Continuous Acquisition of MHC:Peptide Complexes by Recipient Cells Contributes to the Generation of Anti-Graft CD8+ T Cell Immunity.

Understanding the evolution of the direct and indirect pathways of allorecognition following tissue transplantation is essential in the design of tolerance-promoting protocols. On the basis that donor bone marrow-derived antigen-presenting cells are eliminated within days of transplantation, it has been argued that the indirect response represents the major threat to long-term transplant survival, and is consequently the key target for regulation. However, the detection of MHC transfer between cells, and particularly the capture of MHC:peptide complexes by dendritic cells (DCs), led us to propose a third, semidirect, pathway of MHC allorecognition. Persistence of this pathway would lead to sustained activation of direct-pathway T cells, arguably persisting for the life of the transplant. In this study, we focused on the contribution of acquired MHC-class I on recipient DCs during the life span of a skin graft. We observed that MHC-class I acquisition by recipient DCs occurs for at least 1 month following transplantation and may be the main source of alloantigen that drives CD8+ cytotoxic T cell responses. In addition, acquired MHC-class I:peptide complexes stimulate T cell responses in vivo, further emphasizing the need to regulate both pathways to induce indefinite survival of the graft.

Expression of a Chimeric Antigen Receptor Specific for Donor HLA Class I Enhances the Potency of Human Regulatory T Cells in Preventing Human Skin Transplant Rejection.

Regulatory T cell (Treg) therapy using recipient-derived Tregs expanded ex vivo is currently being investigated clinically by us and others as a means of reducing allograft rejection following organ transplantation. Data from animal models has demonstrated that adoptive transfer of allospecific Tregs offers greater protection from graft rejection compared to polyclonal Tregs. Chimeric antigen receptors (CAR) are clinically translatable synthetic fusion proteins that can redirect the specificity of T cells toward designated antigens. We used CAR technology to redirect human polyclonal Tregs toward donor-MHC class I molecules, which are ubiquitously expressed in allografts. Two novel HLA-A2-specific CARs were engineered: one comprising a CD28-CD3ζ signaling domain (CAR) and one lacking an intracellular signaling domain (ΔCAR). CAR Tregs were specifically activated and significantly more suppressive than polyclonal or ΔCAR Tregs in the presence of HLA-A2, without eliciting cytotoxic activity. Furthermore, CAR and ΔCAR Tregs preferentially transmigrated across HLA-A2-expressing endothelial cell monolayers. In a human skin xenograft transplant model, adoptive transfer of CAR Tregs alleviated the alloimmune-mediated skin injury caused by transferring allogeneic peripheral blood mononuclear cells more effectively than polyclonal Tregs. Our results demonstrated that the use of CAR technology is a clinically applicable refinement of Treg therapy for organ transplantation.

Regulatory T cells: tolerance induction in solid organ transplantation.

The concept of regulatory T cell (Treg ) therapy in transplantation is now a reality. Significant advances in science and technology have enabled us to isolate human Tregs , expand them to clinically relevant numbers and infuse them into human transplant recipients. With several Phase I/II trials under way investigating Treg safety and efficacy it is now more crucial than ever to understand their complex biology. However, our journey is by no means complete; results from these trials will undoubtedly provoke both further knowledge and enquiry which, alongside evolving science, will continue to drive the optimization of Treg therapy in the pursuit of transplantation tolerance. In this review we will summarize current knowledge of Treg biology, explore novel technologies in the setting of Treg immunotherapy and address key prerequisites surrounding the clinical application of Tregs in transplantation.

Biomarkers of Tolerance in Kidney Transplantation: Are We Predicting Tolerance or Response to Immunosuppressive Treatment?

We and others have previously described signatures of tolerance in kidney transplantation showing the differential expression of B cell-related genes and the relative expansions of B cell subsets. However, in all of these studies, the index group-namely, the tolerant recipients-were not receiving immunosuppression (IS) treatment, unlike the rest of the comparator groups. We aimed to assess the confounding effect of these regimens and develop a novel IS-independent signature of tolerance. Analyzing gene expression in three independent kidney transplant patient cohorts (232 recipients and 14 tolerant patients), we have established that the expression of the previously reported signature was biased by IS regimens, which also influenced transitional B cells. We have defined and validated a new gene expression signature that is independent of drug effects and also differentiates tolerant patients from healthy controls (cross-validated area under the receiver operating characteristic curve [AUC] = 0.81). In a prospective cohort, we have demonstrated that the new signature remained stable before and after steroid withdrawal. In addition, we report on a validated and highly accurate gene expression signature that can be reliably used to identify patients suitable for IS reduction (approximately 12% of stable patients), irrespective of the IS drugs they are receiving. Only a similar approach will make the conduct of pilot clinical trials for IS minimization safe and hence allow critical improvements in kidney posttransplant management.

Clinical grade manufacturing of human alloantigen-reactive regulatory T cells for use in transplantation.

Regulatory T cell (Treg) therapy has the potential to induce transplantation tolerance so that immunosuppression and associated morbidity can be minimized. Alloantigen-reactive Tregs (arTregs) are more effective at preventing graft rejection than polyclonally expanded Tregs (PolyTregs) in murine models. We have developed a manufacturing process to expand human arTregs in short-term cultures using good manufacturing practice-compliant reagents. This process uses CD40L-activated allogeneic B cells to selectively expand arTregs followed by polyclonal restimulation to increase yield. Tregs expanded 100- to 1600-fold were highly alloantigen reactive and expressed the phenotype of stable Tregs. The alloantigen-expanded Tregs had a diverse TCR repertoire. They were more potent than PolyTregs in vitro and more effective at controlling allograft injuries in vivo in a humanized mouse model.

Whole-body imaging of adoptively transferred T cells using magnetic resonance imaging, single photon emission computed tomography and positron emission tomography techniques, with a focus on regulatory T cells.

Cell-based therapies using natural or genetically modified regulatory T cells (T(regs)) have shown significant promise as immune-based therapies. One of the main difficulties facing the further advancement of these therapies is that the fate and localization of adoptively transferred T(regs) is largely unknown. The ability to dissect the migratory pathway of these cells in a non-invasive manner is of vital importance for the further development of in-vivo cell-based immunotherapies, as this technology allows the fate of the therapeutically administered cell to be imaged in real time. In this review we will provide an overview of the current clinical imaging techniques used to track T cells and T(regs) in vivo, including magnetic resonance imaging (MRI) and positron emission tomography (PET)/single photon emission computed tomography (SPECT). In addition, we will discuss how the finding of these studies can be used, in the context of transplantation, to define the most appropriate T(reg) subset required for cellular therapy.

Donor-specific indirect pathway analysis reveals a B-cell-independent signature which reflects outcomes in kidney transplant recipients.

To investigate the role of donor-specific indirect pathway T cells in renal transplant tolerance, we analyzed responses in peripheral blood of 45 patients using the trans-vivo delayed-type hypersensitivity assay. Subjects were enrolled into five groups-identical twin, clinically tolerant (TOL), steroid monotherapy (MONO), standard immunosuppression (SI) and chronic rejection (CR)-based on transplant type, posttransplant immunosuppression and graft function. The indirect pathway was active in all groups except twins but distinct intergroup differences were evident, corresponding to clinical status. The antidonor indirect pathway T effector response increased across patient groups (TOL < MONO < SI < CR; p < 0.0001) whereas antidonor indirect pathway T regulatory response decreased (TOL > MONO = SI > CR; p < 0.005). This pattern differed from that seen in circulating naïve B-cell numbers and in a cross-platform biomarker analysis, where patients on monotherapy were not ranked closest to TOL patients, but rather were indistinguishable from chronically rejecting patients. Cross-sectional analysis of the indirect pathway revealed a spectrum in T-regulatory:T-effector balance, ranging from TOL patients having predominantly regulatory responses to CR patients having predominantly effector responses. Therefore, the indirect pathway measurements reflect a distinct aspect of tolerance from the recently reported elevation of circulating naïve B cells, which was apparent only in recipients off immunosuppression.

Helicobacter pylori induces in-vivo expansion of human regulatory T cells through stimulating interleukin-1ß production by dendritic cells.

Helicobacter pylori is one of the most common infections in the world. Despite inciting inflammation, immunological clearance of the pathogen is often incomplete. CD4(+) CD25(hi) forkhead box protein 3 (FoxP3(+)) regulatory T cells (T(regs)) are potent suppressors of different types of immune responses and have been implicated in limiting inflammatory responses to H. pylori. Investigating the influence of H. pylori on T(reg) function and proliferation, we found that H. pylori-stimulated dendritic cells (DCs) induced proliferation in T(regs) and impaired their suppressive capability. This effect was mediated by interleukin (IL)-1ß produced by H. pylori-stimulated DCs. These data correlated with in-vivo observations in which H. pylori(+) gastric mucosa contained more T(regs) in active cell division than uninfected stomachs. Inciting local proliferation of T(regs) and inhibiting their suppressive function may represent a mechanism for the chronic gastritis and carcinogenesis attributable to H. pylori.

Relative resistance of human CD4(+) memory T cells to suppression by CD4(+) CD25(+) regulatory T cells.

Successful expansion of functional CD4(+) CD25(+) regulatory T cells (T(reg)) ex vivo under good manufacturing practice conditions has made T(reg) -cell therapy in clinical transplant tolerance induction a feasible possibility. In animals, T(reg) cells home to both transplanted tissues and local lymph nodes and are optimally suppressive if active at both sites. Therefore, they have the opportunity to suppress both naïve and memory CD4(+) CD25(-) T cells (Tresp). Clinical transplantation commonly involves depleting therapy at induction (e.g. anti-CD25), which favors homeostatic expansion of memory T cells. Animal models suggest that T(reg) cells are less suppressive on memory, compared with naïve Tresp that mediate allograft rejection. As a result, in the context of human T(reg) -cell therapy, it is important to define the effectiveness of T(reg) cells in regulating naïve and memory Tresp. Therefore, we compared suppression of peripheral blood naïve and memory Tresp by fresh and ex vivo expanded T(reg) cells using proliferation, cytokine production and activation marker expression (CD154) as readouts. With all readouts, naïve human Tresp were more suppressible by approximately 30% than their memory counterparts. This suggests that T(reg) cells may be more efficacious if administered before or at the time of transplantation and that depleting therapy should be avoided in clinical trials of T(reg) cells.

Restoration of immunogenicity to passenger cell-depleted kidney allografts by the addition of donor strain dendritic cells.

The immunogenicity of long-surviving, enhanced (AS X AUG)F1 renal allografts retransplanted into secondary AS recipients was restored by the injection of small numbers of donor strain dendritic cells derived from afferent lymph. Whereas 1 X 10(4) to 5 X 10(4) dendritic cells were able to trigger an acute rejection response, neither the passenger volume of donor strain blood nor 5 X 10(6) T or B lymphocytes were able to do so, thereby demonstrating more than a 100-fold difference in immunogenic potency. It is concluded that intrarenal dendritic cells provide the major immunogenic stimulus of a kidney allograft. These results suggest that the antigenic strength of major histocompatibility complex-incompatible tissue correlates with the content of donor strain dendritic cells. They also provide further evidence that antigens of the major histocompatibility complex behave like conventional antigens unless they are on the surface of allogeneic dendritic cells.

Immunogenicity of retransplanted rat kidney allografts. Effect of inducing chimerism in the first recipient and quantitative studies on immunosuppression of the second recipient.

It has been previously shown that long surviving, enhanced (AS X AUG)F1 rat kidneys residing in a primary AS recipient are not acutely rejected if transferred into a second AS recipient. The reduced immunogenicity of the retransplanted graft was attributed to a depletion of incompatible passenger cells. It is shown here that if the primary AS recipient is made chimeric by x irradiation and injection of (AS X AUG)F1 bone marrow cells, transfer of the long surviving, enhanced graft into a second AS recipient provokes acute graft rejection comparable to that observed when normal (AS X AUG)F1 kidneys are transplanted into untreated AS recipients. Transplantation of passenger cell-depleted AUG kidneys into AS recipients leads to graft rejection, with a median survival time of 22 d. Treatment of these recipients with as little as 1.5 mg/kg cyclophosphamide for 14 d induces prolonged graft survival. By contrast, five times as much cyclophosphamide treatment is required to induce prolonged survival of normal AUG kidneys (i.e., containing incompatible passenger cells) transplanted to AS recipients. These results confirm that the major alloimmunogenic stimulus of rat kidney grafts is provided by the incompatible passenger cells.

I-A-restricted T cell antigen recognition. Analysis of the roles of A alpha and A beta using DNA-mediated gene transfer.

The contributions of A alpha and A beta chains, and of subregions of A beta, to Ia-restricted recognition of antigen by Th lymphocytes were analyzed using a panel of L cells transfected with various pairs of A alpha b,d, or k genes and recombinant or wild-type A beta b,d, or k genes. The A beta genes included all possible exchanges of the whole NH2-terminal (beta 1) domain or halves of the beta 1 domain among these three allelic A beta genes. The Ia+ L cells derived from such transfections were used as antigen-presenting cells with a 21 member panel of responding Ia-restricted T hybridoma cells of differing nominal antigen specificity and Ia-restriction. Special care was taken to account for quantitative variation in levels of Ia expression throughout the experiments. The results of this analysis reveal that (a) only 2 of the 21 Th cells recognized Ia molecules involving either a nonparental A alpha or a nonparental A beta chain, and in both cases the degeneracy extended to only one of the two other alleles tested. This suggests that allele specific contributions from both A alpha and A beta chains are important in restricted recognition for most, if not all I-A-restricted Th cells. (b) In no case did substitution of the A beta 2 domain from either of the alternative haplotypes lead to any functionally detectable effects, demonstrating that polymorphisms in the A beta 1 domain can entirely account for the restriction imposed on Th cell responses by the entire A beta chain. (c) For 90% of the cells tested, replacement of the NH2-terminal portion of the beta 1 domain with an allogeneic segment led to Ia molecules unable to elicit Th responses. Furthermore, of all the cells permissive of the substitution of one or other half of the beta 1 domain, only two permitted the substitution of sequence from both alternative haplotypes. Taken together, these data strongly suggest that antigen recognition by most, if not all, I-A-restricted Th cells involves contributions from both halves of the A beta 1 domain. These data suggest that the role of I-A molecules in restricted Th cell recognition of antigen depends on conformational determinants unique to a particular combination of polymorphic alpha and beta chains, and that multiple such sites exist on a single Ia molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Early genetic events in T cell development analyzed by in situ hybridization.

In situ hybridization was used to investigate the expression of T cell receptor (TCR) alpha, beta, and gamma mRNAs in developing fetal and adult precursor thymocytes. gamma transcription was observed at the earliest time tested (day 12), followed by beta 12 h later, and TCR alpha on day 16. The early beta transcripts appeared to be from unrearranged or incompletely rearranged (D-J-C) beta loci. V beta region transcription was first detectable on day 14 and transcription of different V beta genes was induced at different times. These results delineate a schedule sequence of TCR gene activation, which begins within 1 d after entry of stem cells into the fetal thymus.

Human T cells cannot act as autonomous antigen-presenting cells, but induce tolerance in antigen-specific and alloreactive responder cells.

The ability of two HLA-DR-expressing human T cell clones to function as antigen-presenting cells (APC) was investigated using highly purified T cells. The results demonstrated that these T cell clones are unable to act as autonomous APC, and that recognition of nominal or alloantigens on the surface of T cells leads to a state of nonresponsiveness. The first observation was that a T cell clone with specificity for the 306-324 peptide of influenza hemagglutinin (HA), and raised from a DR1 responder, exhibited apparent degeneracy of major histocompatibility complex restriction when cultured with peptide in the presence of peripheral blood mononuclear cells (PBMC) expressing a wide variety of structurally unrelated DR types. However, when the PBMC were pulsed with peptide and washed before coculture with the clone, peptide was exclusively recognized with DR1Dw1. This implied that in the presence of soluble peptide the T cells were displaying ligand to each other, and that the third-party APC were providing costimulatory signals. To test the ability of T cells to act as autonomous APC, accessory cell-free preparations of two DR1-restricted clones were cultured with peptide in the presence or the absence of added B cell APC. T cell purity was established by the absence of proliferation in response to the mitogen phytohemagglutinin (PHA). PHA-nonresponsive T cells were completely unable to proliferate in response to peptide alone; furthermore, preculture of the HA-specific clone, in the complete absence of accessory cells, with the same concentration of peptide (1 microgram/ml) that induced optimal proliferation when presented by conventional APC, led to profound nonresponsiveness. The same phenomenon was also observed when two of three anti-DR1 alloreactive T cell clones were precultured with a DR1-expressing T cell clone. The ability of the DR1-expressing clone to induce nonresponsiveness in anti-DR1 clones correlated with recognition of the DR1 alloantigen on the DR1-expressing clone.

Major histocompatibility complex class II-expressing endothelial cells induce allospecific nonresponsiveness in naive T cells.

The role of endothelial cells (EC) in initiating a primary T cell response is of importance in clinical transplantation and autoimmunity since EC are the first allogeneic target encountered by the recipient's immune system and may display tissue-specific autoantigens in the context of an inflammatory response. In this study, we have investigated the antigen-presenting cell function of human umbilical vein-derived EC (HUVEC), depleted of constitutively major histocompatibility complex class II+ cells and induced to express class II molecules by interferon-gamma. The results show that HUVEC do not express B7 but can support proliferation by antigen-specific T cell clones. In contrast, they were unable to initiate a primary alloresponse using three independent HUVEC cultures and MHC class II-mismatched CD4+ T cells from eight donors. The response to HUVEC was reconstituted by trans-costimulation provided by DAP.3 transfectants expressing human B7.1. Coculture of peripheral blood T cells with EC expressing allogeneic DR molecules had markedly different effects on CD45RO+ and RA+ subsets. Subsequent reactivity of the RO+ T cells was unaffected by exposure to EC, indicating a neutral encounter. In contrast, culture with DR+ EC induced allospecific nonresponsiveness in RA+ T cells.

Differential role of naïve and memory CD4 T-cell subsets in primary alloresponses.

The T cell response to major histocompatibility complex (MHC) alloantigens occurs via two main pathways. The direct pathway involves the recognition of intact allogeneic MHC:peptide complexes on donor cells and provokes uniquely high frequencies of responsive T cells. The indirect response results from alloantigens being processed like any other protein antigen and presented as peptide by autologous antigen-presenting cells. The frequencies of T cells with indirect allospecificity are orders of magnitude lower and comparable to other peptide-specific responses. In this study, we explored the contributions of naïve and memory CD4(+) T cells to these two pathways. Using an adoptive transfer and skin transplantation model we found that naive and memory CD4(+) T cells, both naturally occurring and induced by sensitization with multiple third-party alloantigens, contributed equally to graft rejection when only the direct pathway was operative. In contrast, the indirect response was predominantly mediated by the naïve subset. Elimination of regulatory CD4(+)CD25(+) T cells enabled memory cells to reject grafts through the indirect pathway, but at a much slower tempo than for naïve cells. These findings have implications for better targeting of immunosuppression to inhibit immediate and later forms of alloimmunity.

Translational mini-review series on Th17 cells: induction of interleukin-17 production by regulatory T cells.

Uncommitted (naive) CD4(+) T helper cells (Thp) can be induced to differentiate to specific lineages according to the local cytokine milieu, towards T helper type 1 (Th1), Th2, Th17 and regulatory T cell (T(reg)) phenotypes in a mutually exclusive manner. Each phenotype is characterized by unique signalling pathways and expression of specific transcription factors, notably T-bet for Th1, GATA-3 for Th2, forkhead box P3 (FoxP3) for T(regs) and receptor-related orphan receptor (ROR)alpha and RORgammat for Th17 cells. T(regs) and Th17 cells have been demonstrated to arise from common precursors in a reciprocal manner based on exposure to transforming growth factor (TGF)-beta or TGF-beta plus interleukin (IL)-6 and carry out diametrically opposing functions, namely suppression or propagation of inflammation, respectively. However, while epigenetic modifications in Th1 and Th2 differentiated cells prevents their conversion to other phenotypes, Th17 cells generated in vitro using TGF-beta and IL-6 are unstable and can convert to other phenotypes, especially Th1, both in vitro and in vivo. T(regs) are generated from naive precursors both in the thymus (natural, nT(regs)) and in the periphery (induced, iT(regs)). The highly suppressive function of T(regs) enables them to control many inflammatory diseases in animals and makes them particularly attractive candidates for immunotherapy in humans. The stability of the T(reg) phenotype is therefore of paramount importance in this context. Recent descriptions of T(reg) biology have suggested that components of pathogens or inflammatory mediators may subvert the suppressive function of T(regs) in order to allow propagation of adequate immune responses. Unexpectedly, however, a number of groups have now described conversion of T(regs) to the Th17 phenotype induced by appropriate inflammatory stimuli. These observations are particularly relevant in the context of cell therapy but may also explain some of the dysregulation seen in autoimmune diseases. In this paper, we review T(reg) to Th17 conversion and propose some potential mechanisms for this phenomenon.

Author Correction: Diversity of gut microflora is required for the generation of B cell with regulatory properties in a skin graft model.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Natural regulatory T cells: number and function are normal in the majority of patients with lupus nephritis.

CD4(+) CD25(+) regulatory T cells have been shown to be a vital component of the mechanisms that prevent autoreactivity in mice and also in humans. Previous studies have examined CD4(+) CD25(hi) regulatory T cell frequency and function in patients with systemic lupus erythematosus (SLE) with mixed results. We investigated frequency, phenotype and function in 21 patients with SLE and six with inactive disease. We found no reduction in frequency of the CD25(hi) subset, although active disease was associated with an increased proportion of CD4(+) CD25(+) T cells. When examining function, in the majority of individuals suppression was comparable with controls, although cells isolated from one patient with active disease failed to suppress proliferation. On testing the effect of CD25(hi) depletion on the responses of whole peripheral blood mononuclear cells to nucleosomes we found that, where a response was detectable from patients, depletion augmented interferon-gamma secretion, demonstrating intact suppression of responses implicated in the pathogenesis of SLE. Our results did not confirm an association of failure in CD4(+) CD25(hi) regulatory T cell function or a reduction in their frequency with active disease. Instead, perturbations in the CD4(+) CD25(hi) regulatory T cell population may play a role in disease in only a minority of the patients afflicted by the diverse syndromes of SLE.

Leptin protects mice from starvation-induced lymphoid atrophy and increases thymic cellularity in ob/ob mice.

Thymic atrophy is a prominent feature of malnutrition. Forty-eight hours' starvation of normal mice reduced the total thymocyte count to 13% of that observed in freely fed controls, predominantly because of a diminution in the cortical CD4(+)CD8(+) thymocyte subpopulation. Prevention of the fasting-induced fall in the level of the adipocyte-derived hormone leptin by administering exogenous recombinant leptin protected mice from these starvation-induced thymic changes. The ob/ob mouse, which is unable to produce functional leptin because of a mutation in the obese gene, has impaired cellular immunity together with a marked reduction in the size and cellularity of the thymus. We found that ob/ob mice had a high level of thymocyte apoptosis resulting in a ratio of CD4(+)CD8(+) (cortical) to CD4(-)CD8(-) (precursor) thymocytes that was 4-fold lower than that observed in wild-type mice. Peripheral administration of recombinant leptin to ob/ob mice reduced thymocyte apoptosis and substantially increased both thymic cellularity and the CD4(+)CD8(+)/CD4(-)CD8(-) ratio. In contrast, a comparable weight loss in pair-fed PBS-treated ob/ob mice had no impact on thymocyte number. In vitro, leptin protected thymocytes from dexamethasone-induced apoptosis. These data indicate that reduced circulating leptin concentrations are pivotal in the pathogenesis of starvation-induced lymphoid atrophy.