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Pluripotent stem cells are promising candidates for cell-based regenerative therapies. To avoid rejection of transplanted cells, several approaches are being pursued to reduce immunogenicity of the cells or modulate the recipient's immune response. These include gene editing to reduce the antigenicity of cell products, immunosuppression of the host, or using major histocompatibility complex-matched cells from cell banks. In this context, we have investigated the antigenicity of H-Y antigens, a class of minor histocompatibility antigens encoded by the Y chromosome, to assess whether the gender of the donor affects the cell's antigenicity. In a murine transplant model, we show that the H-Y antigen in undifferentiated embryonic stem cells (ESCs), as well as ESC-derived endothelial cells, provokes T- and B cell responses in female recipients.

Zona pellucida 3 (ZP3) expression is classically found in the ZP‐layer of the oocytes, lately shown in ovarian and prostate cancer. A successful ZP3 ovarian cancer immunotherapy in transgenic mice suggested its use as an attractive therapeutic target. The biological role of ZP3 in cancer growth and progression is still unknown. We found that ~88% of the analyzed adenocarcinoma, squamous and small cell lung carcinomas to express ZP3. Knockout of ZP3 in a ZP3‐expressing lung adenocarcinoma cell line, significantly decreased cell viability, proliferation, and migration rates in vitro. Zona pellucida 3 knock out (ZP3‐KO) cell tumors inoculated in vivo in immunodeficient non‐obese diabetic, severe combined immunodeficient mice showed significant inhibition of tumor growth and mitigation of the malignant phenotype. RNA sequencing revealed the deregulation of cell migration/adhesion signaling pathways in ZP3‐KO cells. This novel functional relevance of ZP3 in lung cancer emphasized the suitability of ZP3 as a target in cancer immunotherapy and as a potential cancer biomarker. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the rapidly unfolding coronavirus disease 2019 (COVID-19) pandemic.sup.1,2. Clinical manifestations of COVID-19 vary, ranging from asymptomatic infection to respiratory failure. The mechanisms that determine such variable outcomes remain unresolved. Here we investigated CD4.sup.+ T cells that are reactive against the spike glycoprotein of SARS-CoV-2 in the peripheral blood of patients with COVID-19 and SARS-CoV-2-unexposed healthy donors. We detected spike-reactive CD4.sup.+ T cells not only in 83% of patients with COVID-19 but also in 35% of healthy donors. Spike-reactive CD4.sup.+ T cells in healthy donors were primarily active against C-terminal epitopes in the spike protein, which show a higher homology to spike glycoproteins of human endemic coronaviruses, compared with N-terminal epitopes. Spike-protein-reactive T cell lines generated from SARS-CoV-2-naive healthy donors responded similarly to the C-terminal region of the spike proteins of the human endemic coronaviruses 229E and OC43, as well as that of SARS-CoV-2. This results indicate that spike-protein cross-reactive T cells are present, which were probably generated during previous encounters with endemic coronaviruses. The effect of pre-existing SARS-CoV-2 cross-reactive T cells on clinical outcomes remains to be determined in larger cohorts. However, the presence of spike-protein cross-reactive T cells in a considerable fraction of the general population may affect the dynamics of the current pandemic, and has important implications for the design and analysis of upcoming trials investigating COVID-19 vaccines. A study of patients with COVID-19 and healthy donors found CD4.sup.+ T cells that react to the spike protein of SARS-CoV-2 and human endemic coronaviruses; however, the effect of pre-existing SARS-CoV-2 cross-reactive T cells on clinical outcomes remains to be determined., Author(s): Julian Braun [sup.1] [sup.2] , Lucie Loyal [sup.1] [sup.2] , Marco Frentsch [sup.3] , Daniel Wendisch [sup.4] , Philipp Georg [sup.5] , Florian Kurth [sup.4] [sup.5] [sup.6] , Stefan [...]

Despite extensive research on severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) vaccination responses in healthy individuals, there is comparatively little known beyond antibody titers and T‐cell responses in the vulnerable cohort of patients after allogeneic hematopoietic stem cell transplantation (ASCT). In this study, we assessed the serological response and performed longitudinal multimodal analyses including T‐cell functionality and single‐cell RNA sequencing combined with T cell receptor (TCR)/B cell receptor (BCR) profiling in the context of BNT162b2 vaccination in ASCT patients. In addition, these data were compared to publicly available data sets of healthy vaccinees. Protective antibody titers were achieved in 40% of patients. We identified a distorted B‐ and T‐cell distribution, a reduced TCR diversity, and increased levels of exhaustion marker expression as possible causes for the poorer vaccine response rates in ASCT patients. Immunoglobulin heavy chain gene rearrangement after vaccination proved to be highly variable in ASCT patients. Changes in TCRα and TCRβ gene rearrangement after vaccination differed from patterns observed in healthy vaccinees. Crucially, ASCT patients elicited comparable proportions of SARS‐CoV‐2 vaccine‐induced (VI) CD8+ T‐cells, characterized by a distinct gene expression pattern that is associated with SARS‐CoV‐2 specificity in healthy individuals. Our study underlines the impaired immune system and thus the lower vaccine response rates in ASCT patients. However, since protective vaccine responses and VI CD8+ T‐cells can be induced in part of ASCT patients, our data advocate early posttransplant vaccination due to the high risk of infection in this vulnerable group. [ABSTRACT FROM AUTHOR]

The direct assessment of T helper (T[sub.H])-cell responses specific for antigens is essential to evaluate pathogenic and protective immunity. Presently, analysis and isolation of antigen-specific T[sub.H] cells is restricted to cells that produce cytokines, or can be performed only with a rare selection of specific peptide major histocompatibility complex class II (MHC II) multimers. Here we report a new method that enables the assessment and isolation of T[sub.H] cells specific for a defined antigen according to CD154 expression induced after stimulation in vitro. We show that antigen-induced CD154 expression is highly sensitive and specific for human and mouse antigen-specific T[sub.H] cells. Moreover, the isolation of antigen-specific CD154[sup.+] T[sub.H] cells necessitates only surface staining with antibodies, thereby enabling the fast generation of antigen-specific T[sub.H] cell lines. Our approach allows assessment of T[sub.H] cells with a defined specificity for the combined quantitative and qualitative analysis of T[sub.H]-cell immunity as well as for the isolation of specific T[sub.H] cells for targeted cellular immunotherapies., Author(s): Marco Frentsch [1]; Olga Arbach [1]; Dennis Kirchhoff [2]; Beate Moewes [1]; Margitta Worm [3]; Martin Rothe [1, 3]; Alexander Scheffold [2]; Andreas Thiel [1] Different types of T[sub.H] [...]

A great part of our knowledge on mammalian immunology has been established in laboratory settings. The use of inbred mouse strains enabled controlled studies of immune cell and molecule functions in defined settings. These studies were usually performed in specific-pathogen free (SPF) environments providing standardized conditions. In contrast, mammalians including humans living in their natural habitat are continuously facing pathogen encounters throughout their life. The influences of environmental conditions on the signatures of the immune system and on experimental outcomes are yet not well defined. Thus, the transferability of results obtained in current experimental systems to the physiological human situation has always been a matter of debate. Studies elucidating the diversity of 'wild immunology' imprintings in detail and comparing it with those of 'clean' lab mice are sparse. Here, we applied multidimensional mass cytometry to dissect phenotypic and functional differences between distinct groups of laboratory and pet shop mice as a source for 'wild mice'. For this purpose, we developed a 31-antibody panel for murine leukocyte subsets identification and a 35-antibody panel assessing various cytokines. Established murine leukocyte populations were easily identified and diverse immune signatures indicative of numerous pathogen encounters were classified particularly in pet shop mice and to a lesser extent in quarantine and non-SPF mice as compared to SPF mice. In addition, unsupervised analysis identified distinct clusters that associated strongly with the degree of pathogenic priming, including increased frequencies of activated NK cells and antigen-experienced B- and T-cell subsets. Our study unravels the complexity of immune signatures altered under physiological pathogen challenges and highlights the importance of carefully adapting laboratory settings for immunological studies in mice, including drug and therapy testing. © 2016 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

CD40-CD40 ligand (CD40L) signaling plays multiple indispensable roles in cellular and humoral immunity. Impaired memory T-cell responses in the absence of CD40L have been well documented, but the requirement of this interaction for efficient priming of CD8+ T cells especially under inflammatory conditions has been under debate. In contrast to previous publications, we report here that virus-specific CD8+ T-cell responses as well as viral clearance are affected not only in the memory but also in the effector phase in CD40L−/− mice infected with lymphocytic choriomeningitis virus (LCMV) Armstrong strain. Interestingly, a considerable part of the LCMV-specific effector and memory T cells consists of CD40L+ CD8+ T cells. However, deficiency of CD40L in CD8+ T cells did influence neither the quantity nor the quality of primary T-cell responses in LCMV infection. Virus-specific CD8+ T cells in conditional knockout mice, with a selective deletion of the CD40L in CD8+ T cells, were fully functional regarding cytokine production and efficient pathogen clearance. Thus, our results unambiguously demonstrate that while CD40L is critical to generate effective primary CD8+ T-cell responses also under inflammatory conditions, CD40L expression by CD8+ T cells themselves is dispensable in acute LCMV infection. [ABSTRACT FROM AUTHOR]

CD8+ T cells play an essential role in immunity against intracellular pathogens, with cytotoxicity being considered their major effector mechanism. However, we here demonstrate that a major part of central and effector memory CD8+ T cells expresses CD40L, one key molecule for CD4+ T-cell-mediated help. CD40L+ CD8+ T cells are detectable among human antigen-specific immune responses, including pathogens such as influenza and yellow fever virus. CD40L+ CD8+ T cells display potent helper functions in vitro and in vivo, such as activation of antigen-presenting cells, and exhibit a cytokine expression signature similar to CD4+ T cells and unrelated to cytotoxic CD8+ T cells. The broad occurrence of CD40L+ CD8+ T cells in cellular immunity implicates that helper functions are not only executed by major histocompatibility complex (MHC) class ll-restricted CD4+ helper T cells but are also a common feature of MHC class l-restricted CD8+ T cell responses. Due to their versatile functional capacities, human CD40L+ CD8+ T cells are promising candidate cells for immune therapies, particularly when CD4+ T-cell help or pathogen-associated molecular pattern signals are limited. [ABSTRACT FROM AUTHOR]

CD40 L is one of the key molecules bridging the activation of specific T cells and the maturation of professional and nonprofessional antigen-presenting cells including B cells. CD4+ T cells have been regarded as the major T-cell subset that expresses CD40 L upon cognate activation; however, we demonstrate here that a putative CD8+ helper T-cell subset expressing CD40 L is induced in human and murine CD8+ T cells in vitro and in mice immunized with antigen-pulsed dendritic cells. IL-12 and STAT4-mediated signaling was the major instructive cytokine signal boosting the ability of CD8+ T cells to express CD40 L both in vitro and in vivo. Additionally, TCR signaling strength modulated CD40 L expression in CD8+ T cells after primary differentiation in vitro as well as in vivo. The induction of CD40 L in CD8+ T cells regulated by IL-12 and TCR signaling may enable CD8+ T cells to respond autonomously of CD4+ T cells. Thus, we propose that under proinflammatory conditions, a self-sustaining positive feedback loop could facilitate the efficient priming of T cells stimulated by high affinity peptide displaying APCs. [ABSTRACT FROM AUTHOR]

Natural regulatory T cells (nTreg) play a central role in the induction and maintenance of immunological tolerance. Experimental transplant models and recent clinical trials demonstrate that nTreg can control alloreactivity. To upgrade Treg-based cell therapies to a selective suppression of undesired immune reactions, only the transfer of Ag-specific nTreg represents the appropriate ther-apeutic option. However, Ag-specific nTreg are present at extremely low frequencies in the periphery, and so far appropriate surface markers for their precise identification are missing. In this study, we demonstrate that activated nTreg and activated conventional T cells differ in their 4-1BB and CD40 ligand (CD40L) expression signatures, allowing a clear dissection from each other. Based on the expression of 4-1BB and absence of CD40L expression, human alloantigen-reactive Foxp3+ nTreg can be directly isolated from MLR cultures with high purity. Alloantigen-reactive 4-lBB+CD40L~ nTreg were characterized by a completely demethylated Treg-specific demethylated region and showed alloantigen-specific suppressive properties superior to polyclonal Treg. Impor-tantly, isolated 4-lBB+CD40L~ nTreg maintain the nTreg phenotype and alloantigen-reactivity after in vitro expansion. Our results offer the possibility to simultaneously analyze Ag-specific nTreg and conventional T cells, and to establish cellular therapies with Ag-specific nTreg aiming at a specific inhibition of unwanted immunity. [ABSTRACT FROM AUTHOR]

The direct assessment of T helper (TH)-cell responses specific for antigens is essential to evaluate pathogenic and protective immunity. Presently, analysis and isolation of antigen-specific TH cells is restricted to cells that produce cytokines, or can be performed only with a rare selection of specific peptide major histocompatibility complex class II (MHC II) multimers. Here we report a new method that enables the assessment and isolation of TH cells specific for a defined antigen according to CD154 expression induced after stimulation in vitro. We show that antigen-induced CD154 expression is highly sensitive and specific for human and mouse antigen-specific TH cells. Moreover, the isolation of antigen-specific CD154+ TH cells necessitates only surface staining with antibodies, thereby enabling the fast generation of antigen-specific TH cell lines. Our approach allows assessment of TH cells with a defined specificity for the combined quantitative and qualitative analysis of TH-cell immunity as well as for the isolation of specific TH cells for targeted cellular immunotherapies. [ABSTRACT FROM AUTHOR]