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Humoral allogeneic immunity driven by anti-HLA donor-specific antibodies (DSAs) and antibody-mediated mediated rejection (ABMR) significantly impede prolonged survival of organ allografts after transplantation. Although the importance of T follicular helper (T(FH)) cells in controlling antibody responses has been long established, their role in directing DSA generation leading to ABMR was only recently appreciated in the clinical setting of organ transplantation. In this review, we provide a comprehensive summary of the current knowledge on the biology of human T(FH) cells as well as their circulating (cT(FH)) counterparts and describe their pivotal role in driving humoral alloimmunity. In addition, we discuss the intrinsic effects of current induction therapies and maintenance immunosuppressive drugs as well as of biotherapies on T(FH) cells; and provide future directions and novel opportunities of biotherapeutic targeting of T(FH) cells that have the potential of bringing the prophylactic and curative treatments of ABMR towards personalized and precision medicine.

Current immunosuppressive (IS) regimens used to prevent organ allograft rejection have well-recognized side effects, that include enhanced risk of infection and certain types of cancer, metabolic disorders, cardiovascular disease, renal complications and failure to control chronic allograft rejection. The life-long dependency of patients on these IS agents reflects their inability to induce donor-specific tolerance. Extensive studies in rodent and non-human primate models have demonstrated the ability of adoptively-transferred regulatory immune cells (either regulatory myeloid cells or regulatory T cells) to promote transplant tolerance. Consequently, there is considerable interest in the potential of regulatory immune cell therapy to allow safe minimization/complete withdrawal of immunosuppression and the promotion of organ transplant tolerance in the clinic. Here, we review the properties of regulatory dendritic cells (DCreg) with a focus on the approaches being taken to generate human DCreg for clinical testing. We also document the early phase clinical trials that are underway to assess DCreg therapy in clinical organ transplantation as well as in autoimmune disorders.

EBV is a γ herpesvirus that infects more than 95% of the world’s adult population. Primary EBV infection occurs during childhood when it usually is asymptomatic or during adolescence when it may manifest as infectious mononucleosis (IM). Regardless of the clinical manifestation of primary infection, EBV persists in the B cell compartment of the infected host in a latent state of infection that may be occasionally interrupted by episodic lytic viral reactivations. Although EBV elicits and maintains potent immune responses that successfully keep both its latency and reactivation phases under tight control in healthy humans, the virus may associate with several malignancies in individuals with immunodeficiencies or individuals whose cellular immunity is impaired, because of the growth-transforming function of some of the EBV latent genes, which are true oncogenes. This chapter will discuss the features of innate and adaptive immunity to EBV in healthy and in immunosuppressed organ transplant patients.

Regulatory dendritic cells (DCreg) promote transplant tolerance following their adoptive transfer in experimental animals. We investigated the feasibility, safety, fate, and impact on host T cells of donor monocyte-derived DCreg infused into prospective, living donor liver transplant patients, 7 days before transplantation. The DCreg expressed a tolerogenic gene transcriptional profile, high cell surface programed death ligand-1 (PD-L1):CD86 ratios, high IL-10/no IL-12 productivity and poor ability to stimulate allogeneic T cell proliferation. Target DCreg doses (range 2.5-10 × 106 cells/kg) were achieved in all but 1 of 15 recipients, with no infusion reactions. Following DCreg infusion, transiently elevated levels of donor HLA and immunoregulatory PD-L1, CD39, and CD73 were detected in circulating small extracellular vesicles. At the same time, flow and advanced image stream analysis revealed intact DCreg and "cross-dressing" of host DCs in blood and lymph nodes. PD-L1 co-localization with donor HLA was observed at higher levels than with recipient HLA. Between DCreg infusion and transplantation, T-bethi Eomeshi memory CD8+ T cells decreased, whereas regulatory (CD25hi CD127- Foxp3+ ): T-bethi Eomeshi CD8+ T cell ratios increased. Thus, donor-derived DCreg infusion may induce systemic changes in host antigen-presenting cells and T cells potentially conducive to modulated anti-donor immune reactivity at the time of transplant.

BACKGROUND: Although antibody-mediated rejection (ABMR) has been long recognized as a leading cause of allograft failure after kidney transplantation, the cellular and molecular processes underlying the induction of deleterious donor-specific antibody (DSA) responses remain poorly understood. METHODS: Using high-dimensional flow cytometry, in vitro assays, and RNA sequencing, we concomitantly investigated the role of T follicular helper (T(FH)) cells and B cells during ABMR in 105 kidney transplant recipients. RESULTS: There were 54 patients without DSAs; of those with DSAs, ABMR emerged in 20 patients, but not in 31 patients. We identified proliferating populations of circulating T(FH) cells and activated B cells emerging in blood of patients undergoing ABMR. Although these circulating T(FH) cells comprised heterogeneous phenotypes, they were dominated by activated (ICOS(+)PD-1(+)) and early memory precursor (CCR7(+)CD127(+)) subsets, and were enriched for the transcription factors IRF4 and c-Maf. These circulating T(FH) cells produced large amounts of IL-21 upon stimulation with donor antigen and induced B cells to differentiate into antibody-secreting cells that produced DSAs. Combined analysis of the matched circulating T(FH) cell and activated B cell RNA-sequencing profiles identified highly coordinated transcriptional programs in circulating T(FH) cells and B cells among patients with ABMR, which markedly differed from those of patients who did not develop DSAs or ABMR. The timing of expansion of the distinctive circulating T(FH) cells and activated B cells paralleled emergence of DSAs in blood, and their magnitude was predictive of IgG3 DSA generation, more severe allograft injury, and higher rate of allograft loss. CONCLUSIONS: Patients undergoing ABMR may benefit from monitoring and therapeutic targeting of T(FH) cell–B cell interactions.

Introduction The cellular events that contribute to generation of donor-specific anti-HLA antibodies (DSA) post-kidney transplantation (KTx) are not well understood. Characterization of such mechanisms could allow tailoring of immunosuppression to benefit sensitized patients. Methods We prospectively monitored circulating T follicular helper (cTFH) cells in KTx recipients who received T-cell depleting (thymoglobulin, n = 54) or T-cell nondepleting (basiliximab, n = 20) induction therapy from pre-KTx to 1 year post-KTx and assessed their phenotypic changes due to induction and DSA occurrence, in addition to healthy controls (n = 13), for a total of 307 blood samples. Results Before KTx, patients displayed comparable levels of resting, central memory cTFH cells with similar polarization to those of healthy controls. Unlike basiliximab induction, thymoglobulin induction significantly depleted cTFH cells, triggered lymphopenia-induced proliferation that skewed cTFH cells toward increased Th1 polarization, effector memory, and elevated programmed cell death protein 1 (PD-1)int/hi expression, resembling activated phenotypes. Regardless of induction, patients who developed DSA post-KTx, harbored pre-KTx donor-reactive memory interleukin (IL)-21+ cTFH cells and showed higher % cTFH and lower % of T regulatory (TREG) cells post-KTx resulting in elevated cTFH:TREG ratio at DSA occurrence. Conclusion Induction therapy distinctly shapes cTFH cell phenotype post-KTx. Monitoring cTFH cells before and after KTx may help detect those patients prone to DSA generation post-KTx., Graphical abstract

Memory T cell (Tmem) responses play a critical role in the outcome of allo-transplantation. While the role of the T-box transcription factor Eomesodermin (Eomes) in the maintenance of antigen-specific Tmem is well studied, little is known about Eomes+CD8+T cell responses after transplantation. We evaluated the phenotype and function of allo-reactive Eomes+CD8+T cells in healthy volunteers and kidney transplant patients and their relation to transplant outcome. High Eomes expression by steady-state CD8+T cells correlated with effector and memory phenotype. Following allo-stimulation, the expression of both the T-box proteins Eomes and T-bet by proliferating cells increased significantly, where high expression of Eomes and T-bet correlated with higher incidence of allo-stimulated IFNγ+TNFα+ CD8+T cells. In patients with no subsequent rejection, Eomes but not T-bet expression by donor-stimulated CD8+T cells, increased significantly after transplantation. This was characterized by increased EomeshiT-bet-/lo and decreased Eomes-/loT-bethi CD8+T cell subsets, with no significant changes in the EomeshiT-bethi CD8+T cell subset. No upregulation of exhaustion markers programmed-death-1 (PD-1) and cytotoxic-T-lymphocyte-associated-antigen-4 (CTLA4) by donor-stimulated Eomes+CD8+T cells was observed. Before transplantation, in patients without rejection, there were higher incidences of EomeshiT-bet-/lo, and lower incidences of EomeshiT-bethi and Eomes-/loT-bethi donor-stimulated CD8+T cell subsets, compared to those with subsequent rejection. Overall, our findings indicate that high Eomes expression by allo-stimulated T-bet+CD8+T cells is associated with enhanced effector function, and that an elevated incidence of donor-stimulated CD8+T cells co-expressing high levels of Eomes and T-bet before transplantation, may correlate with an increased incidence of acute cellular rejection.

Dendritic cells (DC) are rare, bone marrow (BM)-derived innate immune cells that critically maintain self-tolerance in the healthy steady-state. Regulatory DC (DCreg) with capacity to suppress allograft rejection and promote transplant tolerance in pre-clinical models can readily be generated from BM precursors or circulating blood monocytes. These DCreg enhance allograft survival via various mechanisms, including promotion of regulatory T cells. In non-human primates receiving minimal immunosuppressive drug therapy (IS), infusion of DCreg of donor origin, one week before transplant, safely prolongs renal allograft survival and selectively attenuates anti-donor CD8+ memory T cell responses in the early post-transplant period. Based on these observations, and in view of the critical need to reduce patient dependence on non-specific IS agents that predispose to cardiometabolic side effects and renal insufficiency, we will conduct a first-in-human safety and preliminary efficacy study of donor-derived DCreg infusion to achieve early (18 months post-transplant) complete IS withdrawal in low-risk, living donor liver transplant recipients receiving standard-of-care IS (mycophenolate mofetil, tacrolimus and steroids). We will test the hypothesis that, although donor-derived DCreg are short-lived, they will induce robust donor-specific T cell hyporesponsiveness. We will examine immunological mechanisms by sequential analysis of blood and tissue samples, incorporating cutting-edge technologies.

To minimize maintenance immunosuppression in upper-extremity transplantation to favor the risk-benefit balance of this procedure.Despite favorable outcomes, broad clinical application of reconstructive transplantation is limited by the risks and side effects of multidrug immunosuppression. We present our experience with upper-extremity transplantation under a novel, donor bone marrow (BM) cell-based treatment protocol ("Pittsburgh protocol").Between March 2009 and September 2010, 5 patients received a bilateral hand (n = 2), a bilateral hand/forearm (n = 1), or a unilateral (n = 2) hand transplant. Patients were treated with alemtuzumab and methylprednisolone for induction, followed by tacrolimus monotherapy. On day 14, patients received an infusion of donor BM cells isolated from 9 vertebral bodies. Comprehensive follow-up included functional evaluation, imaging, and immunomonitoring.All patients are maintained on tacrolimus monotherapy with trough levels ranging between 4 and 12 ng/mL. Skin rejections were infrequent and reversible. Patients demonstrated sustained improvements in motor function and sensory return correlating with time after transplantation and level of amputation. Side effects included transient increase in serum creatinine, hyperglycemia managed with oral hypoglycemics, minor wound infection, and hyperuricemia but no infections. Immunomonitoring revealed transient moderate levels of donor-specific antibodies, adequate immunocompetence, and no peripheral blood chimerism. Imaging demonstrated patent vessels with only mild luminal narrowing/occlusion in 1 case. Protocol skin biopsies showed absent or minimal perivascular cellular infiltrates.Our data suggest that this BM cell-based treatment protocol is safe, is well tolerated, and allows upper-extremity transplantation using low-dose tacrolimus monotherapy.

Th17 and TfH cells are thought to promote tissue inflammation and autoantibody production, respectively, in autoimmune diseases including rheumatoid arthritis (RA). TfH cells that co-express Th17 markers (CXCR5+Th17) encompass both of these pathogenic functions, and are increased in some human autoimmune settings including juvenile dermatomyositis. We investigated CXCR5+Th17 cells in RA subjects with stable or active disease and before and after TNF inhibitor therapy. CXCR5+Th17 cell frequency was increased in RA compared to healthy controls, but other helper T cell subsets were not different. CXCR5+Th17 cells correlated with disease activity in subjects with active RA prior to initiation of TNF inhibitor therapy. Baseline CXCR5+Th17 cells also correlated with numbers of swollen joints as late as one year post-therapy. CXCR5+Th17 cell frequencies were unaltered by TNF blockade and in fact remained remarkably stable within individuals. We conclude that CXCR5+Th17 cells are not a direct target of TNF blockade and therefore cannot serve as a biomarker of current disease activity. However, basal CXCR5+Th17 cell frequency may indicate underlying differences in disease phenotype between patients and predict ultimate success of TNF inhibitor therapy.

The recently described T follicular helper (Tfh) cell is required for the production of high affinity antibody. After contact with follicular dendritic cells, Tfh cells move into the germinal centre and provide help to B cells both by direct B cell-T cell interaction and production of IL-21. This drives proliferation, differentiation, and affinity maturation of the B cells to produce plasma cells capable of secreting high-affinity antibody. Circulating Tfh cells are produced by movement of Tfh cells from lymph nodes after dendritic cell contact. A reduction of Tfh cell-associated molecules is linked with increased expression of other chemokine receptors to form Th1-, Th2-, and Th17-like Tfh cells. These circulating Tfh cells are able to help B cells in vitro and to move into target tissues to support antibody production. Alloantibody production is dependent on T-cell help via the indirect pathway. Antibody-mediated rejection is therefore dependent on Tfh cells. Animal data suggest that Tfh cells and B cells migrate to the allograft and are involved in alloantibody production within a tertiary lymphoid organ. There is some data supporting the same process within human allografts. The requirement for T-cell help provides a potential therapeutic target in the treatment of antibody-mediated rejection.

Purpose The short- and long-term effects of 90 Yttrium microspheres therapy for hepatocellular carcinoma (HCC) on peripheral blood lymphocytes are unknown and were therefore examined. Methods and Materials Ninety-two HCC patients were enrolled in a 90 Yttrium therapy study and routine blood counts were examined as part of standard clinical monitoring. Results We found an early, profound, and prolonged lymphopenia. In a subsequent cohort of 25 additional HCC patients, prospective flow cytometric immune-monitoring analysis was performed to identify specific changes on distinct lymphocyte subsets ( i.e. , CD3, CD4, CD8 T, and CD19 B lymphocytes) and NK cells absolute numbers, in addition to the granulocytes and platelets subsets. We found that the pretreatment lymphocyte subset absolute numbers (with the exception of NK cells) had a tendency to be lower compared with healthy control values, but no significant differences were detected between groups. Posttherapy follow-up revealed that overall, all lymphocyte subsets, except for NK cells, were significantly (>50% from pretherapy values), promptly (as early as 24 h) and persistently (up to 30 months) depleted post- 90 Yttrium microspheres therapy. In contrast, granulocytes increased rapidly (24 h) to compensate for lymphocyte depletion, and remained increased at 1-year after therapy. We further stratified patients into two groups, according to survival at 1 year. We found that lack of recovery of CD19, CD3, CD8, and especially CD4 T cells was linked to poor patient survival. No fungal or bacterial infections were noted during the 30-month follow-up period. Conclusions The results show that lymphocytes (and not granulocytes, platelets, or NK cells) are sensitive to hepatic arterial 90 Yttrium without associated clinical toxicity, and lack of lymphocyte recovery (possibly leading to dysregulation of adaptive cellular immunity) posttherapy indicates poor survival.

BACKGROUND Immunologic monitoring of pediatric transplant (Tx) recipients, who are at increased risk of Epstein-Barr virus (EBV)-driven posttransplant lymphoproliferative disease, is an important goal in clinical transplantation. Here, we investigated the impact of EBV load on T-cell immunity from pediatric Tx recipients, using clinically applicable tests for improved assessment of T-cell immune competence. METHODS Thirty-five asymptomatic pediatric thoracic Tx patients were categorized into three groups according to their EBV load levels as follows: undetectable viral load (UVL), chronic low viral load (LVL) and chronic high viral load (HVL). Global and EBV-specific T-cell immunity were assessed by ATP release using Cylex Immuknow and T Cell Memory assays. RESULTS UVL patients exhibited normal ATP release to Concanavalin A (ConA) and phytohemagglutinin (PHA; 190+/-86 ng/mL, 328+/-163 ng/mL) and detectable EBV-specific (37+/-34 ng/mL) ATP responses. LVL patients displayed significantly stronger responses to ConA (373+/-174 ng/mL), PHA (498+/-196 ng/mL) and EBV (152+/-179 ng/mL), when compared with UVL or to HVL patients (ConA 185+/-114 ng/mL, PHA 318+/-173 ng/mL, and EBV 33+/-42 ng/mL). Moreover, HVL patients displayed significant inverse correlation between CD4+ T-cell ATP levels and EBV loads. CONCLUSIONS Evaluation of global and EBV-specific T-cell immunity provides a rapid assessment of patients' immune competence. It is still unclear whether selective oversuppressed ATP release by CD4+ T cells reflects HVL patients at risk of posttransplant lymphoproliferative disease. Further longitudinal studies will determine the importance of Immuknow test in identifying asymptomatic HVL patients vulnerable to EBV complications.

Dendritic cells (DC) are rare, professional antigen-presenting cells with ability to induce or regulate alloimmune responses. Regulatory DC (DCreg) with potential to down-modulate acute and chronic inflammatory conditions that occur in organ transplantation can be generated in vitro under a variety of conditions. Here, we provide a rationale for evaluation of DCreg therapy in clinical organ transplantation with the goal of promoting sustained, donor-specific hyporesponsiveness, while lowering the incidence and severity of rejection and reducing patients’ dependence on anti-rejection drugs. Generation of donor- or recipient-derived DCreg that suppress T cell responses and prolong transplant survival in rodents or non-human primates has been well-described. Recently, good manufacturing practice (GMP)-grade DCreg have been produced at our Institution for prospective use in human organ transplantation. We briefly review experience of regulatory immune therapy in organ transplantation and describe our experience generating and characterizing human monocyte-derived DCreg. We propose a phase I/II safety study in which the influence of donor-derived DCreg combined with conventional immunosuppression on subclinical and clinical rejection and host alloimmune responses will be examined in detail.

Background Multiple drug immunosuppression has allowed the near elimination of rejection, but without commensurate improvements in longterm graft survival and at the cost of quality of life. We have suggested that transplantation outcomes can be improved by modifying the timing and dosage of immunosuppression to facilitate natural mechanisms of alloengraftment and acquired tolerance. Study design Two therapeutic principles were applied for kidney transplantation: pretransplant recipient conditioning with antilymphoid antibody preparations (Thymoglobulin [Sangstat] or Campath [ILEX Pharmaceuticals]), and minimal posttransplant immunosuppression with tacrolimus monotherapy including "spaced weaning" of maintenance doses when possible. The results in Thymoglobulin- (n = 101) and Campath-pretreated renal transplantation recipients (n = 90) were compared with those in 152 conventionally immunosuppressed recipients in the immediately preceding era. Results Spaced weaning was attempted in more than 90% of the kidney transplant recipients after pretreatment with both lymphoid-depleting agents, and is currently in effect in two-thirds of the survivors. Although there was a much higher rate of acute rejection in the Thymoglobulin-pretreated recipients than in either the Campath-pretreated or historic control recipients, patient and graft survival in both lymphoid depletion groups is at least equivalent to that of historic control patients. In the Thymoglobulin-conditioned patients for whom followups are now 24 to 40 months, chronic allograft nephropathy (CAN) progressed at the same rate as in historic control patients. Selected patients on weaning developed donor-specific nonreactivity. Conclusions After lymphoid depletion, kidney transplantation can be readily accomplished under minimal immunosuppression with less dependence on late maintenance immunosuppression and a better quality of life. Campath was the more effective agent for pretreatment. Guidelines for spaced weaning need additional refinement.

Posttransplant lymphoproliferative disorders (PTLDs) represent life-threatening complications of bone marrow and solid organ transplantation (SOTx). These are B-cell malignancies triggered by Epstein-Barr Virus (EBV) infection in chronically immunosuppressed (IS) recipients. Immunosuppressed EBV seronegative (EBV(-)) organ recipients are at highest risk of developing PTLD owing to the lack of anti-EBV memory T cells to control subsequent EBV challenges. Our aim is to establish effective anti-EBV T-cell generation protocols for prevention or treatment of PTLD encountered in SOTx. We have used autologous dendritic cells (DCs) loaded with apoptotic/necrotic lymphoblastoid cell lines (LCLs) to evaluate the ability of such an approach to activate naïve T cells in vitro. In EBV(-) individuals, both CD8+ and CD4+ T-cell responses were amplified by this approach, as detected by IFN-gamma ELISPOT and cytotoxicity assays. The CD8+ T cells were poly-specific anti-EBNA3 A, -LMP2 and -BMLF1, with uniform reversion to a CD45RO+/RA-phenotype, decreased CD62L expression, and up-regulation of the activation markers CD28 and CD69. Addition of rhIL-12 improved anti-viral T-cell responses and reduced the functional differences observed between EBV(+) and EBV(-) responders. In conclusion, the DC/LCL method promotes cross-presentation of EBV-associated epitopes and may serve as an effective protocol for the adoptive immunotherapy of PTLD in EBV(-) SOTx patients.

Induction with lymphocyte-depleting antibodies is routinely used to prevent rejection but often skews T cells toward memory. It is not fully understood which memory and regulatory T-cell subsets are most affected and how they relate to clinical outcomes.We analyzed T cells from 57 living-donor renal transplant recipients (12 reactive and 45 quiescent) 2.8±1.4 years after alemtuzumab induction. Thirty-four healthy subjects and nine patients with acute cellular rejection (ACR) were also studied.We found that alemtuzumab caused protracted CD4 more than CD8 T-lymphocyte deficiency, increased proportion of CD4 memory T cells, and decreased proportion of CD4 regulatory T cells. Reactive patients exhibited higher proportions of CD4 effector memory T cells (TEM) and CD8 terminally differentiated TEM (TEMRA), with greater CD4 TEM and CD8 TEMRA to regulatory T cell ratios, than quiescent patients or healthy controls. Patients with ongoing ACR had profound reduction in circulating CD8 TEMRA. Mixed lymphocyte assays showed significantly lower T-cell proliferation to donor than third-party antigens in the quiescent group, while reactive and ACR patients exhibited increased effector molecules in CD8 T cells.Our findings provide evidence that T-cell skewing toward TEM may be associated with antigraft reactivity long after lymphodepletion. Further testing of TEM and TEMRA subsets as rejection predictors is warranted.

Posttransplantation lymphoproliferative disorders (PTLD) are life-threatening complications of solid organ transplantation, triggered by EBV infection in chronically immunosuppressed (IS) patients. Our goal is to establish DC-based protocols for adoptive immunotherapy of refractory PTLD, while understanding how the immunosuppressive drug environment may subvert DC-EBV-specific T cell interactions. Type-1 CD8(+) T cells are critical for efficient immune surveillance and control of EBV infection, whereas type-2 or Treg/type-3 responses may provide an environment conductive to disease progression. We have recently reported that chronic IS inhibits DC function in transplant patients. Here, we have analyzed the comparative ability of mature, type-1 polarized DCs (i.e. DC1) generated from quiescent transplant patients or healthy controls, to boost type-1 EBV-specific CD8(+) T cells in vitro. Our results show that unlike healthy controls, where DC1 loaded with MHC class I EBV peptides preferentially reactivate specific type-1 CD8(+) T cells, DC1 generated from transplant patients reactivate EBV-specific CD8(+) T cells that produce both IFN-gamma and IL-10, up-regulate FOXP3 mRNA, and suppress noncognate CD4(+) T-cell proliferation via cell-cell contact. These data support a novel regulatory pathway for anti-EBV T-cell-mediated responses in IS transplant patients, with implications for the design of adoptive immunotherapies in this setting.

Background. Chronic immunosuppressive (IS) therapy impairs normal T-cell immune surveillance and may predispose to opportunistic infections and malignancies that represent life-threatening complication of solid-organ transplantation (SOTx). Our study was designed to ascertain the impact of chronic in vivo administration of IS on the ability of monocyte-derived dendritic cells (MoDC) to differentiate, mature, and function ex vivo. The potential of these cells to be implemented for DC-based adoptive immunotherapy was also considered. Methods. MoDCs were propagated by conventional procedures, their phenotype was analyzed by flow cytometry, and their function was assessed by mixed leukocyte reaction, enzyme-linked immunoadsorbent assay, and ELISPOT assays. Nuclear translocation of nuclear factor (NF)-kB was analyzed by electrophoretic mobility shift assay. Results. Circulating DC1s in peripheral blood were reduced in SOTx patients. MoDCs generated from patients displayed higher endocytic activity versus normal DCs, indicating their comparative immaturity. Patients’ DCs exposed to pro-inflammatory cytokines (tumor necrosis factor-, interleukin [IL]-1, and IL-6) were less able to mature, to stimulate recall antigen (Ag)- or allo–Ag-induced proliferation responses, or to secrete IL-12p70. These deficiencies were associated with a decrease in NF-kB translocation. In contrast, combination of pro-inflammatory cytokines and interferon (IFN)- (a Th1-polarizing factor) augmented patients’ DC1-type function and IL-12p70 production by way of an NF– kB-independent mechanism. Conclusions. Chronic IS restrains DC differentiation, maturation, and function at a transcriptional level; however, type-1 polarizing potential of patients’ DC1 can be augmented ex vivo by a two-signal stimulation provided by pro-inflammatory cytokines and IFN-. These results may have implications for DC-based immunotherapy of malignancies in the transplantation setting.

This chapter explains and illustrates immunological procedures used to characterize different biological fluids. Body fluids are analyzed for a variety of reasons. Some analyses confirm a diagnosis, others identify the cause and nature of an effusion, and still others may have more diverse purposes. Body fluids analysis offers a comprehensive view into monitoring disease progression, therapy effectiveness, and occurrence of complications. Hematologic, chemical/biochemical, cytologic, microbiologic, serologic, molecular, or genetic techniques are commonly used for evaluation of body fluids. Some procedures to remove body fluids pose serious risks to patients. This requires obtaining an informed consent. Specimens should be collected in the correct containers, which are usually dictated by the type of anticoagulant needed. All body fluids should be handled as if contaminated with bloodborne pathogens. Immunological analysis of human body fluids is the focus of the chapter. Analysis of nasal, ear, and seminal fluid is presented.. Guidelines for management of occupational exposure to human blood or body fluids are also presented in the chapter.

Minnie Sarwal and colleagues developed a gene expression assay using peripheral blood samples to detect patients with renal transplant at high risk for acute rejection. Please see later in the article for the Editors' Summary, Background Development of noninvasive molecular assays to improve disease diagnosis and patient monitoring is a critical need. In renal transplantation, acute rejection (AR) increases the risk for chronic graft injury and failure. Noninvasive diagnostic assays to improve current late and nonspecific diagnosis of rejection are needed. We sought to develop a test using a simple blood gene expression assay to detect patients at high risk for AR. Methods and Findings We developed a novel correlation-based algorithm by step-wise analysis of gene expression data in 558 blood samples from 436 renal transplant patients collected across eight transplant centers in the US, Mexico, and Spain between 5 February 2005 and 15 December 2012 in the Assessment of Acute Rejection in Renal Transplantation (AART) study. Gene expression was assessed by quantitative real-time PCR (QPCR) in one center. A 17-gene set—the Kidney Solid Organ Response Test (kSORT)—was selected in 143 samples for AR classification using discriminant analysis (area under the receiver operating characteristic curve [AUC] = 0.94; 95% CI 0.91–0.98), validated in 124 independent samples (AUC = 0.95; 95% CI 0.88–1.0) and evaluated for AR prediction in 191 serial samples, where it predicted AR up to 3 mo prior to detection by the current gold standard (biopsy). A novel reference-based algorithm (using 13 12-gene models) was developed in 100 independent samples to provide a numerical AR risk score, to classify patients as high risk versus low risk for AR. kSORT was able to detect AR in blood independent of age, time post-transplantation, and sample source without additional data normalization; AUC = 0.93 (95% CI 0.86–0.99). Further validation of kSORT is planned in prospective clinical observational and interventional trials. Conclusions The kSORT blood QPCR assay is a noninvasive tool to detect high risk of AR of renal transplants. Please see later in the article for the Editors' Summary, Editors' Summary Background Throughout life, the kidneys filter waste products (from the normal breakdown of tissues and food) and excess water from the blood to make urine. If the kidneys stop working for any reason, the rate at which the blood is filtered decreases, and dangerous amounts of creatinine and other waste products build up in the blood. The kidneys can fail suddenly (acute kidney failure) because of injury or poisoning, but usually failing kidneys stop working gradually over many years (chronic kidney disease). Chronic kidney disease is very common, especially in people who have high blood pressure or diabetes and in elderly people. In the UK, for example, about 20% of people aged 65–74 years have some degree of chronic kidney disease. People whose kidneys fail completely (end-stage kidney disease) need regular dialysis (hemodialysis, in which blood is filtered by an external machine, or peritoneal dialysis, which uses blood vessels in the abdominal lining to do the work of the kidneys) or a renal transplant (the surgical transfer of a healthy kidney from another person into the patient's body) to keep them alive. Why Was This Study Done? Our immune system protects us from pathogens (disease-causing organisms) by recognizing specific molecules (antigens) on the invader's surface as foreign and initiating a sequence of events that kills the invader. Unfortunately, the immune system sometimes recognizes kidney transplants as foreign and triggers transplant rejection. The chances of rejection can be minimized by “matching” the antigens on the donated kidney to those on the tissues of the kidney recipient and by giving the recipient immunosuppressive drugs. However, acute rejection (rejection during the first year after transplantation) affects about 20% of kidney transplants. Acute rejection needs to be detected quickly and treated with a short course of more powerful immunosuppressants because it increases the risk of transplant failure. The current “gold standard” method for detecting acute rejection if the level of creatinine in the patient's blood begins to rise is to surgically remove a small piece (biopsy) of the transplanted kidney for analysis. However, other conditions can change creatinine levels, acute rejection can occur without creatinine levels changing (subclinical acute rejection), and biopsies are invasive. Here, the researchers develop a noninvasive test for acute kidney rejection called the Kidney Solid Organ Response Test (kSORT) based on gene expression levels in the blood. What Did the Researchers Do and Find? For the Assessment of Acute Rejection in Renal Transplantation (AART) study, the researchers used an assay called quantitative polymerase chain reaction (QPCR) to measure the expression of 43 genes whose expression levels change during acute kidney rejection in blood samples collected from patients who had had a kidney transplant. Using a training set of 143 samples and statistical analyses, the researchers identified a 17-gene set (kSORT) that discriminated between patients with and without acute rejection detected by kidney biopsy. The 17-gene set correctly identified 39 of the samples taken from 47 patients with acute rejection as being from patients with acute rejection, and 87 of 96 samples from patients without acute rejection as being from patients without acute rejection. The researchers validated the gene set using 124 independent samples. Then, using 191 serial samples, they showed that the gene set was able to predict acute rejection up to three months before detection by biopsy. Finally, the researchers used 100 blood samples to develop an algorithm (a step-wise calculation) to classify patients as being at high or low risk of acute rejection. What Do These Findings Mean? These findings describe the early development of a noninvasive tool (kSORT) that might, eventually, help clinicians identify patients at risk of acute rejection after kidney transplantation. kSORT needs to be tested in more patients before being used clinically, however, to validate its predictive ability, particularly given that the current gold standard test against which it was compared (biopsy) is far from perfect. An additional limitation of kSORT is that it did not discriminate between cell-mediated and antibody-mediated immune rejection. These two types of immune rejection are treated in different ways, so clinicians ideally need a test for acute rejection that indicates which form of immune rejection is involved. The authors are conducting a follow-up study to help determine whether kSORT can be used in clinical practice to identify acute rejection and to identify which patients are at greatest risk of transplant rejection and may require biopsy. Additional Information Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001759. The US National Kidney and Urologic Diseases Information Clearinghouse provides links to information about all aspects of kidney disease; the US National Kidney Disease Education Program provides resources to help improve the understanding, detection, and management of kidney disease (in English and Spanish) The UK National Health Service Choices website provides information for patients on chronic kidney disease and about kidney transplants, including some personal stories The US National Kidney Foundation, a not-for-profit organization, provides information about chronic kidney disease and about kidney transplantation (in English and Spanish) The not-for-profit UK National Kidney Federation provides support and information for patients with kidney disease and for their carers, including information and personal stories about kidney donation and transplantation World Kidney Day, a joint initiative between the International Society of Nephrology and the International Federation of Kidney Foundations, aims to raise awareness about kidneys and kidney disease MedlinePlus provides links to additional resources about kidney diseases, kidney failure, and kidney transplantation; the MedlinePlus encyclopedia has a page about transplant rejection

In efforts to minimize the chronic administration of immunosuppression (IS) drugs in transplantation and autoimmune disease, various cell-based tolerogenic therapies, including the use of regulatory or tolerogenic dendritic cells (tolDC) have been developed. These DC-based therapies aim to harness the inherent immunoregulatory potential of these professional antigen-presenting cells. In this short review, we describe both the demonstrated tolerogenic properties, and current limitations of rapamycin-conditioned DC (RAPA-DC). RAPA-DC are generated through inhibition of the integrative kinase mammalian target of rapamycin (mTOR) by the immunosuppressive macrolide rapamycin during propagation of monocyte-derived DC. Consistent with the characteristics of tolDC, murine RAPA-DC display resistance to phenotypic maturation induced by pro-inflammatory stimuli; exhibit the ability to migrate to secondary lymphoid tissue (important for ‘cross-presentation’ of antigen to T cells), and enrich for naturally-occurring CD4+ regulatory T cells. In rodent models, delivery of recipient-derived RAPA-DC pulsed with donor antigen prior to organ transplantation can prolong allogeneic heart-graft survival indefinitely, especially when combined with a short course of IS. These encouraging data support ongoing efforts to develop RAPA-DC for clinical testing. When compared to murine RAPA-DC however, human RAPA-DC have proven only partially resistant to maturation triggered by pro-inflammatory cytokines, and display heterogeneity in their impact on effector T-cell expansion and function. In total, the evidence suggests the need for more in-depth studies to better understand the mechanisms by which mTOR controls human DC function. These studies may facilitate the development of RAPA-DC therapy alone or together with agents that preserve/enhance their tolerogenic properties as clinical immunoregulatory vectors.