In Vitro Induction of Human Regulatory T Cells Using Conditions of Low Tryptophan Plus Kynurenines.

Thymic regulatory T cells (tTregs) and induced regulatory T cells (iTregs) suppress murine acute graft-versus-host disease (GVHD). Previously, we demonstrated that the plasmacytoid dendritic cell indoleamine 2,3-dioxygenase (IDO) fosters the in vitro development of human iTregs via tryptophan depletion and kynurenine (Kyn) metabolites. We now show that stimulation of naïve CD4+ T cells in low tryptophan (low Trp) plus Kyn supports human iTreg generation. In vitro, low Trp + Kyn iTregs and tTregs potently suppress T effector cell proliferation equivalently but are phenotypically distinct. Compared with tTregs or T effector cells, bioenergetics profiling reveals that low Trp + Kyn iTregs have increased basal glycolysis and oxidative phosphorylation and use glutaminolysis as an energy source. Low Trp + Kyn iTreg viability was reliant on interleukin (IL)-2 in vitro. Although in vivo IL-2 administration increased low Trp + Kyn iTreg persistence on adoptive transfer into immunodeficient mice given peripheral blood mononuclear cells to induce GVHD, IL-2-supported iTregs did not improve recipient survival. We conclude that low Trp + Kyn create suppressive iTregs that have high metabolic needs that will need to be addressed before clinical translation.

Reconstitution of immune cell populations in multiple sclerosis patients after autologous stem cell transplantation.

Multiple sclerosis is an inflammatory T cell-mediated autoimmune disease. In a Phase II clinical trial, high-dose immunosuppressive therapy combined with autologous CD34+ haematopoietic stem cell transplant resulted in 69·2% of subjects remaining disease-free without evidence of relapse, loss of neurological function or new magnetic resonance imaging (MRI) lesions to year 5 post-treatment. A combination of CyTOF mass cytometry and multi-parameter flow cytometry was used to explore the reconstitution kinetics of immune cell subsets in the periphery post-haematopoietic cell transplant (HSCT) and the impact of treatment on the phenotype of circulating T cells in this study population. Repopulation of immune cell subsets progressed similarly for all patients studied 2 years post-therapy, regardless of clinical outcome. At month 2, monocytes and natural killer (NK) cells were proportionally more abundant, while CD4 T cells and B cells were reduced, relative to baseline. In contrast to the changes observed at earlier time-points in the T cell compartment, B cells were proportionally more abundant and expansion in the proportion of naive B cells was observed 1 and 2 years post-therapy. Within the T cell compartment, the proportion of effector memory and late effector subsets of CD4 and CD8 T cells was increased, together with transient increases in proportions of CD45RA-regulatory T cells (Tregs ) and T helper type 1 (Th1 cells) and a decrease in Th17·1 cells. While none of the treatment effects studied correlated with clinical outcome, patients who remained healthy throughout the 5-year study had significantly higher absolute numbers of memory CD4 and CD8 T cells in the periphery prior to stem cell transplantation.

B Cell Receptor Genes Associated With Tolerance Identify a Cohort of Immunosuppressed Patients With Improved Renal Allograft Graft Function.

We previously reported that two B cell receptor genes, IGKV1D-13 and IGKV4-1, were associated with tolerance following kidney transplantation. To assess the potential utility of this "signature," we conducted a prospective, multicenter study to determine the frequency of patients predicted tolerant within a cohort of patients deemed to be candidates for immunosuppressive minimization. At any single time point, 25-30% of patients were predicted to be tolerant, while 13.7% consistently displayed the tolerance "signature" over the 2-year study. We also examined the relationship of the presence of the tolerance "signature" on drug use and graft function. Contrary to expectations, the frequency of predicted tolerance was increased in patients receiving tacrolimus and reduced in those receiving corticosteroids, mycophenolate mofetil, or Thymoglobulin as induction. Surprisingly, patients consistently predicted to be tolerant displayed a statistically and clinically significant improvement in estimated glomerular filtration rate that increased over time following transplantation. These findings indicate that the frequency of patients consistently predicted to be tolerant is sufficiently high to be clinically relevant and confirm recent findings by others that immunosuppressive agents impact putative biomarkers of tolerance. The association of a B cell-based "signature" with graft function suggests that B cells may contribute to the function/survival of transplanted kidneys.

Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney-Bone Marrow Transplantation to Induce Transplantation Tolerance.

We examined tolerance mechanisms in patients receiving HLA-mismatched combined kidney-bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high-throughput sequencing of T cell receptor-ß hypervariable regions of DNA from peripheral blood regulatory T cells (Tregs) and CD4 non-Tregs revealed marked early enrichment of Tregs (CD3+ CD4+ CD25high CD127low Foxp3+ ) in blood that resulted from peripheral proliferation (Ki67+ ), possibly new thymic emigration (CD31+ ), and, in one tolerant subject, conversion from non-Tregs. Among recovering conventional T cells, central memory CD4+ and CD8+ cells predominated. A large proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptor sequencing were detected in the peripheral blood and were not donor-reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia-driven peripheral proliferation in the early posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells.

A role for plasma cell targeting agents in immune tolerance induction in autoimmune disease and antibody responses to therapeutic proteins.

Antibody responses to life saving therapeutic protein products, such as enzyme replacement therapies (ERT) in the setting of lysosomal storage diseases, have nullified product efficacy and caused clinical deterioration and death despite treatment with immune-suppressive therapies. Moreover, in some autoimmune diseases, pathology is mediated by a robust antibody response to endogenous proteins such as is the case in pulmonary alveolar proteinosis, mediated by antibodies to Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF). In this work, we make the case that in such settings, when the antibody response is high titered, sustained, and refractory to immune suppressive treatments, the antibody response is mediated by long-lived plasma cells which are relatively unperturbed by immune suppressants including rituximab. However, long-lived plasma cells can be targeted by proteasome inhibitors such as bortezomib. Recent reports of successful reversal of antibody responses with bortezomib in the settings of ERT and Thrombotic Thrombocytopenic Purpura (TTP) argue that the safety and efficacy of such plasma cell targeting agents should be evaluated in larger scale clinical trials to delineate the risks and benefits of such therapies in the settings of antibody-mediated adverse effects to therapeutic proteins and autoantibody mediated pathology.

Selective Sparing of Human Tregs by Pharmacologic Inhibitors of the Phosphatidylinositol 3-Kinase and MEK Pathways.

Phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated (MEK) signaling are central to the survival and proliferation of many cell types. Multiple lines of investigation in murine models have shown that control of the PI3K pathway is particularly important for regulatory T cell (Treg) stability and function. PI3K and MEK inhibitors are being introduced into the clinic, and we hypothesized that pharmacologic inhibition of PI3K, and possibly MEK, in mixed cultures of human mononuclear cells would preferentially affect CD4(+) and CD8(+) lymphocytes compared with Tregs. We tested this hypothesis using four readouts: proliferation, activation, functional suppression, and signaling. Results showed that Tregs were less susceptible to inhibition by both δ and α isoform-specific PI3K inhibitors and by an MEK inhibitor compared with their conventional CD4(+) and CD8(+) counterparts. These studies suggest less functional reliance on PI3K and MEK signaling in Tregs compared with conventional CD4(+) and CD8(+) lymphocytes. Therefore, the PI3K and MEK pathways are attractive pharmacologic targets for transplantation and treatment of autoimmunity.

Ex Vivo Costimulatory Blockade to Generate Regulatory T Cells From Patients Awaiting Kidney Transplantation.

Short-term outcomes of kidney transplantation have improved dramatically, but chronic rejection and regimen-related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless, the immunomodulatory effects of end-stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study, we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of ex vivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover, these Tregs maintain a CD3(+) CD4(+) CD25(+) CD127(lo) surface phenotype, high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg-specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple, brief Treg production strategy in clinical trials of mismatched kidney transplantation.

Longitudinal studies of a B cell-derived signature of tolerance in renal transplant recipients.

Biomarkers of transplant tolerance would enhance the safety and feasibility of clinical tolerance trials and potentially facilitate management of patients receiving immunosuppression. To this end, we examined blood from spontaneously tolerant renal transplant recipients and patients enrolled in two interventional tolerance trials using flow cytometry and gene expression profiling. Using a previously reported tolerant cohort as well as newly identified tolerant patients, we confirmed our previous finding that tolerance was associated with increased expression of B cell-associated genes relative to immunosuppressed patients. This was not accounted for merely by an increase in total B cell numbers, but was associated with the increased frequencies of transitional and naïve B cells. Moreover, serial measurements of gene expression demonstrated that this pattern persisted over several years, although patients receiving immunosuppression also displayed an increase in the two most dominant tolerance-related B cell genes, IGKV1D-13 and IGLL-1, over time. Importantly, patients rendered tolerant via induction of transient mixed chimerism, and those weaned to minimal immunosuppression, showed similar increases in IGKV1D-13 as did spontaneously tolerant individuals. Collectively, these findings support the notion that alterations in B cells may be a common theme for tolerant kidney transplant recipients, and that it is a useful monitoring tool in prospective trials.

Effector cell signature in peripheral blood following nasal allergen challenge in grass pollen allergic individuals.

BACKGROUND: Several studies have demonstrated the time course of inflammatory mediators in nasal fluids following nasal allergen challenge (NAC), whereas the effects of NAC on cells in the periphery are unknown. We examined the time course of effector cell markers (for basophils, dendritic cells and T cells) in peripheral blood after nasal grass pollen allergen challenge. METHODS: Twelve participants with seasonal allergic rhinitis underwent a control (diluent) challenge followed by NAC after an interval of 14 days. Nasal symptoms and peak nasal inspiratory flow (PNIF) were recorded along with peripheral basophil, T-cell and dendritic cell responses (flow cytometry), T-cell proliferative responses (thymidine incorporation), and cytokine expression (FluoroSpot assay). RESULTS: Robust increases in nasal symptoms and decreases in PNIF were observed during the early (0-1 h) response and modest significant changes during the late (1-24 h) response. Sequential peaks in peripheral blood basophil activation markers were observed (CD107a at 3 h, CD63 at 6 h, and CD203c(bright) at 24 h). T effector/memory cells (CD4(+) CD25(lo) ) were increased at 6 h and accompanied by increases in CD80(+) and CD86(+) plasmacytoid dendritic cells (pDCs). Ex vivo grass antigen-driven T-cell proliferative responses and the frequency of IL-4(+) CD4(+) T cells were significantly increased at 6 h after NAC when compared to the control day. CONCLUSION: Basophil, T-cell, and dendritic cell activation increased the frequency of allergen-driven IL-4(+) CD4(+) T cells, and T-cell proliferative responses are detectable in the periphery after NAC. These data confirm systemic cellular activation following a local nasal provocation.

Lymphodepletion and homeostatic proliferation: implications for transplantation.

Control of the alloimmune response requires elimination and/or suppression of alloreactive immune cells. Lymphodepleting induction therapies are increasingly used to accomplish this goal, both as part of tolerance induction protocols or to reduce the requirements for maintenance immunosuppression in the peritransplant setting. However, it is well recognized that lymphopenia induces compensatory proliferation of immune cells, generally termed ``homeostatic proliferation,'' which favors the emergence of memory T cells. Paradoxically therefore, the result may be a situation that favors graft rejection and/or makes tolerance difficult to achieve or sustain. Yet all depletion is not alike, particularly with respect to the timing of reconstitution and the types of cells that repopulate the host. Thus, to design more effective induction strategies it is important to understand the homeostatic mechanisms, which exist to maintain a balanced repertoire of naïve and memory T and B cells in the periphery and how they respond to lymphodepletion. Here we will review the biology of homeostatic proliferation stimulated by lymphopenia, the effects of specific depleting agents on reconstitution of the T- and B-cell immune repertoire, drawing from both from animal models and human experience, and potential strategies to enhance allodepletion while minimizing the adverse effects of homeostatic proliferation.

Critical role of proinflammatory cytokine IL-6 in allograft rejection and tolerance.

The proinflammatory cytokine IL-6 plays an important role in controlling T-cell differentiation, especially the development of Th17 and regulatory T cells. To determine the function of IL-6 in regulating allograft rejection and tolerance, BALB/c cardiac grafts were transplanted into wild-type or IL-6-deficient C57BL/6 mice. We observed that production of IL-6 and IFN-γ was upregulated during allograft rejection in untreated wild-type mice. In IL-6-deficient mice, IFN-γ production was greater than that observed in wild-type controls, suggesting that IL-6 production affects Th1/Th2 balance during allograft rejection. CD28-B7 blockade by CTLA4-Ig inhibited IFN-γ production in C57BL/6 recipients, but had no effect on the production of IL-6. Although wild-type C57BL/6 recipients treated with CTLA4-Ig rejected fully MHC-mismatched BALB/c heart transplants, treatment of IL-6-deficient mice with CTLA4-Ig resulted in graft acceptance. Allograft acceptance appeared to result from the combined effect of costimulatory molecule blockade and IL-6-deficiency, which limited the differentiation of effector cells and promoted the migration of regulatory T cells into the grafts. These data suggest that the blockade of IL-6, or its signaling pathway, when combined with strategies that inhibit Th1 responses, has a synergistic effect on the promotion of allograft acceptance. Thus, targeting the effects of IL-6 production may represent an important part of costimulation blockade-based strategies to promote allograft acceptance and tolerance.

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.

Paradoxical functions of B7: CD28 costimulation in a MHC class II-mismatched cardiac transplant model.

Blockade of the B7: CD28 costimulatory pathway has emerged as a promising therapy to prevent allograft rejection. However, this pathway has also been demonstrated to be important for the generation and maintenance of regulatory T cells. In this study, we investigated the role of the B7: CD28 pathway in the 'bm12 into B6' MHC class II-mismatched vascularized cardiac transplant model of chronic rejection. Allograft rejection was remarkably accelerated in B6 background B7DKO and CD28KO recipients compared with B6 wild-type (WT) recipients. Allograft rejection was associated with a significantly enhanced Th1/Th2 alloreactivity and marked reduction in the ratio of regulatory T cells to CD4(+) effector/memory cells. We noted that administration of anti-B7-1 and anti-B7-2 mAb prior to transplantation also accelerated allograft rejection. Furthermore, depleting CD25(+) cells in B6 WT recipients of bm12 hearts prior to transplant also precipitated rejection at a similar rate. Neither B7/CD28 deficiency nor CD25 depletion affected graft survival in single MHC class I-mismatched (bm1 into B6) recipients. This study highlights the paradoxical functions of B7: CD28 costimulation in a MHC class II-mismatched model, in which the B7: CD28 pathway is demonstrated to be important in preventing rejection through the generation and maintenance of Tregs.

CD4+ CD25+ FOXP3+ regulatory T cells increase de novo in kidney transplant patients after immunodepletion with Campath-1H.

Campath-1H (Alemtuzumab) is an effective immunodepletion agent used in renal transplantation. To evaluate its influence on T lymphocytes during repletion, we analyzed peripheral blood from Campath-1H-treated renal allograft recipients for the presence of FOXP3(+) regulatory T (Treg) cells. Flow cytometry demonstrated that CD4(+)CD25(+)FOXP3(+) lymphocytes increased significantly within the CD4(+) T-cell population, skewing Treg/Teff (T effector) ratios for up to several years. In contrast, Treg levels in patients treated with anti-CD25 (Basiliximab) and maintained on CsA demonstrated a sustained decrease. The increase in Tregs in Campath-1H treated patients developed independent of maintenance immunosuppression. Importantly, the increase in Tregs was not fully explained by their homeostatic proliferation, increased thymic output, or Treg sparing, suggesting de novo generation/expansion. Consistent with this, in vitro stimulation of PBMCs with Campath-1H, with or without anti-CD3, activation led to an increase in CD4(+)CD25(+)FOXP3(+) cells that had suppressive capabilities. Together, these data suggest that Campath-1H promotes an increase in peripheral Tregs and may act as an intrinsic generator of Tregs in vivo.

Conditional gene expression: a new tool for the transplantologist.

The ability to generate genetically manipulated mice has revolutionized the study of development, cell biology, immunobiology and transplantation. Conventional gene targeting approaches lead to inactivation of the target gene in all tissues. This approach often has unintended consequences, such as embryonic lethality, which preclude studying the originally intended tissue. Newer approaches allowing conditional gene expression in a tissue-specific or temporally controlled fashion have the advantage of normal development with gene deletion only in the desired tissues. While nuances to these techniques continue to be developed, the underlying concepts remain consistent. This minireview focuses on the use of conditional gene targeting in mice using the Cre-loxP system and drug inducible gene expression using the tetracycline system.

Signaling through CD28 and CTLA-4 controls two distinct forms of T cell anergy.

Primary T cell proliferative responses to TCR ligation plus CD28 costimulation are surprisingly heterogeneous. Many cells that enter G1 fail to progress further through the cell cycle, and some of these cells subsequently fail to divide upon restimulation, even in the presence of IL-2. Such IL-2-refractory anergy is distinct from the IL-2-reversible anergy induced by TCR occupancy in the absence of CD28 costimulation. Here, we focus on the contributions of cell cycle progression and costimulatory (CD28/CTLA-4) signals in the regulation of anergy. We show that CD28 costimulation is not sufficient for anergy avoidance and that activated T cells must progress through the cell cycle in order to escape anergy. Induction of this "division-arrest" form of anergy requires CTLA-4 signaling during the primary response. Also, cell division per se is not sufficient for anergy avoidance: the few T cells that undergo multiple rounds of cell division during overt CD28 costimulatory blockade do not escape the ultimate induction of clonal anergy. Anergy avoidance by primary T cells is thus a multistep process: in order to participate in a productive immune response, an individual T cell activated through its antigen receptor must receive CD28 costimulation and progress through the cell cycle. Anergy may be induced either through a combination of CTLA-4 signaling and the failure of cell cycle progression, or through a proliferation-independent mechanism in which TCR ligation occurs in the absence of CD28.

A closer look at homeostatic proliferation of CD4+ T cells: costimulatory requirements and role in memory formation.

Ag-specific proliferation of CD4+ T cells is regulated, in part, by costimulatory signals through CD28. The proliferative response during primary activation is an important determinant of the ability of the T cell to respond to Ag re-encounter. Proliferation of mature CD4+ T cells during lymphopenia (homeostatic proliferation) requires interaction with endogenous peptide MHC. However, the role of costimulation during homeostatic proliferation is unclear, as is the ability of homeostatic proliferation to regulate secondary T cell responses. Using a TCR transgenic system and serial adoptive transfers we find that homeostatic proliferation of CD4+ T cells occurs for at least 5 wk after adoptive transfer into recombination-activating gene (RAG)-/- recipients. Two discrete populations of proliferating T cells can be resolved, one that is highly proliferative and dependent on CD28 signaling, and the other that contains cells undergoing low levels of CD28-independent proliferation. Importantly, naive CD4+ T cells that have undergone homeostatic proliferation acquire both phenotypic and functional characteristics of true memory cells. These studies indicate that functional memory T cells can be generated by encounters with endogenous Ags only. This mechanism of T cell regeneration is possibly active during lymphopenia due to viral infections, such as HIV, transplantation, or cancer therapy, and may explain selected autoimmune diseases.

CD28-independent costimulation of T cells in alloimmune responses.

T cell costimulation by B7 molecules plays an important role in the regulation of alloimmune responses. Although both B7-1 and B7-2 bind CD28 and CTLA-4 on T cells, the role of B7-1 and B7-2 signaling through CTLA-4 in regulating alloimmune responses is incompletely understood. To address this question, we transplanted CD28-deficient mice with fully allogeneic vascularized cardiac allografts and studied the effect of selective blockade of B7-1 or B7-2. These mice reject their grafts by a mechanism that involves both CD4(+) and CD8(+) T cells. Blockade of CTLA-4 or B7-1 significantly accelerated graft rejection. In contrast, B7-2 blockade significantly prolonged allograft survival and, unexpectedly, reversed the acceleration of graft rejection caused by CTLA-4 blockade. Furthermore, B7-2 blockade prolonged graft survival in recipients that were both CD28 and CTLA-4 deficient. Our data indicate that B7-1 is the dominant ligand for CTLA-4-mediated down-regulation of alloimmune responses in vivo and suggest that B7-2 has an additional receptor other than CD28 and CTLA-4 to provide a positive costimulatory signal for T cells.

Differential NF-kappaB and IkappaB gene expression during development of cardiac allograft rejection versus CD154 monoclonal antibody-induced tolerance.

BACKGROUND: The Rel/NF-kappaB transcription factor pathway, regulated by IkappaB proteins, is considered central to immune responses, although there are surprisingly few in vivo data concerning alloresponses. METHODS: We undertook analysis of NF-kappaB and IkappaB mRNA intracardiac allograft expression, and NF-kappaB nuclear translocation, during acute rejection versus CD154 monoclonal antibody (mAb)-induced tolerance induction in fully MHC-disparate mice. RESULTS: Intragraft expression of all nine NF-kappaB and IkappaB genes increased during development of rejection, and nuclear translocation of p50, p52, and p65 was detected. CD154 mAb therapy decreased mRNA levels of all nine NF-kappaB and IkappaB genes, and impaired nuclear translocation of p50, p52, and p65 NF-kappaB proteins. However, prolonged survival could not be induced by CD154 mAb in p50- or p52-deficient allograft recipients, indicating an absolute requirement for expression of these genes in CD154 mAb-induced tolerance. CONCLUSIONS: We conclude that, whereas blanket approaches to NF-kappaB suppression are unlikely to be effective strategies for tolerance induction, a better understanding of the roles of individual NF-kappaB and IkappaB genes may allow development of more precise and effective therapies.