Data analysis as a source of variability of the HLA-peptide multimer assay: from manual gating to automated recognition of cell clusters.

Multiparameter flow cytometry is an indispensable method for assessing antigen-specific T cells in basic research and cancer immunotherapy. Proficiency panels have shown that cell sample processing, test protocols and data analysis may all contribute to the variability of the results obtained by laboratories performing ex vivo T cell immune monitoring. In particular, analysis currently relies on a manual, step-by-step strategy employing serial gating decisions based on visual inspection of one- or two-dimensional plots.

Alpha-synuclein levels in blood plasma decline with healthy aging.

There is unequivocal evidence that alpha-synuclein plays a pivotal pathophysiological role in neurodegenerative diseases, and in particular in synucleinopathies. These disorders present with a variable extent of cognitive impairment and alpha-synuclein is being explored as a biomarker in CSF, blood serum and plasma. Considering key events of aging that include proteostasis, alpha-synuclein may not only be useful as a marker for differential diagnosis but also for aging per se.

HLA antibody specification using single-antigen beads--a technical solution for the prozone effect.

Background: Substantial progress in human leukocyte antigen antibody specification has been made by the introduction of Luminex single-antigen bead (SAB) assays. This progress was impaired when it turned out that this method is prone to a prozone effect leading to false-negative results in the case of high antibody titers. Testing serum and ethylenediaminetetraacetic acid (EDTA) plasma of one patient in parallel, we observed the prozone effect with the serum sample only.

Promiscuous survivin peptide induces robust CD4+ T-cell responses in the majority of vaccinated cancer patients.

CD4(+) T cells have been shown to be crucial for the induction and maintenance of cytotoxic T cell responses and to be also capable of mediating direct tumor rejection. Therefore, the anticancer therapeutic efficacy of peptide-based vaccines may be improved by addition of HLA class II epitopes to stimulate T helper cells. Survivin is an apoptosis inhibiting protein frequently overexpressed in tumors.

Analysis of tumor antigen-specific T cells and antibodies in cancer patients treated with radiofrequency ablation.

Radiofrequency (RF) ablation is a minimally invasive technique routinely applied for the treatment of primary and secondary liver tumors. It induces cell death by thermal coagulative necrosis of tumor tissues, whereas cellular metabolism can still take place in a transition zone surrounding the necrotic area. An increase in heat shock protein expression occurs shortly after treatment, suggesting that the induction of activating signals may stimulate the host immune system.

Involvement of CD91 and scavenger receptors in Hsp70-facilitated activation of human antigen-specific CD4+ memory T cells.

Hsp70 plays several roles in the adaptive immune response. Based on the ability to interact with diverse peptides, extracellular Hsp70:peptide complexes exert profound effects both in autoimmunity and in tumor rejection by evoking potent T cell responses to the chaperoned peptide. The interaction with receptors on APC represents the basis for the immunological functions of Hsp70 and a critical point where the immune response can be regulated.

[AB0-incompatible kidney transplantation--case 13/2009].

Unlabelled: PATIENT HISTORY AND CLINICAL FINDINGS: A 49-year old patient with chronic kidney disease was referred for evaluation of living donor kidney transplantation from his spouse in a blood group incompatible setting.

Clinical Investigations: Patient blood group was 0, donor blood group was A, subtype A. recipient isoagglutinin titer against donor erythrocytes was 1:64.

Ex vivo detection of adenovirus specific CD4+ T-cell responses to HLA-DR-epitopes of the Hexon protein show a contracted specificity of T(HELPER) cells following stem cell transplantation.

Human adenovirus (HAdV) is a cause of significant morbidity and mortality in immunocompromised patients, especially after stem cell transplantation (SCT). Viral clearance has been attributed to CD4(+) T-cell responses against the Hexon-protein, but the frequency of specific T(HELPER) cells is extremely low or not detectable ex vivo and preference for different CD4(+) T-cell epitopes is variable among individuals. We therefore analyzed 44 healthy donors and 6 SCT-recipients for Hexon-specific CD4(+)-responses ex vivo, to identify epitopes which would be broadly applicable.

Simultaneous infiltration of polyfunctional effector and suppressor T cells into renal cell carcinomas.

Renal cell carcinoma is frequently infiltrated by cells of the immune system. This makes it important to understand interactions between cancer cells and immune cells so they can be manipulated to bring clinical benefit. Here, we analyze subsets and functions of T lymphocytes infiltrating renal cell tumors directly ex vivo following mechanical disaggregation and without any culture step.

Novel multi-peptide vaccination in Hla-A2+ hormone sensitive patients with biochemical relapse of prostate cancer.

Background: A phase I/II trial was conducted to assess feasibility and tolerability of tumor associated antigen peptide vaccination in hormone sensitive prostate carcinoma (PC) patients with biochemical recurrence after primary surgical treatment.

Methods: Nineteen HLA-A2 positive patients with rising PSA without detectable metastatic disease or local recurrence received 11 HLA-A*0201-restricted and two HLA class II synthetic peptides derived from PC tumor antigens subcutaneously for 18 months or until PSA progression. The vaccine was emulgated in montanide ISA51 and combined with imiquimod, GM-CSF, mucin-1-mRNA/protamine complex, local hyperthermia or no adjuvant.

HLA ligand profiles of primary renal cell carcinoma maintained in metastases.

In recent years, several approaches have been taken in the peptide-based immunotherapy of metastatic renal cell carcinoma (RCC), although little is known about HLA presentation on metastases compared to primary tumor and normal tissue of RCC. In this study we compared primary tumor, normal tissue and metastases with the aim of identifying similarities and differences between these tissues. We performed this comparison for two RCC patients on the level of the HLA ligandome using mass spectrometry and for three patients on the level of the transcriptome using oligonucleotide microarrays.

Essential differences in ligand presentation and T cell epitope recognition among HLA molecules of the HLA-B44 supertype.

Human leukocyte antigens (HLA) have long been grouped into supertypes to facilitate peptide-based immunotherapy. Analysis of several hundreds of peptides presented by all nine antigens of the HLA-B44 supertype (HLA-B*18, B*37, B*40, B*41, B*44, B*45, B*47, B*49 and B*50) revealed unique peptide motifs for each of them. Taking all supertype members into consideration only 25 out of 670 natural ligands were found on more than one HLA molecule.

Identification of HLA-A*01- and HLA-A*02-restricted CD8+ T-cell epitopes shared among group B enteroviruses.

Acute enteroviral infections ranging from meningitis, pancreatitis to myocarditis are common and normally well controlled by the host immune system comprising virus-specific CD8+ cytotoxic T lymphocytes (CTL). However, in some patients enteroviruses and especially coxsackieviruses of group B are capable of inducing severe chronic forms of diseases such as chronic myocarditis. Currently, it is not known whether divergences in the CTL-related immune response may contribute to the different outcome and course of enterovirus myocarditis.

A cryptic vascular endothelial growth factor T-cell epitope: identification and characterization by mass spectrometry and T-cell assays.

Vascular endothelial growth factor (VEGF) is involved in various physiologic processes, such as angiogenesis or wound healing, but is also crucial in pathologic events, such as tumor growth. Thus, clinical anti-VEGF treatments have been developed that could already show beneficial effects for cancer patients. In this article, we describe the first VEGF-derived CD8(+) T-cell epitope.

Potent costimulation of human CD8 T cells by anti-4-1BB and anti-CD28 on synthetic artificial antigen presenting cells.

The in vitro generation of cytotoxic T lymphocytes (CTLs) for anticancer immunotherapy is a promising approach to take patient-specific therapy from the bench to the bedside. Two criteria must be met by protocols for the expansion of CTLs: high yield of functional cells and suitability for good manufacturing practice (GMP). The antigen presenting cells (APCs) used to expand the CTLs are the key to achieving both targets but they pose a challenge: Unspecific stimulation is not feasible because only memory T cells are expanded and not rare naïve CTL precursors; in addition, antigen-specific stimulation by cell-based APCs is cumbersome and problematic in a clinical setting.

Is there a predisposition for the development of autoimmune diseases in patients with fibromyalgia? Retrospective analysis with long term follow-up.

The objectives of the study were to evaluate the prevalence of antinuclear antibodies (ANA) in patients with fibromyalgia (FM) and the probability of the development of clinically overt connective tissue diseases. Four hundred and fifty FM patients were compared to 129 healthy matched blood donors. ANA testing was performed by immunofluorescence on rat tissue sections; in case of highly positive results, ANA were specified further by ELISA and immunodiffusion.

HAGE, a cancer/testis antigen with potential for melanoma immunotherapy: identification of several MHC class I/II HAGE-derived immunogenic peptides.

There remains a need to identify novel epitopes of potential tumour target antigens for use in immunotherapy of cancer. Here, several melanoma tissues and cell lines but not normal tissues were found to overexpress the cancer-testis antigen HAGE at the mRNA and protein level. We identified a HAGE-derived 15-mer peptide containing a shorter predicted MHC class I-binding sequence within a class II-binding sequence.

Distorted relation between mRNA copy number and corresponding major histocompatibility complex ligand density on the cell surface.

The major histocompatibility complex (MHC) presents peptides derived from degraded cellular proteins to T-cells and is thus crucial for triggering specific immune responses against viral infections or cancer. Up to now, there has been no evidence for a correlation between levels of mRNA (the "transcriptome") and the density of MHC-peptide complexes (the "MHC ligandome") on cells. Because such dependences are of intrinsic importance for the detailed understanding of translation efficiency and protein turnover and thus for systems biology in general and for tumor immunotherapy in practical application, we quantitatively analyzed the levels of mRNA and corresponding MHC ligand densities in samples of renal cell carcinomas and their autologous normal kidney tissues.

Unexpected abundance of HLA class II presented peptides in primary renal cell carcinomas.

Purpose: To elicit a long-lasting antitumor immune response, CD8+ and CD4+ T cells should be activated. We attempted to isolate HLA-DR-presented peptides directly from dissected solid tumors, in particular from renal cell carcinoma, to identify MHC class II ligands from tumor-associated antigens (TAA) for their use in peptide-based immunotherapy.

Experimental Design: Tumor specimens were analyzed by immunohistochemical staining for their HLA class II expression.

The heat shock protein Hsp70 enhances antigen-specific proliferation of human CD4+ memory T cells.

Heat shock proteins (HSP) can interact with a wide variety of peptides and the resulting HSP:peptide complexes are known to be highly immunogenic. The ability of HSP:peptide complexes to elicit CD8+ T cell responses by cross-presentation of exogenous antigen via MHC class I is well known. In contrast, their role in the activation of CD4+ T cells is less clearly defined, although several recent studies in mice and T cell lines suggest an involvement of HSP in the presentation of antigenic peptides via MHC class II.

A noninvasive strategy for screening prospective blood donors for anemia.

Background: The reliability of capillary hemoglobin (Hb) as an indicator for eligibility to donate blood is discussed controversially. Therefore, a noninvasive alternative with acceptable predictive values was established and evaluated.

Study Design And Methods: Donor candidates were selected according to their Hb level.

High frequencies of functionally impaired cytokeratin 18-specific CD8+ T cells in healthy HLA-A2+ donors.

Combining cell surface phenotyping with functional analysis, human CD8+ T cells have been divided into several subsets which are being studied extensively in diverse physiological situations, such as viral infection, cancer and ageing. In particular, so-called terminally differentiated effector cells possess a CD45RA+ CCR7- CD27- CD28- phenotype, contain perforin and, in different models, have been shown to exert direct ex vivo killing and to release interleukins upon both antigen-nonspecific and -specific stimulation. Using HLA class I multimers, we have identified a high frequency of peripheral CD8+ T cells that recognize a peptide derived from the self protein cytokeratin 18 presented by the HLA-A*0201 molecule.

Cytotoxic minor histocompatibility antigen HA-1-specific CD8+ effector memory T cells: artificial APCs pave the way for clinical application by potent primary in vitro induction.

Induction of cytotoxic T lymphocytes (CTLs) for treatment of relapsed leukemia after allogeneic stem-cell transplantation is hindered by the laborious and time-consuming procedure of generating dendritic cells for antigen presentation. Artificial antigen-presenting cells (aAPCs) offer the advantage of being readily available in sufficient numbers, thus allowing for a highly standardized in vitro induction of CTLs. We generated aAPCs coated with anti-CD28 antibody (Ab) and either high-density (HD) or low-density (LD) major histocompatibility complex (MHC) class I molecules loaded with HA-1(H), a nonapeptide derived from the hematopoiesis-restricted minor histocompatibility antigen HA-1.

Comprehensive analysis of thiopurine S-methyltransferase phenotype-genotype correlation in a large population of German-Caucasians and identification of novel TPMT variants.

The thiopurine S-methyltransferase (TPMT) genetic polymorphism has a significant clinical impact on the toxicity of thiopurine drugs. It has been proposed that the identification of patients who are at high risk for developing toxicity on the basis of genotyping could be used to individualize drug treatment. In the present study, phenotype-genotype correlation of 1214 healthy blood donors was investigated to determine the accuracy of genotyping for correct prediction of different TPMT phenotypes.

Cytotoxic activity of natural killer cells lacking killer-inhibitory receptors for self-HLA class I molecules against autologous hematopoietic stem cells in healthy individuals.

Killer-inhibitory receptors (KIR) are receptors for self-HLA class I molecules, which are expressed on natural killer (NK) cells and small subsets of T-lymphocytes. KIR receptors that do not bind to self-HLA class I have been implicated in the pathogenesis of pure red-cell aplasia and other autoimmune diseases. However, NK cells whose inhibitory receptors lack any apparent self-ligand can also be found in healthy individuals.