree of humoral tolerance was caused by clonal deletion of alloreactive specificities from the primary B cell repertoire. Thus, de novo B cell compartment development at the time of transplantation is of critical importance in recipient repertoire "remodeling" to a humoral tolerant state.

PKC-θ is a Drug Target for Prevention of T Cell-Mediated Autoimmunity and Allograft Rejection

Protein kinase C theta (PKC-θ) is a key kinase in mediating T cell receptor (TCR) signals. PKC-θ activated by T cell receptor (TCR) engagement translocates to immunological synapses and regulates the activation of transcriptional factors NFκB, AP-1, and NFAT. These transcription factors then activate target genes such as IL-2. T cells deficient in PKC-θ display defects in T cell activation, survival, activation-induced cell death, and the differentiation into inflammatory T cells, such as Th2 and Th17 cells both in vitro and in vivo. Since these effector T helper cells are responsible for mediating autoimmunity, selective inhibition of PKC-θ is considered a treatment for prevention of autoimmune diseases and allograft rejection.

Store-Operated Ca2+ Entry through ORAI1 Is Critical for T Cell-Mediated Autoimmunity and Allograft Rejection

ORAI1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel, which is responsible for store-operated Ca(2+) entry in lymphocytes. A role for ORAI1 in T cell function in vivo has been inferred from in vitro studies of T cells from human immunodeficient patients with mutations in ORAI1 and Orai1(-/-) mice, but a detailed analysis of T cell-mediated immune responses in vivo in mice lacking functional ORAI1 has been missing. We therefore generated Orai1 knock-in mice (Orai1(KI/KI)) expressing a nonfunctional ORAI1-R93W protein. Homozygosity for the equivalent ORAI1-R91W mutation abolishes CRAC channel function in human T cells resulting in severe immunodeficiency. Homozygous Orai1(KI/KI) mice die neonatally, but Orai1(KI/KI) fetal liver chimeric mice are viable and show normal lymphocyte development. T and B cells from Orai1(KI/KI) mice display severely impaired store-operated Ca(2+) entry and CRAC channel function resulting in a strongly reduced expression of several key cytokines including IL-2, IL-4, IL-17, IFN-γ, and TNF-α in CD4(+) and CD8(+) T cells. Cell-mediated immune responses in vivo that depend on Th1, Th2, and Th17 cell function were severely attenuated in ORAI1-deficient mice. Orai1(KI/KI) mice lacked detectable contact hypersensitivity responses and tolerated skin allografts significantly longer than wild-type mice. In addition, T cells from Orai1(KI/KI) mice failed to induce colitis in an adoptive transfer model of inflammatory bowel disease. These findings reaffirm the critical role of ORAI1 for T cell function and provide important insights into the in vivo functions of CRAC channels for T cell-mediated immunity.

Interleukin-17 Promotes Early Allograft Inflammation

Acute cellular rejection of organ transplants is executed by donor-reactive T cells, which are dominated by interferon-gamma-producing cells. As interferon-gamma is dispensable for graft destruction, we evaluated the contribution of interleukin-17A (IL-17) to intragraft inflammation in major histocompatibility complex-mismatched heart transplants. A/J (H-2(a)) cardiac allografts placed into wild-type BALB/c (H-2(d)) mice induced intragraft IL-17 production on day 2 after transplant. Allografts placed into BALB/c IL-17(-/-) recipients demonstrated diminished production of the chemokines CXCL1 and CXCL2 and delayed neutrophil and T cell recruitment. However, by day 7 after transplant, allografts from IL-17(-/-) and wild-type recipients had comparable levels of cellular infiltration. The priming of donor-specific T cells was not affected by the absence of IL-17, and the kinetics of cardiac allograft rejection were similar in wild-type and IL-17(-/-) recipients. In contrast, IL-17(-/-) mice depleted of CD8 T cells rejected A/J allografts in a delayed fashion compared with CD8-depleted wild-type recipients. Although donor-reactive CD4 T cells were efficiently activated in both groups, the infiltration of effector T cells into allografts was impaired in IL-17(-/-) recipients. Our data indicate that locally produced IL-17 amplifies intragraft inflammation early after transplantation and promotes tissue injury by facilitating T cell recruitment into the graft. Targeting the IL-17 signaling network in conjunction with other graft-prolonging therapies may decrease this injury and improve the survival of transplanted organs.

Progress towards the clinical use of CD3 monoclonal antibodies in the treatment of autoimmunity

A major problem in the field of clinical transplantation, as well as in autoimmunity, is that conventional treatments rely on chronic immunosuppression that is not specific for the antigens involved and that increases the risk of infections and tumours. A major need and challenge is, therefore, to achieve 'operational tolerance', namely an inhibition of pathogenic responses in the absence of chronic immunosuppression. Here we review data showing that monoclonal antibodies to the CD3 complex, the signal transducing element of the T cell receptor, promote immune tolerance. This strategy has been the matter of extensive experimental studies in models of autoimmunity and has recently led to a successful clinical translation. Results from controlled trials in autoimmune insulin-dependent diabetes showed that CD3 monoclonal antibodies afford long-term effects following a short administration. The present challenge is to build on these results, first, to set the use of CD3 monoclonal antibodies as an established therapy in well selected subsets of patients with autoimmunity, and second, given the similarities of immune mechanisms underlying T cell-mediated autoimmune diseases and allograft rejection, to address if and how this therapeutic strategy could be extended to organ transplantation in the not-too-distant future.

Donor Deficiency of Decay-Accelerating Factor Accelerates Murine T Cell-Mediated Cardiac Allograft Rejection

Decay-accelerating factor (DAF) is a cell surface regulator that accelerates the dissociation of C3/C5 convertases and thereby prevents the amplification of complement activation on self cells. In the context of transplantation, DAF has been thought to primarily regulate antibody-mediated allograft injury, which is in part serum complement-dependent. Based on our previously delineated link between DAF and CD4 T cell responses, we evaluated the effects of donor Daf1 (the murine homolog of human DAF) deficiency on CD8 T cell-mediated cardiac allograft rejection. MHC-disparate Daf1(-/-) allografts were rejected with accelerated kinetics compared with wild-type grafts. The accelerated rejection predominantly tracked with DAF's absence on bone marrow-derived cells in the graft and required allograft production of C3. Transplantation of Daf1(-/-) hearts into wild-type allogeneic hosts augmented the strength of the anti-donor (direct pathway) T cell response, in part through complement-dependent proliferative and pro-survival effects on alloreactive CD8 T cells. The accelerated allograft rejection of Daf1(-/-) hearts occurred in recipients lacking anti-donor Abs. The results reveal that donor DAF expression, by controlling local complement activation on interacting T cell APC partners, regulates the strength of the direct alloreactive CD8(+) T cell response. The findings provide new insights into links between innate and adaptive immunity that could be exploited to limit T cell-mediated injury to an allograft following transplantation.

Renal allograft rejection: The contribution of chemokines to the adhesion and retention of αE(CD103)β7 integrin-expressing intratubular T cells

Recruitment of activated T cells to the tubules is a defining feature of cell-mediated renal allograft rejection. Many of these intratubular T cells express the αE(CD103)β7 integrin, potentially allowing adhesion to epithelial cells which express the only defined counter-receptor, E-cadherin. However, the potential of rejection-associated intratubular chemokines to modulate the adhesive function of this integrin has not been investigated. This study demonstrated that CCL7 is expressed within the tubules during renal allograft rejection. Modelling with CD103-expressing MOLT-16 T cells demonstrated chemotactic responses to the chemokines CXCL10, CXCL12, CCL5 and, most significantly, CCL7 ( p < 0.001); these responses were consistent with the expression of CXCR3, CXCR4 and CCR1 by these cells. A solid-phase adhesion assay showed little background binding of MOLT-16 cells to immobilised human E-cadherin.Fc fusion protein but αEβ7 integrin-specific adhesion was greatly increased by the addition of either Mn 2+ or 10 nM CCL7 ( p < 0.01 or < 0.001, respectively). Treatment of activated human peripheral T cells with TGFβ 1 for 3 days induced the expression of CD103 on a mean 53% of these cells; a similar proportion of CD103+ and CD103− T cells within these cultures expressed receptors for the chemokine CCL7. CD103+ T cell fractions were sorted from mitogen- or alloantigen-activated, TGFβ 1-treated T cell cultures and also showed specific enhancement of adhesion to E-cadherin.Fc fusion protein following stimulation with Mn 2+ or 10 nM CCL7 ( p < 0.01 in all cases); CD103− T cells were not adherent under any conditions. Together these data suggest that although the αEβ7 integrin is induced on activated intratubular T cells by the presence of TGFβ, the adhesive function of this integrin is promoted by the presence of chemokines such as CCL7, which are also expressed within tubules during renal allograft rejection.

A Critical Role for Protein Kinase C-θ-Mediated T Cell Survival in Cardiac Allograft Rejection

Protein kinase C (PKC)-theta mediates the critical TCR signals required for T cell activation. Previously, we have shown that in response to TCR stimulation, PKC-theta-/- T cells undergo apoptosis due to greatly reduced levels of the anti-apoptotic molecule, Bcl-xL. In this study, we demonstrate that PKC-theta-regulated expression of Bcl-xL is essential for T cell-mediated cardiac allograft rejection. Rag1-/- mice reconstituted with wild-type T cells readily rejected fully mismatched cardiac allografts, whereas Rag1-/- mice reconstituted with PKC-theta-/- T cells failed to promote rejection. Transgenic expression of Bcl-xL in PKC-theta-/- T cells was sufficient to restore cardiac allograft rejection, suggesting that PKC-theta-regulated survival is required for T cell-mediated cardiac allograft rejection in this adoptive transfer model. In contrast to adoptive transfer experiments, intact PKC-theta-/- mice displayed delayed, but successful cardiac allograft rejection, suggesting the potential compensation for PKC-theta function. Finally, a subtherapeutic dose of anti-CD154 Ab or CTLA4-Ig, which was not sufficient to prevent cardiac allograft rejection in the wild-type mice, prevented heart rejection in the PKC-theta-/- mice. Thus, in combination with other treatments, inhibition of PKC-theta may facilitate achieving long-term survival of allografts.

Banff Schema for Grading Pancreas Allograft Rejection: Working Proposal by a Multi-Disciplinary International Consensus Panel

Accurate diagnosis and grading of rejection and other pathological processes are of paramount importance to guide therapeutic interventions in patients with pancreas allograft dysfunction. A multi-disciplinary panel of pathologists, surgeons and nephrologists was convened for the purpose of developing a consensus document delineating the histopathological features for diagnosis and grading of rejection in pancreas transplant biopsies. Based on the available published data and the collective experience, criteria for the diagnosis of acute cell-mediated allograft rejection (ACMR) were established. Three severity grades (I/mild, II/moderate and III/severe) were defined based on lesions known to be more or less responsive to treatment and associated with better- or worse-graft outcomes, respectively. The features of chronic rejection/graft sclerosis were reassessed, and three histological stages were established. Tentative criteria for the diagnosis of antibody-mediated rejection were also characterized, in anticipation of future studies that ought to provide more information on this process. Criteria for needle core biopsy adequacy and guidelines for pathology reporting were also defined. The availability of a simple, reproducible, clinically relevant and internationally accepted schema for grading rejection should improve the level of diagnostic accuracy and facilitate communication between all parties involved in the care of pancreas transplant recipients.

A critical role for PKC‐theta‐mediated T cell survival in cardiac allograft rejection

PKC‐theta mediates the critical TCR signals required for T cell activation. Previously we have shown that in response to TCR stimulation, PKC‐theta−/− T cells undergo apoptosis due to greatly reduced levels of the anti‐apoptotic molecule, Bcl‐xL. Here we demonstrate that PKC‐theta‐regulated expression of Bcl‐xL is essential for T cell‐mediated cardiac allograft rejection. Rag1−/− mice reconstituted with wild type (WT) T cells readily rejected fully mismatched cardiac allografts, whereas, Rag1−/− mice reconstituted with PKC‐theta−/− T cells failed to promote rejection. Transgenic expression of Bcl‐xL in PKC‐theta−/− T cells was sufficient to restore cardiac allograft rejection, suggesting that PKC‐theta‐regulated survival is required for T cell‐mediated cardiac allograft rejection in this adoptive transfer model. In contrast to adoptive transfer experiments, intact PKC‐theta−/− mice displayed delayed, but successful cardiac allograft rejection, suggesting the potential compensation for PKC‐theta function. Finally, a sub‐therapeutic dose of anti‐CD154 antibody, that was not sufficient to prevent cardiac allograft rejection in the WT mice, prevented heart rejection in the PKC‐theta−/− mice. Thus, in combination with other treatments, inhibition of PKC‐theta may facilitate achieving long‐term survival of allografts.

OX40 costimulation turns off Foxp3+ Tregs

OX40 is a recently identified T-cell costimulatory molecule that belongs to the TNF/TNFR superfamily. OX40 can be expressed by both activated T effector cells and Foxp3(+) Tregs. It is well known that OX40 delivers a potent costimulatory signal to T effector cells, but very little is known about the role of OX40 in regulating the suppressor properties of Foxp3(+) Tregs and the de novo generation of new inducible Foxp3(+) Tregs from T effector cells. In the present study, we found, by using a newly created foxp3gfp knockin model, that OX40 was dispensable for the genesis and suppressor functions of naturally arising CD4(+)Foxp3(+) Tregs, but stimulating OX40 on the Foxp3(+) Tregs abrogated their ability to suppress T effector cell proliferation, IFN-gamma production, and T effector cell-mediated allograft rejection. OX40 costimulation did not significantly affect proliferation and survival of the naturally arising Foxp3(+) Tregs, but profoundly inhibited Foxp3 gene expression. Importantly, OX40 costimulation to T effector cells prevented the induction of new inducible Foxp3(+) Tregs from T effector cells. Our study identified OX40 as a key negative regulator of Foxp3(+) Tregs and may have important clinical implications in models of transplantation and autoimmunity.

Suppression of Memory CD8 T Cell Generation and Function by Tryptophan Catabolism

Dendritic cell-derived indoleamine 2,3-dioxygenase (IDO) suppresses naive T cell proliferation and induces their apoptosis by catalyzing tryptophan, and hence is essential for the maintenance of peripheral tolerance. However, it is not known whether memory T cells are subject to the regulation by IDO-mediated tryptophan catabolism, as memory T cells respond more rapidly and vigorously than their naive counterparts and are resistant to conventional costimulatory blockade. In this study, we present the evidence that memory CD8+ T cells are susceptible to tryptophan catabolism mediated by IDO. We found that overexpression of IDO in vivo attenuated the generation of both central memory CD8+ T cells (T(CM)) and effector memory CD8+ T cells (T(EM)) while suppressing IDO activity promoted their generation. Moreover, IDO overexpression suppressed the effector function of T(CM) cells or T(CM) cell-mediated allograft rejection as well as their proliferation in vivo. Interestingly, T(CM) cells were resistant to apoptosis induced by tryptophan catabolism. However, IDO overexpression did not suppress the effector function of T(EM) cells or T(EM) cell-mediated allograft rejection, suggesting that T(EM) cells, unlike T(CM) cells, do not require tryptophan for their effector function once they are generated. This study provides insight into the mechanisms underlying the differential regulation of memory T cell responsiveness and has clinical implications for vaccination or tolerance induction.

Immunomodulatory Properties of FK734, a Humanized Anti-CD28 Monoclonal Antibody With Agonistic and Antagonistic Activities

We describe immunomodulatory effects of FK734, a humanized version of a mouse anti-human CD28 mAb (clone TN228), in vitro and in a chimeric human-mouse model of allograft rejection. Cytokine production and proliferation were assessed in a mixed lymphocyte reaction containing FK734, human T cells, and endothelial cells or monocytes. FK734 was also administered to SCID mice engrafted with human skin and adoptively transferred with human peripheral blood mononuclear cells allogeneic to the skin graft. In vitro, FK734 enhanced secretion of interleukin-2 and interferon-gamma as well as proliferation of CD4+ and CD8+ T cells stimulated by allogeneic human leukocyte antigen (HLA)-DR+ human umbilical vein endothelial cells (which lack B7 molecules and FcgammaRs) or by blood monocytes (which express low levels of B7 molecules and FcgammaRs) compared with control mAb, but these effects were significantly smaller than those provided by mAb 28.2, a stimulatory mouse anti-human CD28 mAb, at comparable concentrations. However, FK734 generally inhibited cytokine secretion and T cell proliferation in cocultures with human umbilical vein endothelial cells transduced to express CD86. In vivo using SCID/beige mice bearing human skin with adoptively transferred peripheral blood mononuclear cells, administration of FK734 protected human endothelial cell-lined microvessels, significantly but incompletely reducing endothelial cell injury and T cell infiltration into the graft one or two weeks later. FK734 is a partial agonist of CD28 signaling that can reduce human T cell alloresponses in the presence of strong costimulation by B7 molecules in vitro and can reduce T cell-mediated skin allograft rejection in vivo.

Tolerance induction by third-party “off-the-shelf” CD4 +CD25 + Treg cells

Recent reports have shown that donor or host CD4(+)CD25(+) Treg cells can be used to control GVHD or graft rejection following allogeneic BMT in mice. In the present study we investigated the potential of third-party Treg cells compared to donor-type cells to facilitate BM allografting. Graft rejection is assessed in a mouse model of T cell-mediated BM allograft rejection. Lethally irradiated C3H mice are transplanted at day 2 after irradiation with T cell-depleted Balb/Nude BM. Graft rejection is induced by purified host-type T cells infused one day prior to BMT. Cells tested for their facilitating activity are added to the T cell-depleted BM allograft. Naïve or ex vivo-expanded third-party Treg cells can effectively enhance engraftment of T cell-depleted BM allografts, exhibiting reactivity in vitro and in vivo similar to that found for donor-type Treg cells. The use of third-party Treg cells in contrast to donor-type cells could allow advanced preparation of a large bank of Treg cells (off-the-shelf), with all the appropriate quality controls required for cell therapy.

A role for cytokine measurement in therapeutic monitoring of immunosuppressive drugs following lung transplantation

The strength of the allo-immune response to a transplanted organ [1,2] currently mandates combined pharmacological immunosuppression to prevent rejection. The calcineurine inhibitor Cyclosporine remains an important component of immunosuppression regimens post lung transplantation with the International Registry of Heart and Lung Transplantation recording 50% of patients receiving this drug. Cyclosporine requires therapeutic drug monitoring with the aim of maximizing efficacy whilst minimizing the risk of toxicity. Historically the trough (predose or Co) level has been used to guide the dose of cyclosporin and the dose range in lung transplantation has been determined by rather empirical review of studies in kidney transplantation [3]. A survey conducted in 1994–95 showed that most lung transplant centres however, recommended a higher range of target concentrations during the early post operative period [4]. The recommended therapeutic blood concentrations however, are broad and may vary according to the clinical situation and the choice of other immunosuppressive agents being administered. Relatively recent data has supported the use of therapeutic drug monitoring of cyclosporin by the use of whole blood drawn two hours after administration of the dose (C2) with data showing a better prediction of clinical effect [5]. Nevertheless therapeutic drug monitoring has certain general limitations with the concentration measured in blood, not necessarily the concentration of the drug at the site of action. The alternative approach, as discussed in a study by Hodge and colleagues in the January 2005 issue of Clinical and Experimental Immunology is to measure the therapeutic action of the drug either directly or indirectly [6]. In general the approaches involve in vitro assessment of T cell activation by measuring cytokine release from peripheral circulating cells either in the resting or stimulated state [7–9]. Whilst these techniques have great potential, it is not yet established whether they are predictive of clinical events. Moreover the complexity of the allo-immune response may make it difficult to model using such assays. There is also a problem regarding which compartment of the immune system should one study in order to give the best biological information. Most studies use peripheral circulating cells rather than intragraft immune cells which may be more relevant in terms of effecting graft injury. In a broad sense, the process of immune cell-mediated allograft rejection is regulated by an array of cytokines. Some of these, for example the chemokines, are produced by stressed graft cells and activated immune cells whilst others, such as IL-2 and IL-4, are produced by activated immune cells. Given the importance of this range of cytokines, it is perhaps surprising that immunosuppressive calcineurin inhibitors such as Cyclosporin A (Cs-A) are effective as they directly regulate the expression of only a very limited range of cytokines. This includes blockade of de novo expression of the T cell growth factor IL-2 following the activation of resting T cells [10] and up-regulation of the expression of TGF-β by a range of parenchymal cells [11]. Clearly the analysis of cytokine expression has the potential to provide clinically useful information about the state of allograft rejection or acceptance. However, a number of issues must first be addressed, including:

Non-Invasive Monitoring of Renal Transplant Recipients: Urinary Excretion of Soluble Adhesion Molecules and of the Complement-Split Product C4d

Background: The number of inducible adhesion molecules known to be involved in cell-mediated allograft rejection is still increasing. In addition, recent data describe complement activation during acute humoral allograft rejection. The aim of this study was to assess whether specific molecules from either pathway are excreted into urine and whether they can provide useful diagnostic tools for the monitoring of renal transplant recipients. Methods: Urinary concentrations of soluble adhesion molecules (sICAM-1, sVCAM-1) and of the complement degradation product C4d were determined by standardized ELISA technique in 75 recipients of renal allografts and 29 healthy controls. Patient samples were assigned to four categories according to clinical criteria: group 1: acute steroid-sensitive rejection (ASSR, n = 14), group 2: acute steroid-resistant rejection (ASRR, n = 12), group 3: chronic allograft dysfunction (CAD, n = 20) and group 4: stable graft function (SGF, n = 29). Results: All patients with rejection episodes (groups 1–3) had significantly higher values of urinary sC4d compared with healthy controls and patients with stable graft function (p < 0.05). The urinary levels of sVCAM-1 were significantly higher in group 2 (ASRR) compared with all other groups (p < 0.001). Uniformly low amounts of s-VCAM-1 and complement-split product C4d were excreted by healthy controls (group 0). In contrast, urinary sICAM-1 concentration in healthy controls was almost as high as in group 2 (ASRR) whereas patients with a stable functioning graft (group 4) excreted significantly less sICAM-1 (p < 0.05). Conclusion: The evaluation of sVCAM-1 and sC4d excretion in urine can provide a valuable tool with regard to the severity and type of allograft rejection. With respect to long-term allograft survival, serial measurements of these markers should have the potential to detect rejection episodes and prompt immediate treatment.