Operational tolerance is not always permanent: A 10-year prospective study in pediatric liver transplantation recipients.

Immunosuppression withdrawal can be safely performed in select liver transplantation recipients, but the long-term outcomes and sustainability of tolerance have not been well studied. We completed a 10-year prospective, observational study of 18 pediatric liver transplantation recipients with operational tolerance to (1) assess the sustainability of tolerance over time, (2) compare the clinical characteristics of patients who maintained versus lost tolerance, (3) characterize liver histopathology findings in surveillance liver biopsies; and (4) describe immunologic markers in patients with tolerance. Comparator patients from two clinical phenotype groups termed "stable" and "nontolerant" patients were used as controls. Of the 18 patients with operational tolerance, the majority of patients were males (n = 14, 78%) who were transplanted for cholestatic liver disease (n = 12, 67%). Median age at transplantation was 1.9 (range, 0.6-8) years. Median time after transplantation that immunosuppression had been discontinued was 13.1 (range, 2.9-22.1) years. As many as 11 (61%) maintained tolerance for a median of 10.4 (range, 1.9-22.1) years, whereas 7 (39%) lost tolerance after a median of 3.2 (range, 1.5-18.6) years. Populations of T regulatory cells (%CD4+ CD25hi CD127lo ) were significantly higher in patients with tolerance (p = 0.02). Our results emphasize that spontaneous operational tolerance is a dynamic and nonpermanent state. It is therefore essential for patients who are clinically stable off immunosuppression to undergo regular follow-up and laboratory monitoring, as well as surveillance biopsies to rule out subclinical rejection.

A model for harmonizing flow cytometry in clinical trials.

Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.

Serum creatinine detection by a conducting-polymer-based electrochemical sensor to identify allograft dysfunction.

Kidney transplant recipients who have abnormally high creatinine levels in their blood often have allograft dysfunction secondary to rejection. Creatinine has become the preferred marker for renal dysfunction and is readily available in hospital clinical settings. We developed a rapid and accurate polymer-based electrochemical point-of-care (POC) assay for creatinine detection from whole blood to identify allograft dysfunction. The creatinine concentrations of 19 blood samples from transplant recipients were measured directly from clinical serum samples by the conducting polymer-based electrochemical (EC) sensor arrays. These measurements were compared to the traditional clinical laboratory assay. The time required for detection was <5 min from sample loading. Sensitivity of the detection was found to be 0.46 mg/dL of creatinine with only 40 µL sample in the creatinine concentration range of 0 mg/dL to 11.33 mg/dL. Signal levels that were detected electrochemically correlated closely with the creatinine blood concentration detected by the UCLA Ronald Reagan Medical Center traditional clinical laboratory assay (correlation coefficient = 0.94). This work is encouraging for the development of a rapid and accurate POC device for measuring creatinine levels in whole blood.

Associations between single nucleotide polymorphisms in double-stranded DNA repair pathway genes and familial breast cancer.

PURPOSE: DNA damage recognition and repair play a major role in risk for breast cancer. We investigated 104 single nucleotide polymorphisms (SNP) in 17 genes whose protein products are involved in double-stranded break repair (DSBR). EXPERIMENTAL DESIGN: We used a case-control design. Both the case individuals affected with breast cancer or with both breast and ovarian cancers and the controls had similar familial risk of breast cancer and were participants in a high-risk cancer registry. RESULTS: We found that 12 of the polymorphisms are associated with breast or breast and ovarian cancers, most notably rs16888927, rs16888997, and rs16889040, found in introns of RAD21, suggesting that SNPs in other genes in the DSBR pathway in addition to BRCA1 and BRCA2 may affect breast cancer risk. CONCLUSIONS: SNPs within or near several DSBR DNA repair pathway genes are associated with breast cancer in individuals from a high-risk population. In addition, our study reemphasizes the unique perspective that recruitment of cases and controls from family cancer registries has for gene discovery studies.

Temporal expression of cytokines and B-cell phenotypes during mechanical circulatory support.

BACKGROUND: Allosensitization during mechanical circulatory support (MCS) is a well-described phenomenon, although its mechanism remains unknown. Although immune-mediated interactions from devices or blood transfusions have been proposed, the role of inflammation in this development is less clear. This study was undertaken to further investigate the temporal association of cytokines and B-cell phenotypes in the MCS population. METHODS: Adult patients who received the Heartmate II (Thoratec, Pleasanton, Calif) at our center between September 2012 and March 2015 were prospectively followed after device implantation. Blood draws for anti-human leukocyte antigen (HLA) antibody, cytokine expression, and B-cell immunophenotyping were performed before implantation and for 3 weeks postoperatively. Time courses for cytokines and B-cell subsets were expressed using visual representations of median levels as heat maps, and mixed modeling analysis was used to model changes with time and patient factors. RESULTS: Twenty patients who received the Heartmate II (Thoratec) were analyzed during the study period. Four patients showed measureable levels of anti-HLA antibody during the follow-up period, although 3 of these had evidence of antibodies preoperatively. Analysis of cytokine trends revealed early (interleukin [IL]-6, IL-8, and IL-10) and late peaking (IL-3, IL-4, fibroblast growth factor 2, and CD40L) patterns. Upregulation of switched memory, transitional, and plasma blast B cells occurred over time. Right ventricular assist device use and low Interagency Registry for Mechanically Assisted Circulatory Support score were associated with decreased mature naive and increased antibody-secreting cells. CONCLUSIONS: MCS device implantation was associated with increased inflammatory cytokines and maturation of B-cell phenotypes. No patients developed de novo HLA antibodies, whereas several showed increases in anti-HLA antibody levels detected before implantation. This suggests that inflammation and maturation of existing sensitized B cells might play an important role in the pathogenesis of allosensitization in MCS.

T cell dysfunction and patient age are associated with poor outcomes after mechanical circulatory support device implantation.

Immunologic impairment may contribute to poor outcomes after implantation of mechanical circulatory support device (MCSD), with infection often as a terminal event. The study of immune dysfunction is of special relevance given the growing numbers of older patients with heart disease. The aim of the study was to define which immunologic characteristics are associated with development of adverse clinical outcomes after MCSD implantation. We isolated peripheral blood mononuclear cells (PBMC) from patients pre- and up to 20 days post-MCSD implantation and analyzed them by multiparameter flow cytometry for T cell dysfunction, including terminal differentiation, exhaustion, and senescence. We used MELD-XI and SOFA scores measured at each time point as surrogate markers of clinical outcome. Older patients demonstrated increased frequencies of terminally differentiated T cells as well as NKT cells. Increased frequency of terminally differentiated and immune senescent T cells were associated with worse clinical outcome as measured by MELD-XI and SOFA scores, and with progression to infection and death. In conclusion, our data suggest that T cell dysfunction, independently from age, is associated with poor outcomes after MCSD implantation, providing a potential immunologic mechanism behind patient vulnerability to multiorgan dysfunction and death. This noninvasive approach to PBMC evaluation holds promise for candidate evaluation and patient monitoring.

Differences in Proinflammatory Cytokines and Monocyte Subtypes in Older as Compared With Younger Kidney Transplant Recipients.

BACKGROUND: The number of elderly patients with end-stage kidney disease requiring kidney transplantation continues to grow. Evaluation of healthy older adults has revealed proinflammatory changes in the immune system, which are posited to contribute to age-associated illnesses via "inflamm-aging." Immunologic dysfunction is also associated with impaired control of infections. Whether these immunologic changes are found in older kidney transplant recipients is not currently known, but may have important implications for risk for adverse clinical outcomes. METHODS: Three months after transplant, innate immune phenotype was evaluated by flow cytometry from 60 kidney transplant recipients (22 older [≥60 years] and 38 younger [<60 years old]). Multiplex cytokine testing was used to evaluate plasma cytokine levels. Younger patients were matched to older patients based on transplant type and induction immune suppression. RESULTS: Older kidney transplant recipients demonstrated decreased frequency of intermediate monocytes (CD14++CD16+) compared with younger patients (1.2% vs 3.3%, P = 0.007), and a trend toward increased frequency of proinflammatory classical monocytes (CD14++CD16-) (94.5% vs 92.1%) (P = 0.065). Increased levels of interferon-gamma (IFN-γ) were seen in older patients. CONCLUSIONS: In this pilot study of kidney transplant recipients, we identified differences in the innate immune system in older as compared with younger patients, including increased levels of IFN-γ. This suggests that age-associated nonspecific inflammation persists despite immune suppression. The ability to apply noninvasive testing to transplant recipients will provide tools for patient risk stratification and individualization of immune suppression regimens to improve outcomes after transplantation.

Increased Frequency of BK Virus-Specific Polyfunctional CD8+ T Cells Predict Successful Control of BK Viremia After Kidney Transplantation.

BACKGROUND: BK virus infection remains an important cause of loss of allograft function after kidney transplantation. We sought to determine whether polyfunctional T cells secreting multiple cytokines simultaneously, which have been shown to be associated with viral control, could be detected early after start of BK viremia, which would provide insight into the mechanism of successful antiviral control. METHODS: Peripheral blood mononuclear cells collected during episodes of BK viral replication were evaluated by multiparameter flow cytometry after stimulation by overlapping peptide pools of BK virus antigen to determine frequency of CD8+ and CD4+ T cells expressing 1 or more cytokines simultaneously, as well as markers of T-cell activation, exhaustion, and maturation. RESULTS: BK virus controllers, defined as those with episodes of BK viremia of 3 months or less, had an 11-fold increase in frequency of CD8+ polyfunctional T cells expressing multiple cytokines, as compared with patients with prolonged episodes of BK viremia. Patients with only low level BK viremia expressed low frequencies of polyfunctional T cells. Polyfunctional T cells were predominantly of the effector memory maturation subtype and expressed the cytotoxicity marker CD107a. CONCLUSIONS: Noninvasive techniques for immune assessment of peripheral blood can provide insight into the mechanism of control of BK virus replication and may allow for future patient risk stratification and customization of immune suppression at the onset of BK viremia.

Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.

Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.

A novel flow assay for the detection of cytokine secreting alloreactive T cells: application to immune monitoring.

The direct and indirect allorecognition pathways play an important role in graft rejection. We hypothesized that the presence of alloreactive memory T cells in the recipient's circulation increases the risk of rejection after transplantation. The objective of this study was to develop a noninvasive, immune monitoring tool that simultaneously measures donor-specific responses via both the direct and indirect recognition pathways. Our laboratory developed a whole blood flow cytometric cytokine secretion assay to identify interferon (IFN)-gamma secreting memory T cells in whole blood of renal transplant patients. The assay readily detected IFN-gamma producing CD3+ T cells in response to recall antigens tetanus toxoid, purified protein derivative, and alloantigens in whole blood from healthy controls. Analysis of sequential posttransplant blood samples from 19 renal allograft recipients showed that alloimmune responses were higher in transplant recipients who had undergone acute rejection than in those without acute rejection episodes. In addition, patients showing increased creatinine levels 3 months after transplantation were more likely to exhibit alloimmune responses than recipients with stable graft function. The flow cytokine secretion assay provides a reliable and simple method for identification of patients at risk of acute rejection and early graft dysfunction.

Specific binding and magnetic concentration of CD8+ T-lymphocytes on electrowetting-on-dielectric platform.

In the quest to create a low-power portable lab-on-a-chip system, we demonstrate the specific binding and concentration of human CD8+ T-lymphocytes on an electrowetting-on-dielectric (EWOD)-based digital microfluidic platform using antibody-conjugated magnetic beads (MB-Abs). By using a small quantity of nonionic surfactant, we enable the human cell-based assays with selective magnetic binding on the EWOD device in an air environment. High binding efficiency (∼92%)of specific cells on MB-Abs is achieved due to the intimate contact between the cells and the magnetic beads (MBs) produced by the circulating flow within the small droplet. MBs have been used and cells manipulated in the droplets actuated by EWOD before; reported here is a cell assay of a clinical protocol on the EWOD device in air environment. The present technique can be further extended to capture other types of cells by suitable surface modification on the MBs.

TNFalpha blockade in human diseases: mechanisms and future directions.

Tumor necrosis factor-alpha (TNFalpha) antagonists have shown remarkable efficacy in a variety of immune-mediated inflammatory diseases (IMIDs). Therapeutic scope and limitations of these agents are reviewed in a recently published article in the Journal. In spite of their therapeutic popularity, little is known about their modes of action in vivo and factors that limit their scope of therapeutic use. Intriguingly, while all TNFalpha antagonists including blocking antibodies and soluble receptors are effective in certain IMIDs, only anti-TNFalpha antibodies are effective in other IMIDs. Early efforts at understanding how TNFalpha antagonists act in IMIDs centered on their ability to neutralize soluble TNFalpha or to block TNF receptors from binding to their ligands. Subsequent studies suggested a role of complement-mediated lysis or antibody-dependent cell cytotoxicity in their therapeutic effects. More recent models postulate that TNFalpha blockers may act by affecting intracellular signaling, with the end result being a hastened cell cycle arrest, apoptosis, suppression of cytokine production, or improved Treg cell function. TNFalpha antagonists can also modulate the functions of myofibroblasts and osteoclasts, which might explain how TNFalpha antagonists reduce tissue damage in chronic IMIDs. Focusing on the human therapeutic experience, this analytical review will review the biology of mechanisms of action, the limiting factors contributing to disease restriction in therapeutic efficacy, and the mechanism and frequency of treatment-limiting adverse responses of TNFalpha antagonists. It is hoped that the overview will address the needs of clinicians to decide on optimal use, spur clinical innovation, and incite translational researchers to set priorities for in vivo human investigations.

TNFalpha blockade in human diseases: an overview of efficacy and safety.

Tumor necrosis factor-alpha (TNFalpha) antagonists including antibodies and soluble receptors have shown remarkable efficacy in various immune-mediated inflammatory diseases (IMID). As experience with these agents has matured, there is an emerging need to integrate and critically assess the utility of these agents across disease states and clinical sub-specialties. Their remarkable efficacy in reducing chronic damage in Crohn's disease and rheumatoid arthritis has led many investigators to propose a new, 'top down' paradigm for treating patients initially with aggressive regimens to quickly control disease. Intriguingly, in diseases such as rheumatoid arthritis and asthma, anti-TNFalpha agents appear to more profoundly benefit patients with more chronic stages of disease but have a relatively weaker or little effect in early disease. While the spectrum of therapeutic efficacy of TNFalpha antagonists widens to include diseases such as recalcitrant uveitis and vasculitis, these agents have failed or even exacerbated diseases such as heart failure and multiple sclerosis. Increasing use of these agents has also led to recognition of new toxicities as well as to understanding of their excellent long-term tolerability. Disconcertingly, new cases of active tuberculosis still occur in patients treated with all TNFalpha antagonists due to lack of compliance with recommendations to prevent reactivation of latent tuberculosis infection. These safety issues as well as guidelines to prevent treatment-associated complications are reviewed in detail in this article. New data on mechanisms of action and development of newer TNFalpha antagonists are discussed in a subsequent article in the Journal. It is hoped that these two review articles will stimulate a fresh assessment of the priorities for research and clinical innovation to improve and extend therapeutic use and safety of TNFalpha antagonism.

RNA interference elucidates the role of focal adhesion kinase in HLA class I-mediated focal adhesion complex formation and proliferation in human endothelial cells.

Ligation of class I molecules by anti-HLA Ab stimulates an intracellular signaling cascade resulting in endothelial cell (EC) survival and proliferation, and has been implicated in the process of chronic allograft rejection and transplant-associated vasculopathy. In this study, we used small interfering RNA blockade of focal adhesion kinase (FAK) protein to determine its role in class I-mediated organization of the actin cytoskeleton, cell survival, and cell proliferation in primary cultures of human aortic EC. Knockdown of FAK appreciably inhibited class I-mediated phosphorylation of Src at Tyr(418), p85 PI3K, and Akt at both Thr(308) and Ser(473) sites. FAK knockdown also reduced class I-mediated phosphorylation of paxillin at Try(118) and blocked class I-induced paxillin assembly into focal contacts. FAK small interfering RNA completely abrogated class I-mediated formation of actin stress fibers. Interestingly, FAK knockdown did not modify fibroblast growth factor receptor expression induced by class I ligation. However, FAK knockdown blocked HLA class I-stimulated cell cycle proliferation in the presence and absence of basic fibroblast growth factor. This study shows that FAK plays a critical role in class I-induced cell proliferation, cell survival, and focal adhesion assembly in EC and may promote the development of transplant-associated vasculopathy.

Effects of prostratin on T-cell activation and human immunodeficiency virus latency.

Human immunodeficiency virus (HIV) replication is linked to cellular gene transcription and requires target cell activation. The latent reservoir of HIV-1 in quiescent T cells is thought to be a major obstacle to clearance of infection by highly active antiretroviral therapy (HAART). Thus, identification of agents that can induce expression of latent virus may, in the presence of HAART, allow elimination of the infected cells by the immune response. We previously used the SCID-hu (Thy/Liv) mouse model to establish that activation-inducible HIV can be generated at high frequency during thymopoiesis. Latently infected mature thymocytes can be exported into the periphery, providing an efficient primary cell model to determine cellular activation signals that induce renewed expression of latent virus. Here we characterized the effects of prostratin, a non-tumor-promoting phorbol ester, on primary human peripheral blood lymphocytes (PBLs) and assessed its ability to reactivate latent HIV infection from thymocytes and PBLs in the SCID-hu (Thy/Liv) model. Prostratin stimulation alone did not induce proliferation of quiescent PBLs; however, it could provide a secondary signal in the context of T-cell receptor stimulation or a primary activation signal in the presence of CD28 stimulation to induce T-cell proliferation. While prostratin alone was not sufficient to allow de novo HIV infection, it efficiently reactivated HIV expression from latently infected cells generated in the SCID-hu mouse. Our data indicate that prostratin alone is able to specifically reactivate latent virus in the absence of cellular proliferation, making it an attractive candidate for further study as an adjunctive therapy for the elimination of the latent HIV reservoir.

Interleukin-7 induces expression of latent human immunodeficiency virus type 1 with minimal effects on T-cell phenotype.

Latent human immunodeficiency virus type 1 (HIV-1) persists even in patients treated with antiretroviral therapy. New treatment strategies are therefore needed to eradicate this latent viral reservoir without reducing immune cell function. We characterize the interleukin-7 (IL-7)-induced stimulation of primary human T cells and thymocytes and demonstrate, using the SCID-hu model, that IL-7 induces substantial expression of latent HIV while having minimal effects on the cell phenotype. Thus, IL-7 is a viable candidate to activate expression of latent HIV and may facilitate immune clearance of latently infected cells.