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Immunotherapies targeting cancer-specific neoantigens have revolutionized the treatment of cancer patients. Recent evidence suggests that epigenetic therapies synergize with immunotherapies, mediated by the de-repression of endogenous retroviral element (ERV)-encoded promoters, and the initiation of transcription. Here, we use deep RNA sequencing from cancer cell lines treated with DNA methyltransferase inhibitor (DNMTi) and/or Histone deacetylase inhibitor (HDACi), to assemble a de novo transcriptome and identify several thousand ERV-derived, treatment-induced novel polyadenylated transcripts (TINPATs). Using immunopeptidomics, we demonstrate the human leukocyte antigen (HLA) presentation of 45 spectra-validated treatment-induced neopeptides (t-neopeptides) arising from TINPATs. We illustrate the potential of the identified t-neopeptides to elicit a T-cell response to effectively target cancer cells. We further verify the presence of t-neopeptides in AML patient samples after in vivo treatment with the DNMT inhibitor Decitabine. Our findings highlight the potential of ERV-derived neoantigens in epigenetic and immune therapies. Epigenetic therapies are known to synergize with immunotherapies through the de-repression of endogenous retroviral element (ERV)-encoded promoters. Here the authors identify treatment-induced neoantigens and validate their ability to induce T cell response and anti-tumor effects in vitro and in patient samples. [ABSTRACT FROM AUTHOR]

Common flow cytometry-based methods used for functional assessment of antigen-specific T cells rely on de novo expression of intracellular cytokines or cell surface activation induced markers. They come with some limitations such as complex experimental setting, loss of cell viability and often high unspecific background which impairs assay sensitivity. We have previously shown that staining of activated ß2-integrins either with multimers of their ligand ICAM-1 or with a monoclonal antibody can serve as a functional marker detectable on T cells after minutes (CD8+) or few hours (CD4+) of activation. Here, we present a simple method for detection of activated ß2-integrins in combination with established cell surface activation induced markers. We observed that activated ß2-integrins were still detectable after 14 hours of stimulation, allowing their detection together with CD137 and CD154. Combinatorial gating of cells expressing activated ß2-integrins and CD137 or CD154 reduced background in unstimulated samples, increasing the signal-to-noise ratio and allowing improved assessment of low-frequency T cell responses. Extracellular staining of these markers highly correlated with production of intracellular cytokines IL-2, TNF or IFNg in CD4+ and CD8+ T cells. As an exemplary application, SARS-CoV-2 spike-specific T cell responses were assessed in individuals after COVID-19 vaccination. This method should be useful for epitope discovery projects and for the simultaneous monitoring of lowfrequency antigen-specific CD4+ and CD8+ T cell responses in various physiological situations. [ABSTRACT FROM AUTHOR]

Elongated peptides (EPs), containing possibly one or multiple epitope/s, are increasingly used for the screening of antigen-specific CD8+ and CD4+ cell responses. Here, we present an in vitro protocol that allows the amplification of antigen-specific cells and the subsequent functional analysis of both T cell types using EPs. Known viral-derived epitopes were elongated to 20 mer EPs on the N-, C-, and both termini for HLA class I binders, or on the N- and C- termini for HLA class II binders. With EP stimulation only, the percentage of responding CD8+ T cells was dependent on the elongation site of the EP, whereas CD4+ T cell responses were completely lost in 22% of the tests performed ex vivo. A short-term amplification step plus the addition of a TLR3 agonist (Poly-ICLC) together with an increased EP concentration improved markedly the detection of CD8+ and CD4+ T cell reactivities. [ABSTRACT FROM AUTHOR]

Simple Summary: The immunopeptidome represents the entirety of peptides that are presented on the surface of cells on human leukocyte antigen (HLA) molecules and are recognized by the T-cell receptors of CD4+ and CD8+ T-cells. Malignant cells present tumor-associated antigens essential for tumor immune surveillance, which can be targeted by T-cell-based immunotherapy approaches. For ovarian carcinomas, various tumor-associated antigens, such as Mucin-16 and Mesothelin, have been described. The aim of our study is to analyze immunopeptidome-defined tumor antigen presentation in ovarian carcinoma patients in relation to clinical characteristics and disease outcomes to define potential biomarkers. Our work demonstrates the central role of HLA-DR-restricted peptide presentation of the tumor antigen Mesothelin and of CD4+ T-cell responses for tumor immune surveillance, and underlines Mesothelin as a prime target antigen for novel immunotherapeutic approaches for ovarian carcinoma patients. T-cell recognition of HLA-presented antigens is central for the immunological surveillance of malignant disease and key for the development of novel T-cell-based immunotherapy approaches. In recent years, large-scale immunopeptidome studies identified naturally presented tumor-associated antigens for several malignancies. Regarding ovarian carcinoma (OvCa), Mucin-16 (MUC16) and Mesothelin (MSLN) were recently described as the top HLA class I- and HLA class II-presented tumor antigens, respectively. Here, we investigate the role and impact of immunopeptidome-presented tumor antigens on the clinical outcomes of 39 OvCa patients with a follow-up time of up to 50 months after surgery. Patients with a HLA-restricted presentation of high numbers of different MSLN-derived peptides on their tumors exhibited significantly prolonged progression-free (PFS) and overall survival (OS), whereas the presentation of MUC16-derived HLA class I-restricted peptides had no impact. Furthermore, a high HLA-DRB gene expression was associated with increased PFS and OS. In line, in silico prediction revealed that MSLN-derived HLA class II-presented peptides are predominantly presented on HLA-DR allotypes. In conclusion, the correlation of MSLN tumor antigen presentation and HLA-DRB gene expression with prolonged survival indicates a central role of CD4+ T-cell responses for tumor immune surveillance in OvCa, and highlights the importance of immunopeptidome-guided tumor antigen discovery. [ABSTRACT FROM AUTHOR]

Both B cells and T cells are involved in an effective immune response to SARS‐CoV‐2, the disease‐causing virus of COVID‐19. While B cells—with the indispensable help of CD4+ T cells—are essential to generate neutralizing antibodies, T cells on their own have been recognized as another major player in effective anti‐SARS‐CoV‐2 immunity. In this report, we provide insights into the characteristics of individual HLA‐A*02:01‐ and HLA‐A*24:02‐restricted SARS‐CoV‐2‐reactive TCRs, isolated from convalescent COVID‐19 patients. We observed that SARS‐CoV‐2‐reactive T‐cell populations were clearly detectable in convalescent samples and that TCRs isolated from these T cell clones were highly functional upon ectopic re‐expression. The SARS‐CoV‐2‐reactive TCRs described in this report mediated potent TCR signaling in reporter assays with low nanomolar EC50 values. We further demonstrate that these SARS‐CoV‐2‐reactive TCRs conferred powerful T‐cell effector function to primary CD8+ T cells as evident by a robust anti‐SARS‐CoV‐2 IFN‐γ response and in vitro cytotoxicity. We also provide an example of a long‐lasting anti‐SARS‐CoV‐2 memory response by reisolation of one of the retrieved TCRs 5 months after initial sampling. Taken together, these findings contribute to a better understanding of anti‐SARS‐CoV‐2 T‐cell immunity and may contribute to paving the way toward immunotherapeutics approaches targeting SARS‐CoV‐2. [ABSTRACT FROM AUTHOR]

Reactivation of Human Cytomegalovirus (HCMV) and Human Adenovirus (HAdV) in immunocompromised patients following stem cell transplantation (SCT) or solid organ transplantation (SOT) is associated with high morbidity and mortality. The adoptive transfer of virus-specific CD8+ and CD4+ T cells has been shown to re-establish the antiviral T-cell response and improve clinical outcome. The viral load in immunocompromised patients can efficiently be reduced solely by the infusion of virus-specific CD4+ T cells. The identification of CD4+ T-cell epitopes has mainly focused on a limited number of viral proteins that were characterized as immunodominant. Here, we used in silico prediction to determine promiscuous CD4+ T-cell epitopes from the entire proteomes of HCMV and HAdV. Immunogenicity testing with enzyme-linked immuno spot (ELISpot) assays and intracellular cytokine staining (ICS) revealed numerous novel CD4+ T-cell epitopes derived from a broad spectrum of viral antigens. We identified 17 novel HCMV-derived and seven novel HAdV-derived CD4+ T-cell epitopes that were recognized by > 50% of the assessed peripheral blood mononuclear cell (PBMC) samples. The newly identified epitopes were pooled with previously published, retested epitopes to stimulate virus-specific memory T cells in PBMCs from numerous randomly selected blood donors. Our peptide pools induced strong IFNγ secretion in 46 out of 48 (HCMV) and 31 out of 31 (HAdV) PBMC cultures. In conclusion, we applied an efficient method to screen large viral proteomes for promiscuous CD4+ T-cell epitopes to improve the detection and isolation of virus-specific T cells in a clinical setting. [ABSTRACT FROM AUTHOR]

T cells keep it up: A major focus of investigation for researches studying severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is how long immunological memory persists after infection. In this study, Bilich et al. specifically explored the kinetics of SARS-CoV-2–specific T cell responses in two cohorts of patients up to 6 months after infection. The authors found that, whereas antibody responses wane, T cell responses to SARS-CoV-2 antigens remain consistent or increase over time. These findings are supported by other studies and suggest that vaccines that elicit T cell responses may be essential for providing long-term protection against SARS-CoV-2 infection. Long-term immunological memory to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for the development of population-level immunity, which is the aim of vaccination approaches. Reports on rapidly decreasing antibody titers have led to questions regarding the efficacy of humoral immunity alone. The relevance of T cell memory after coronavirus disease 2019 (COVID-19) remains unclear. Here, we investigated SARS-CoV-2 antibody and T cell responses in matched samples of COVID-19 convalescent individuals up to 6 months after infection. Longitudinal analysis revealed decreasing and stable spike- and nucleocapsid-specific antibody responses, respectively. In contrast, functional T cell responses remained robust, and even increased, in both frequency and intensity. Single peptide mapping of T cell diversity over time identified open reading frame–independent, dominant T cell epitopes mediating long-term SARS-CoV-2 T cell responses. Identification of these epitopes may be fundamental for COVID-19 vaccine design. [ABSTRACT FROM AUTHOR]

We have previously shown that conformational change in the β2-integrin is a very early activation marker that can be detected with fluorescent multimers of its ligand intercellular adhesion molecule (ICAM)-1 for rapid assessment of antigen-specific CD8+ T cells. In this study, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4+ T cells using a monoclonal antibody (clone m24 Ab) specific for the open, high-affinity conformation of the β2-integrin. The kinetics of β2-integrin activation was different on CD4+ and CD8+ T cells (several hours vs. few minutes, respectively); however, m24 Ab readily stained both cell types 4–6 h after antigen stimulation. With this protocol, we were able to monitor ex vivo effector and memory CD4+ and CD8+ T cells specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), Epstein–Barr virus (EBV), and hepatitis B virus (HBV) in whole blood or cryopreserved peripheral blood mononuclear cells (PBMCs) of infected or vaccinated individuals. By costaining β2-integrin with m24 and CD154 Abs, we assessed extremely low frequencies of polyfunctional CD4+ T cell responses. The novel assay used in this study allows very sensitive and simultaneous screening of both CD4+ and CD8+ T cell reactivities, with versatile applicability in clinical and vaccination studies. [ABSTRACT FROM AUTHOR]

Although the CNS is immune privileged, continuous search for pathogens and tumours by immune cells within the CNS is indispensable. Thus, distinct immune-cell populations also cross the blood-brain barrier independently of inflammation/under homeostatic conditions. It was previously shown that effector memory T cells populate healthy CNS parenchyma in humans and, independently, that CCR5-expressing lymphocytes as well as CCR5 ligands are enriched in the CNS of patients with multiple sclerosis. Apart from the recently described CD8+ CNS tissue-resident memory T cells, we identified a population of CD4+CCR5high effector memory cells as brain parenchyma-surveilling cells. These cells used their high levels of VLA-4 to arrest on scattered VCAM1, their open-conformation LFA-1 to crawl preferentially against the flow in search for sites permissive for extravasation, and their stored granzyme K (GZMK) to induce local ICAM1 aggregation and perform trans-, rather than paracellular diapedesis through unstimulated primary brain microvascular endothelial cells. This study included peripheral blood mononuclear cell samples from 175 healthy donors, 29 patients infected with HIV, with neurological symptoms in terms of cognitive impairment, 73 patients with relapsing-remitting multiple sclerosis in remission, either 1-4 weeks before (n = 29), or 18-60 months after the initiation of natalizumab therapy (n = 44), as well as white matter brain tissue of three patients suffering from epilepsy. We here provide ex vivo evidence that CCR5highGZMK+CD4+ effector memory T cells are involved in CNS immune surveillance during homeostasis, but could also play a role in CNS pathology. Among CD4+ T cells, this subset was found to dominate the CNS of patients without neurological inflammation ex vivo. The reduction in peripheral blood of HIV-positive patients with neurological symptoms correlated to their CD4 count as a measure of disease progression. Their peripheral enrichment in multiple sclerosis patients and specific peripheral entrapment through the CNS infiltration inhibiting drug natalizumab additionally suggests a contribution to CNS autoimmune pathology. Our transcriptome analysis revealed a migratory phenotype sharing many features with tissue-resident memory and Th17.1 cells, most notably the transcription factor eomesodermin. Knowledge on this cell subset should enable future studies to find ways to strengthen the host defence against CNS-resident pathogens and brain tumours or to prevent CNS autoimmunity. [ABSTRACT FROM AUTHOR]

Immediate β2-integrin activation upon T cell receptor stimulation is critical for effective interaction between T cells and their targets and may therefore be used for the rapid identification and isolation of functional T cells. We present a simple and sensitive flow cytometry-based assay to assess antigen-specific T cells using fluorescent intercellular adhesion molecule (ICAM)-1 multimers that specifically bind to activated β2-integrins. The method is compatible with surface and intracellular staining; it is applicable for monitoring of a broad range of virus-, tumor-, and vaccine-specific CD8+ T cells, and for isolating viable antigen-reacting cells. ICAM-1 binding correlates with peptide-MHC multimer binding but, notably, it identifies the fraction of antigen-specific CD8+ T cells with immediate and high functional capability (i.e., expressing high levels of cytotoxic markers and cytokines). Compared with the currently available methods, staining of activated β2-integrins presents the unique advantage of requiring activation times of only several minutes, therefore delivering functional information nearly reflecting the in vivo situation. Hence, the ICAM-1 assay is most suitable for rapid and precise monitoring of functional antigen-specific T cell responses, including for patient samples in a variety of clinical settings, as well as for the isolation of functional T cells for adoptive cell-transfer immunotherapies. [ABSTRACT FROM AUTHOR]

Glioblastoma is the most frequent malignant primary brain tumor. In a hierarchical tumor model, glioblastoma stem-like cells (GSC) play a major role in tumor initiation and maintenance as well as in therapy resistance and recurrence. Thus, targeting this cellular subset may be key to effective immunotherapy. Here, we present a mass spectrometry-based analysis of HLA-presented peptidomes of GSC and glioblastoma patient specimens. Based on the analysis of patient samples (n = 9) and GSC (n = 3), we performed comparative HLA peptidome profiling against a dataset of normal human tissues. Using this immunopeptidome-centric approach we could clearly delineate a subset of naturally presented, GSC-associated HLA ligands, which might serve as highly specific targets for T cell-based immunotherapy. In total, we identified 17 antigens represented by 41 different HLA ligands showing natural and exclusive presentation both on GSC and patient samples. Importantly, in vitro immunogenicity and antigen-specific target cell killing assays suggest these peptides to be epitopes of functional CD8+ T cell responses, thus rendering them prime candidates for antigen-specific immunotherapy of glioblastoma. [ABSTRACT FROM AUTHOR]

Recent studies suggest that CLL is an immunogenic disease, which might be effectively targeted by antigen-specific T-cell-based immunotherapy. However, CLL is associated with a profound immune defect, which might represent a critical limitation for mounting clinically effective antitumor immune responses. As several studies have demonstrated that lenalidomide can reinforce effector T-cell responses in CLL, the combination of T-cell-based immunotherapy with the immunomodulatory drug lenalidomide represents a promising approach to overcome the immunosuppressive state in CLL. Antigen-specific immunotherapy also requires the robust presentation of tumor-associated HLA-presented antigens on target cells. We thus performed a longitudinal study of the effect of lenalidomide on the HLA ligandome of primary CLL cells in vitro. We showed that lenalidomide exposure does not affect absolute HLA class I and II surface expression levels on primary CLL cells. Importantly, semi-quantitative mass spectrometric analyses of the HLA peptidome of three CLL patient samples found only minor qualitative and quantitative effects of lenalidomide on HLA class I- and II-restricted peptide presentation. Furthermore, we confirmed stable presentation of previously described CLL-associated antigens under lenalidomide treatment. Strikingly, among the few HLA ligands showing significant modulation under lenalidomide treatment, we identified upregulated IKZF-derived peptides, which may represent a direct reflection of the cereblon-mediated effect of lenalidomide on CLL cells. Since we could not observe any relevant influence of lenalidomide on the established CLL-associated antigen targets of anticancer T-cell responses, this study validates the suitability of lenalidomide for the combination with antigen-specific T-cell-based immunotherapies. [ABSTRACT FROM AUTHOR]

Graft-versus-host disease (GVHD) represents the major nonrelapse complication of allogeneic hematopoietic cell transplantation. Although rare, the CNS and the eye can be affected. In this study, manifestation in the retina as part of the CNS and T-cell epitopes recognized by the allogeneic T cells were evaluated. In 2 of 6 patients with posttransplantation retina diseases and 6 of 22 patients without ocular symptoms, antigen-specific T-cell responses against retinaspecific epitopes were observed. No genetic differences between donor and recipient could be identified indicating T-cell activation against self-antigens (graft versus self). Transplantation of a preexisting immunity and cross-reactivity with ubiquitous epitopes was excluded in family donors and healthy individuals. In summary, an immunological reaction against retina cells represents a mechanism of graft-versus-host interaction following hematopoietic cell transplantation. [ABSTRACT FROM AUTHOR]

There are limitations in pre-clinical settings using mice as a basis for clinical development in humans. In cancer, similarities exist between humans and dogs; thus, the dog patient can be a link in the transition from laboratory research on mouse models to clinical trials in humans. Knowledge of the peptides presented on MHC molecules is fundamental for the development of highly specific T cell-based immunotherapies. This information is available for human MHC molecules but is absent for the canine MHC. In the present study, we characterized the binding motif of dog leukocyte antigen (DLA) class I allele DLA-88*50101, using human C1R and K562 transfected cells expressing the DLA-88*50101 heavy chain. MHC class I immunoaffinity-purification revealed 3720 DLA-88*50101 derived peptides, which enabled the determination of major anchor positions. The characterized binding motif of DLA-88*50101 was similar to HLA-A*02:01. Peptide binding analyses on HLA-A*02:01 and DLA-88*50101 via flow cytometry showed weak binding of DLA-88*50101 derived peptides to HLA-A*02:01, and vice versa. Our results present for the first time a detailed peptide binding motif of the canine MHC class I allelic product DLA-88*50101. These data support the goal of establishing dogs as a suitable animal model for the evaluation and development of T cell-based cancer immunotherapies, benefiting both dog and human patients. [ABSTRACT FROM AUTHOR]

Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms’ Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8–1.4 x 106). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1126-134 (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1950-958 epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients. [ABSTRACT FROM AUTHOR]

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. It is therefore operator dependent and subjective. In the context of continuing efforts to support inter-laboratory T cell assay harmonization, the CIMT Immunoguiding Program organized its third proficiency panel dedicated to the detection of antigen-specific CD8 T cells by HLA-peptide multimer staining. We first assessed the contribution of manual data analysis to the variability of reported T cell frequencies within a group of laboratories staining and analyzing the same cell samples with their own reagents and protocols. The results show that data analysis is a source of variation in the multimer assay outcome. To evaluate whether an automated analysis approach can reduce variability of proficiency panel data, we used a hierarchical statistical mixture model to identify cell clusters. Challenges for automated analysis were the need to process non-standardized data sets from multiple centers, and the fact that the antigen-specific cell frequencies were very low in most samples. We show that this automated method can circumvent difficulties inherent to manual gating strategies and is broadly applicable for experiments performed with heterogeneous protocols and reagents. [ABSTRACT FROM AUTHOR]

T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific 64Cu-monoclonal antibody (mAb)-TCR complex enables a stable labeling of T cells. The TCR-mAb complex was internalized within 24 h, whereas antigen recognition was not impaired. Harmful effects of the label on the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images.We were able tofollow and quantify the specific homing of systemically applied 64Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayed-type hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of naïve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover. [ABSTRACT FROM AUTHOR]

The lifelong generation of αβT cells enables us to continuously build immunity against pathogens and malignancies despite the loss of thymic function with age. Homeostatic proliferation of post-thymic naïve and memory T cells and their transition into effector and long-lived memory cells balance the decreasing output of naïveT cells, and recent research suggests that also αβT-cell development independent from the thymus may occur. However, the sites and mechanisms of extrathymic T-cell development are not yet understood in detail. γδT cells represent a small fraction of the overall T-cell pool, and are endowed with tremendous phenotypic and functional plasticity. γδT cells that express the Vδ1 gene segment are a minor population in human peripheral blood but predominate in epithelial (and inflamed) tissues. Here, we characterize a CD4+ peripheral Vδ1+ γδT-cell subpopulation that expresses stem-cell and progenitor markers and is able to develop into functional abT cells ex vivo in a simple culture system and in vivo. The route taken by this process resembles thymic T-cell development. However, it involves the re-organization of the Vδ1+ γδTCR into the abTCR as a consequence ofTCR-γ chain downregulation and the expression of surfaceVδ1+Vβ+TCR components, whichwe believe function as surrogate pre-TCR.This transdifferentiation process is readily detectable in vivo in inflamed tissue. Our study provides a conceptual framework for extrathymicT-cell development and opens up a new vista in immunology that requires adaptive immune responses in infection, autoimmunity, and cancer to be reconsidered. [ABSTRACT FROM AUTHOR]

Every cancer is different and cancer cells differ from normal cells, in particular, through genetic alterations. HLA molecules on the cell surface enable T lymphocytes to recognize cellular alterations as antigens, including mutations, increase in gene product copy numbers or expression of genes usually not used in the adult organism. The search for cancer-associated antigens shared by many patients with a particular cancer has yielded a number of hits used in clinical vaccination trials with indication of survival benefit. Targeting cancer-specific antigens, which are exclusively expressed on cancer cells and not on normal cells, holds the promise for much better results and perhaps even a cure. Such antigens, however, may specifically appear in very few patients or may be mutated appearing just in one patient. Therefore, to target these in a molecularly defined way, the approach has to be individualized. [ABSTRACT FROM AUTHOR]

The HLA-DR15 haplotype confers the largest part of the genetic risk to develop multiple sclerosis, a prototypic CD4+ T cell-mediated autoimmune disease. The mechanisms how certain HLA-class II molecules functionally contribute to autoimmune diseases are still poorly understood, but probably involve shaping an autoimmune-prone T cell repertoire during central tolerance in the thymus and subsequently maintaining or even expanding it in the peripheral immune system. Self-peptides that are presented by disease-associated HLA-class II molecules most likely play important roles during both processes. Here, we examined the functional involvement of the HLA-DR15 haplotype in autologous proliferation in multiple sclerosis and the contribution of HLA-DR15 haplotype-derived self-peptides in an in vitro system. We observe increased autologous T cell proliferation in patients with multiple sclerosis in relation to the multiple sclerosis risk-associated HLA-DR15 haplotype. Assuming that the spectrum of self-peptides that is presented by the two HLA-DR15 allelic products is important for sustaining autologous proliferation we performed peptide elution and identification experiments from the multiple sclerosis-associated DR15 molecules and a systematic analysis of a DR15 haplotype-derived self-peptide library. We identify HLA-derived self-peptides as potential mediators of altered autologous proliferation. Our data provide novel insights about perturbed T cell repertoire dynamics and the functional involvement of the major genetic risk factor, the HLA-DR15 haplotype, in multiple sclerosis. [ABSTRACT FROM AUTHOR]

Adenoviral infections are a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric patients. Adoptive transfer of donor-derived human adenovirus (HAdV)-specific T-cells represents a promising treatment option. However, the difficulty in identifying and selecting rare HAdV-specific T-cells, and the short time span between patients at high risk for invasive infection and viremia are major limitations. We therefore developed an IL-15-driven 6 to 12 day short-term protocol for in vitro detection of HAdV-specific T cells, as revealed by known MHC class I multimers and a newly identified adenoviral CD8 T-cell epitope derived from the E1A protein for the frequent HLA-type A*02∶01 and IFN-γ. Using this novel and improved diagnostic approach we observed a correlation between adenoviral load and reconstitution of CD8+ and CD4+ HAdV-specific T-cells including central memory cells in HSCT-patients. Adaption of the 12-day protocol to good manufacturing practice conditions resulted in a 2.6-log (mean) expansion of HAdV-specific T-cells displaying high cytolytic activity (4-fold) compared to controls and low or absent alloreactivity. Similar protocols successfully identified and rapidly expanded CMV-, EBV-, and BKV-specific T-cells. Our approach provides a powerful clinical-grade convertible tool for rapid and cost-effective detection and enrichment of multiple virus-specific T-cells that may facilitate broad clinical application. [ABSTRACT FROM AUTHOR]

Peptides presented at the cell surface reflect the protein content of the cell; those on HLA class I molecules comprise the critical peptidome elements interacting with CD8 T lymphocytes. We hypothesize that peptidomes from ex vivo tumour samples encompass immunogenic tumour antigens. Here, we uncover >6000 HLA-bound peptides from HLA-A*02+ glioblastoma, of which over 3000 were restricted by HLA-A*02. We prioritized in-depth investigation of 10 glioblastoma-associated antigens based on high expression in tumours, very low or absent expression in healthy tissues, implication in gliomagenesis and immunogenicity. Patients with glioblastoma showed no T cell tolerance to these peptides. Moreover, we demonstrated specific lysis of tumour cells by patients’ CD8+ T cells in vitro. In vivo, glioblastoma-specific CD8+ T cells were present at the tumour site. Overall, our data show the physiological relevance of the peptidome approach and provide a critical advance for designing a rational glioblastoma immunotherapy. The peptides identified in our study are currently being tested as a multipeptide vaccine (IMA950) in patients with glioblastoma. [ABSTRACT FROM AUTHOR]

The article presents research on the tumor antigen-specific T cells and antibodies of cancer patients treated with radiofrequency (RF) ablation. RF ablation is a technique used for the treatment of primary and secondary liver tumors by inducing cell death through thermal coagulative necrosis. An increase in heat shock protein is observed after the RF treatment suggesting the stimulation of the host immune system. It concludes that RF ablation can provide conditions for activating tumor-antigen specific immune responses.

Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme capable of inhibiting a destructive maternal T cell response against allogeneic fetuses. Expression of IDO is evident in tumours and is thought to enable escape from immunologically mediated rejection. Consequently, clinical trials using an inhibitor of IDO, 1-methyltryptophan (1MT), have been initiated. However, a review of the current literature indicates that we are far from understanding the biological relevance of IDO expression during tumorigenesis. A better understanding of IDO biology is needed to comprehend the effect of IDO inhibitors and to provide a rationale for their therapeutic application in cancer. [ABSTRACT FROM AUTHOR]

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. However, synthetic artificial APCs which can be loaded reproducibly with MHC-peptide monomers and antibodies specific for costimulatory molecules could resolve these problems. The purpose of this study was to investigate the potential of complex synthetic artificial APCs in triggering the costimulatory molecules CD28 and 4-1BB on the T cell. Anti-4-1BB antibodies were added to an established system of microbeads coated with MHC-peptide monomers and anti-CD28. Triggering via CD28 and 4-1BB resulted in strong costimulatory synergy. The quantitative ratio between these signals determined the outcome of the stimulation with optimal results when anti-4-1BB and anti-CD28 were applied in a 3:1 ratio. Functional CTLs of an effector memory subtype (CD45RA− CCR7−) were generated in high numbers. We present a highly defined APC platform using off-the-shelf reagents for the convenient generation of large numbers of antigen-specific CTLs. [ABSTRACT FROM AUTHOR]

Glucocorticoid-induced TNF-related protein (GITR) has been shown to stimulate T cell-mediated antitumor immunity in mice. However, the functional relevance of GITR and its ligand (GITRL) for non-T cells has yet to be fully explored. In addition, recent evidence suggests that GITR plays different roles in mice and humans. We studied the role of GITR-GTRL interaction in human tumor immunology and report for the first time that primary gastrointestinal cancers and tumor cell lines of different histological origin express substantial levels of GITRL. Signaling through GITRL down-regulated the expression of the immunostimulatory molecules CD40 and CD54 and the adhesion molecule EpCAM, and induced production of the immunosuppressive cytokine TGF-β by tumor cells. On NK cells, GITR is constitutively expressed and up-regulated following activation. Blocking GITR-GITRL interaction in cocultures of tumor cells and NK cells substantially increased cytotoxicity and IFN-γ production of NK cells demonstrating that constitutive expression of GITRL by tumor cells diminishes NK cell antitumor immunity. GITRD-Ig fusion protein or cell surface-expressed GITRL did not induce apoptosis in NK cells, but diminished nuclear localized c-Rel and RelB, indicating that GITR might negatively modulate NK cell NF-κB activity. Taken together, our data indicate that tumor-expressed GITRL mediates immunosubversion in humans. [ABSTRACT FROM AUTHOR]

Differences in the cleavage specificities of constitutive proteasomes and immunoproteasomes significantly affect the generation of MHC class I ligands and therefore the activation of CD8-positive T cells. Based on these findings, we investigated whether proteasomal specificity also influences CD8-positive T cells during thymic selection by peptides derived from self proteins. We find that one of the self peptides responsible for positive selection of ovalbumin-specific OT-1 T cells, which is derived from the f-actin capping protein (Cpα1), is efficiently generated only by immunoproteasomes. Furthermore. OT-1 mice backcrossed onto low molecular mass protein 7 (LMP7)-deficient mice show a 50% reduction of OT-1 cells. This deficiency is also observed after transfer of BM from OT-1 mice in LMP7-deficient mice and can be corrected by the injection of the Cpα1 peptide. Interestingly, WT and LMP7-deficient mice mount comparable immune responses to the ovalbumin-derived epitope SIINFEKL. However, their cytotoxic T lymphocytes (CTL) differ in the use of T cell receptor Vβ genes. CTL derived from WT mice use Vβ8 or VβS (the latter is also used by OT-1 cells), whereas SIINFEKL-specific CTL from LMP7-deficient mice are exclusively Vβ8-positive. Taken together, our experiments provide strong evidence that proteasomal specificity shapes the repertoire of I cells participating in antigen-specific immune responses. [ABSTRACT FROM AUTHOR]

The single nucleotide polymorphism (SNP)–derived Epitope Prediction program (SNEP) is now available to the public. It predicts minor histocompatibility antigens (miHAgs), which are T-cell epitopes containing polymorphic spots, from proteins listed in the SWISS-PROT database. SNEP recognizes polymorphisms (termed VARIANT or CONFLICT in SWISS-PROT) and predicts potential T-cell epitopes within a chosen distance around the polymorphic residue. The prediction algorithm is based on the SYFPEITHI T-cell epitope prediction program. SNEP is able to search for proteins according to their accession numbers, sequence stretches or gene names, for example. The predictions are available for several human leucocyte antigen class I and class II allelic products, which allow for a rapid and precise evaluation of potential miHAgs within polymorphic antigens. [ABSTRACT FROM AUTHOR]

The tumor-associated antigens CEA, MUC1 and Her2/neu are broadly expressed in gastrointestinal tumors, and are attractive candidates for targeting by T-cell-based immunotherapy. However, little is known about the natural cytotoxic T-cell response of patients suffering from colorectal or gastric carcinoma against these three as well as other antigens. Using a quantitative reverse transcription-polymerase chain reaction-based assay for IFN-γ, we analyzed the CD8+ T-cell repertoire present in the blood of HLA-A2+ gastrointestinal tumor survivors against five known epitopes derived from CEA, MUC1 and Her2/neu. The results show that most of the patients (16 from 22 tested) have detectable, peripheral CD8+ T cells directed against at least one of these three proteins. Interestingly, the majority of these patients reacts to the two MUC1-derived HLA-A*0201 epitopes tested (14 from 16), demonstrating that this protein represents one dominant target for CD8+ T cells in gastrointestinal cancer. [ABSTRACT FROM AUTHOR]

MHC-peptide complexes mediate key functions in adaptive immunity. In a classical view, MHC-I molecules present peptides from intracellular source proteins, whereas MHC-II molecules present antigenic peptides from exogenous and membrane proteins. Nevertheless, substantial crosstalk between these two pathways has been observed. We investigated the influence of autophagy on the MHC-II ligandome and demonstrated that peptide presentation is altered considerably upon induction of autophagy. The presentation of peptides from intracellular and lysosomal source proteins was strongly increased on MHC-II in contrast with peptides from membrane and secreted proteins. In addition, autophagy influenced the MHC-II antigen-processing machinery. Our study illustrates a profound influence of autophagy on the class II peptide repertoire and suggests that this finding has implications for the regulation of CD4+ T cell-mediated processes. [ABSTRACT FROM AUTHOR]

NY-ESO-1 is a SEREX-defined cancer-testis antigen of which several MHC I, but only few MHC II–restricted epitopes have been identified. Searching for highly promiscuous MHC II–restricted peptides that might be suitable as a CD4+ stimulating vaccine for many patients, we used the SYFPEITHI algorithm and identified an NY-ESO-1–derived pentadecamer epitope (p134–148) that induced specific CD4+ T-cell responses restricted to the HLA-DRB1 subtypes *0101, *0301, *0401, and *0701 that have a cumulative prevalence of 40% in the Caucasian population. The DR restriction of the p134–148 pentadecamer was demonstrated by inhibition with an HLA-DR antibody and a functional peptide displacement titration assay with the pan-DR-binding T-helper epitope PADRE as the competitor. The natural processing and presentation of this epitope was demonstrated by recognition of an NY-ESO-1+ melanoma cell line by T cells with specificity for p134–148. The pentadecamer p134–148 was able to induce CD4+ responses in 4/38 cancer patients tested. However, no strict correlation was found between CD4+ T-cell responses against p134–148 reactivity and anti-NY-ESO-1 antibody titers in the serum of patients, suggesting that CD4+ and B-cell responses are regulated independently. In conclusion, p134–148 holds promise as a broadly applicable peptide vaccine for patients with NY-ESO-1–positive neoplasms. [ABSTRACT FROM AUTHOR]

Despite worldwide eradication of naturally occurring variola virus, smallpox remains a potential threat to both civilian and military populations. New, safe smallpox vaccines are being developed, and there is an urgent need for methods to evaluate vaccine efficacy after immunization. Here we report the identification of an immunodominant HLA-A[sup *]0201-restricted epitope that is recognized by cytotoxic CD8[sup +] T cells and conserved among Orthopoxvirus species including variola virus. This finding has permitted analysis and monitoring of epitope-specific T cell responses after immunization and demonstration of the identified T cell specificity in an A[sup *]0201-positive human donor. Vaccination of transgenic mice allowed us to compare the immunogenicity of several vaccinia viruses including highly attenuated, replication-deficient modified vaccinia virus Ankara (MVA). MVA vaccines elicited levels of CD8[sup +] T cell responses that were comparable to those induced by the replication-competent vaccinia virus strains. Finally, we demonstrate that MVA vaccination is fully protective against a lethal respiratory challenge with virulent vaccinia virus strain Western Reserve. Our data provide a basis to rationally estimate immunogenicity of safe, second-generation poxvirus vaccines and suggest that MVA may be a suitable candidate. [ABSTRACT FROM AUTHOR]

Adoptive transfer of donor-derived human cytomegalovirus (HCMV)-specific T-cell clones can restore protective immunity after stem cell transplantation. Ex vivo induction of HCMV-specific T cells using HCMV-infected fibroblasts as stimulator cells confines this approach to HCMV-seropositive donors and requires the presence of infectious virus during the stimulation procedure. In this study, we describe a potential alternative strategy to generate HCMV-specific T cells ex vivo for adoptive immunotherapy. Generation of HCMV-specific cytotoxic T lymphocytes (CTLs) ex vivo was investigated using peptide-pulsed dendritic cells as antigen-presenting cells. HCMV-specific T cells were generated and sufficiently expanded for adoptive immunotherapy in 6 out of 14 HCMV-seropositive and 2 out of 11 HCMV-seronegative donors. The CTLs recognized HCMV-infected autologous fibroblasts. No lysis was observed with either non-infected autologous or HLA-mismatched infected fibroblasts. Staining with tetrameric HLA/peptide complexes revealed significant enrichment for peptide-specific T cells of up to 28% and > 90% of CD8+ T cells after three and five specific stimulations respectively. In addition, the expansion rates indicated that ex vivo generation of > 1 × 109 HCMV-specific T cells was possible after 6–7 weeks when cultures were initiated with 1–5 × 106 responder cells. Thus, the approach with peptide-pulsed DCs to generate HCMV-specific CTLs is feasible for clinical application after allogeneic stem cell transplantation. [ABSTRACT FROM AUTHOR]

The first version of PAProC (Prediction Algorithm for Proteasomal Cleavages) is now available to the general public. PAProC is a prediction tool for cleavages by human and yeast proteasomes, based on experimental cleavage data. It will be particularly useful for immunologists working on antigen processing and the prediction of major histocompatibility complex class I molecule (MHC I) ligands and cytotoxic T-lymphocyte (CTL) epitopes. Likewise, in cases in which proteasomal protein degradation has been indicated in disease, PAProC can be used to assess the general cleavability of disease-linked proteins. On its web site (http://www.paproc.de), background information and hyperlinks are provided for the user (e.g., to SYFPEITHI, the database for the prediction of MHC I ligands). [ABSTRACT FROM AUTHOR]

We describe the results of two vaccinations of a self-experimenting healthy volunteer with SARS-CoV-2-derived peptides performed in March and April 2020, respectively. The first set of peptides contained eight peptides predicted to bind to the individual's HLA molecules. The second set consisted of ten peptides predicted to bind promiscuously to several HLA-DR allotypes. The vaccine formulation contained the new TLR 1/2 agonist XS15 and was administered as an emulsion in Montanide as a single subcutaneous injection. Peripheral blood mononuclear cells isolated from blood drawn before and after vaccinations were assessed using Interferon-γ ELISpot assays and intracellular cytokine staining. We detected vaccine-induced CD4 T cell responses against six out of 11 peptides predicted to bind to HLA-DR after 19 days, following vaccination, for one peptide already at day 12. We used these results to support the design of a T-cell-inducing vaccine for application in high-risk patients, with weakened lymphocyte performance. Meanwhile, an according vaccine, incorporating T cell epitopes predominant in convalescents, is undergoing clinical trial testing. [ABSTRACT FROM AUTHOR]

• Single-dose CoVac-1 induces long-lasting T cell responses in 100% of participants. • Long-term follow-up shows good tolerability and no immune-related side effects. • Magnitude of T cell responses correlates with severity of injection site reactions. • Most CoVac-1-induced peptide-specific immunoglobulin G antibodies peaked at month 3. • Very low SARS-CoV-2 infection rate (8.3 %) in the study population. T cell immunity is key for the control of viral infections including SARS-CoV-2, in particular with regard to immune memory and protection against arising genetic variants. We recently evaluated a peptide-based SARS-CoV-2 T cell activator termed CoVac-1 in a first-in-human trial in healthy adults. Here, we report on long-term safety and efficacy data of CoVac-1 until month 12. CoVac-1 is well tolerated without long-term immune-related side effects and induces long-lasting anti-viral T cell responses in 100% of study participants, with potent expandability of clusters of differentiation (CD4+) and CD8+ T cells targeting multiple different CoVac-1 T cell epitopes. T cell responses were associated with stronger injection site reaction. Beyond induction of T cell immunity, 89% of subjects developed CoVac-1-specific immunoglobulin G antibodies which associated with the intensity of the T cell response, indicating that CoVac-1-specific CD4+ T cells support the induction of B-cell responses. Vaccination with approved COVID-19 vaccines boosted CoVac-1-specific T cell responses. Overall, a low SARS-CoV-2 infection rate (8.3%) was observed. Together, a single application of CoVac-1 elicits long-lived and broad SARS-CoV-2-specific T cell immunity, which further supports the current evaluation of our T cell activator in patients with congenital or acquired B-cell defects. [ABSTRACT FROM AUTHOR]

Combination of radiotherapy with immunotherapy has become an attractive concept for the treatment of cancer. The objective of this study was to assess the effect of curative, normofractionated radiotherapy on peripheral immune lymphocytes in prostate cancer patients, in order to propose a rationale for scheduling of normofractionated radiotherapy with T-cell based immunotherapy. In a prospective study (clinicaltrials.gov: NCT01376674), eighteen patients with localized prostate cancer were treated with radiotherapy with or without hormonal therapy. Irradiation volumes encompassed prostate and, in select cases, elective pelvic nodal regions. Blood samples were collected from all patients before, during, and after radiotherapy, as well as from 6 healthy individuals as control. Normofractionated radiotherapy of prostate cancer over eight weeks had a significant influence on the systemic immune status of patients compared to healthy controls. Absolute leukocyte and lymphocyte counts decreased during treatment as did peripheral blood immune subsets (T cells, CD8+ and naïve CD4+ T cells, B cells). Regulatory T cells and NK cells increased. Proliferation of all immune cells except regulatory T cells increased during RT. Most of these changes were transient. Importantly, the functionality of T lymphocytes and the frequency of antigen-specific CD8+ T cells were not affected during therapy. Our data indicate that combination of normofractionated radiotherapy with immunotherapy might be feasible for patients with prostate cancer. Conceptually, beginning with immunotherapy early during the course of radiotherapy could be beneficial, as the percentage of T cells is highest, the percentage of regulatory T cells is lowest, and as the effects of radiotherapy did not completely subside 3 months after end of radiotherapy. [ABSTRACT FROM AUTHOR]

Despite recent advances in immunotherapy of renal cell carcinoma (RCC), peptide vaccination strategies still lack an approach for personalized peptide vaccination that takes intra- and inter-tumoral heterogeneity and biological characteristics into account. In this study, we use an immunoprecipitation and mass spectrometry-based approach supplemented by network analysis of HLA ligands to target this goal. By analyzing HLA-presented peptides for HLA class I and II of 11 malignant and 6 non-malignant kidney tissue samples, more than 2,700 peptides and 1,600 corresponding source proteins were identified. A high overlap with HLA ligands derived from peripheral blood mononuclear cells (PBMCs) was detected most likely due to tumor-infiltrating inflammatory cells and therefore excluded from network analysis. Subsequent biological function analysis of HLA ligands by the GeneMANIA online platform showed enrichment for well established, but also novel, pathways and biological processes involved in carcinogenesis of RCC almost exclusively in malignant tissue samples. By exploring source proteins involved in these overrepresented pathways, we verified various known tumor-associated antigens (TAAs) for RCC (e.g., CA9, EGLN3, IGFBP3, MMP7, PAX2, VEGFA, or EGFR) but could also detect novel TAAs for RCC (e.g., PLOD2, LOX, ENPEP, or TGFBI). Some of these new TAAs had already been shown to elicit a T cell response in cancer patients. Thus, network analysis of HLA ligands provided a new platform for implementing personalized, multipeptide vaccines with potentially synergistic antitumor effects. [ABSTRACT FROM PUBLISHER]

Effective antigen-specific cancer immunotherapy requires exact knowledge of tumor-associated epitopes that can act as rejection antigens. Although the current paradigm views mutation-derived neoantigens as the most promising targets, we have recently demonstrated that leukemia-specific T-cell responses associated with survival benefit in CLL patients target a panel of non-mutated tumor-associated antigens. [ABSTRACT FROM PUBLISHER]

Summary Skin is constantly exposed to bacteria and antigens, and cutaneous innate immune sensing orchestrates adaptive immune responses. In its absence, skin pathogens can expand, entering deeper tissues and leading to life-threatening infectious diseases. To characterize skin-driven immunity better, we applied living bacteria, defined lipopeptides, and antigens cutaneously. We found suppression of immune responses due to cutaneous infection with Gram-positive S. aureus , which was based on bacterial lipopeptides. Skin exposure to Toll-like receptor (TLR)2-6-binding lipopeptides, but not TLR2-1-binding lipopeptides, potently suppressed immune responses through induction of Gr1 + CD11b + myeloid-derived suppressor cells (MDSCs). Investigating human atopic dermatitis, in which Gram-positive bacteria accumulate, we detected high MDSC amounts in blood and skin. TLR2 activation in skin resident cells triggered interleukin-6 (IL-6), which induced suppressive MDSCs, which are then recruited to the skin suppressing T cell-mediated recall responses such as dermatitis. Thus, cutaneous bacteria can negatively regulate skin-driven immune responses by inducing MDSCs via TLR2-6 activation. [ABSTRACT FROM AUTHOR]

Abstract: The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release assay (CRA). This end point assay, however, shows several disadvantages including the inevitable use of radioactivity. Based on electrical impedance measurements (using the xCELLigence system), we have established a label-free assay, facilitating the real-time monitoring of T-cell-mediated cytotoxicity. The coculture of peptide-specific T-cell lines with peptide-loaded target cells reproducibly led to a decrease in impedance due to induced apoptosis and detachment of target cells. Comparing our results to the standard CRA assay, we could demonstrate that impedance-based measurements show comparable results after short incubation periods (6h) but outperform the CRA both in reproducibility and sensitivity after prolonged incubation (24h), enabling the detection of target cell lysis with an effector to target ratio as low as 0.05:1. The impedance-based assay represents a valuable and highly sensitive tool for label-free real-time high throughput analysis of T-cell-mediated cytotoxicity. [Copyright &y& Elsevier]

Abstract: The etiology of sarcoidosis remains unknown. Recently, by mass spectrometric sequencing of peptides eluted from HLA-DR molecules of bronchoalveolar lavage (BAL) cells from DRB1⁎0301pos patients, we identified potential self-antigens in sarcoidosis. The aim of the present study was to investigate the capacity of selected peptides to stimulate lung and blood T cells of sarcoidosis patients using an interferon-γ ELISPOT assay. In peripheral blood, there were strong T cell responses to a peptide derived from the cytoskeletal protein vimentin in 6 out of 11 DRB1⁎0301pos patients with active disease but not in patients with other HLA types. BAL T cell responses against peptides derived from ATP synthase or from lysyl-tRNA synthetase were detected in DRB1⁎0301pos as well as DRB1⁎0301neg patients. By using antigenic peptides presented in vivo in the lungs of sarcoidosis patients, we have identified blood and lung T cell autoimmune responses that may help sustain the inflammation in this disease. [Copyright &y& Elsevier]

Vitiligo is a skin and hair disorder characterized by circumscribed depigmented lesions due to lack of melanocytes in the respective areas. It has been suggested that vitiligo is caused by an autoimmune-mediated destruction of melanocytes. Recently, the presence of a high frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in the peripheral blood of patients with vitiligo was reported. Our study examines the frequency of melanocyte-specific cytotoxic T lymphocytes in vitiligo patients and its relationship to disease activity. Thirty-two patients with moderate to active vitiligo and 17 control subjects were included. Melanocyte specific reactive CD8+ T cells were identified by enzyme-linked immunospot assay after stimulation with five peptides from gp100, four peptides from MelanA/MART1, and two peptides from tyrosinase. In selected patients, intracellular interferon-γ staining for the detection of specific reactive CD8+ T cells was additionally performed. In seven of 10 patients (70%) with actively progressive disease CD8+ T cells directed against melanocyte epitopes were detected, whereas only in four of 22 patients (18%) with moderate disease activity such specific reactivity was found. MelanA/MART1 peptides were immunodominant in nine patients reacting against EAAGIGILTV and three patients reacting against ILTVILGVL. Intracellular interferon-γ staining confirmed the findings obtained by the enzyme-linked immunospot technique. The present study supports the hypothesis that vitiligo is a cytotoxic T lymphocyte-mediated autoimmune disease. The presence of melanocyte-specific reactive CD8+ T cells seems to be closely related to disease activity. [ABSTRACT FROM AUTHOR]