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Post-transplant lymphoproliferative disorder is a life-threatening complication of immunosuppression following transplantation mediated by failure of T cells to control Epstein-Barr virus (EBV)-infected and transformed B cells. Typically, a modification or reduction of immunosuppression is recommended, but insufficiently defined thus far. In order to help delineate this, we characterized EBV-antigen-specific T cells and lymphoblastoid cell lines from healthy donors and in patients with a kidney transplant in the absence or presence of the standard immunosuppressants tacrolimus, cyclosporin A, prednisolone, rapamycin, and mycophenolic acid. Phenotypes of lymphoblastoid cell-lines and T cells, T cell-receptor-repertoire diversity, and T-cell reactivity upon co-culture with autologous lymphoblastoid cell lines were analyzed. Rapamycin and mycophenolic acid inhibited lymphoblastoid cell-line proliferation. T cells treated with prednisolone and rapamycin showed nearly normal cytokine production. Proliferation and the viability of T cells were decreased by mycophenolic acid, while tacrolimus and cyclosporin A were strong suppressors of T-cell function including their killing activity. Overall, our study provides a basis for the clinical decision for the modification and reduction of immunosuppression and adds information to the complex balance of maintaining anti-viral immunity while preventing acute rejection. Thus, an immunosuppressive regime based on mTOR inhibition and reduced or withdrawn calcineurin inhibitors could be a promising strategy for patients with increased risk of or manifested EBV-associated post-transplant lymphoproliferative disorder., (Copyright © 2022 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

While virus-specific antibodies are broadly recognized as correlates of protection, virus-specific T cells are important for direct clearance of infected cells. Failure to generate hepatitis B virus (HBV)-specific antibodies is well-known in patients with end-stage renal disease. However, whether and to what extent HBV-specific cellular immunity is altered in this population and how it influences humoral immunity is not clear. To address it, we analyzed HBV-reactive T cells and antibodies in hemodialysis patients post vaccination. 29 hemodialysis patients and 10 healthy controls were enrolled in a cross-sectional study. Using multiparameter flow cytometry, HBV-reactive T cells were analyzed and functionally dissected based on granzyme B, interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and IL-4 expression. Importantly, HBV-reactive CD4 + T cells were detected not only in all patients with sufficient titers but also in 70% of non-responders. Furthermore, a correlation between the magnitude of HBV-reactive CD4 + T cells and post-vaccination titers was observed. In summary, our data showed that HBV-reactive polyfunctional T cells were present in the majority of hemodialysis patients even if humoral immunity failed. Further studies are required to confirm their in vivo antiviral capacity. The ability to induce vaccine-reactive T cells paves new ways for improved vaccination and therapy protocols., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

The prevailing 'division of labor' concept in cellular immunity is that CD8 + T cells primarily utilize cytotoxic functions to kill target cells, while CD4 + T cells exert helper/inducer functions. Multiple subsets of CD4 + memory T cells have been characterized by distinct chemokine receptor expression. Here, we demonstrate that analogous CD8 + memory T-cell subsets exist, characterized by identical chemokine receptor expression signatures and controlled by similar generic programs. Among them, Tc2, Tc17 and Tc22 cells, in contrast to Tc1 and Tc17 + 1 cells, express IL-6R but not SLAMF7, completely lack cytotoxicity and instead display helper functions including CD40L expression. CD8 + helper T cells exhibit a unique TCR repertoire, express genes related to skin resident memory T cells (T RM ) and are altered in the inflammatory skin disease psoriasis. Our findings reveal that the conventional view of CD4 + and CD8 + T cell capabilities and functions in human health and disease needs to be revised.

Reactivation of the BK polyomavirus is known to lead to severe complications in kidney transplant patients. The current treatment strategy relies on decreasing the immunosuppression to allow the immune system to clear the virus. Recently, we demonstrated a clear association between the resolution of BKV reactivation and reconstitution of BKV-specific CD4 + T-cells. However, which factors determine the duration of viral infection clearance remains so far unclear. Here we apply a combination of in-depth multi-parametric flow cytometry and NGS-based CDR3 beta chain receptor repertoire analysis of BKV-specific T-cells to a cohort of 7 kidney transplant patients during the clinical course of BKV reactivation. This way we followed TCR repertoires at single clone levels and functional activity of BKV-specific T-cells during the resolution of BKV infection. The duration of BKV clearance did not depend on the number of peripheral blood BKV-specific T-cells nor on a few immunodominant BKV-specific T-cell clones. Rather, the T-cell receptor repertoire diversity and exhaustion status of BKV-specific T-cells affected the duration of viral clearance: high clonotype diversity and lack of PD1 and TIM3 exhaustion markers on BKV-specific T-cells was associated with short clearance time. Our data thus demonstrate how the diversity and the exhaustion state of the T-cells can determine the clinical course of BKV infection.

Influenza vaccination is a common approach to prevent seasonal and pandemic influenza. Pre-existing antibodies against close viral strains might impair antibody formation against previously unseen strains-a process called original antigenic sin. The role of this pre-existing cellular immunity in this process is, despite some hints from animal models, not clear. Here, we analyzed cellular and humoral immunity in healthy individuals before and after vaccination with seasonal influenza vaccine. Based on influenza-specific hemagglutination inhibiting (HI) titers, vaccinees were grouped into HI-negative and -positive cohorts followed by in-depth cytometric and TCR repertoire analysis. Both serological groups revealed cross-reactive T-cell memory to the vaccine strains at baseline that gave rise to the majority of vaccine-specific T-cells post vaccination. On the contrary, very limited number of vaccine-specific T-cell clones was recruited from the naive pool. Furthermore, baseline quantity of vaccine-specific central memory helper T-cells and clonotype richness of this population directly correlated with the vaccination efficacy. Our findings suggest that the deliberate recruitment of pre-existing cross-reactive cellular memory might help to improve vaccination outcome.

Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses in humans are poorly understood. Here, we identify the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungi are induced by cross-reactivity to C. albicans. Intestinal inflammation expands total C. albicans and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, especially during acute allergic bronchopulmonary aspergillosis. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identify heterologous immunity to a single, ubiquitous member of the microbiota as a central mechanism for systemic induction of human anti-fungal Th17 responses and as a potential risk factor for pulmonary inflammatory diseases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

About 8 % of the human genome consists of human endogenous retroviruses (HERVs), which are relicts of ancient exogenous retroviral infections incurred during evolution. Although the majority of HERVs have functional gene defects or epigenetic modifications, many of them are still able to produce retroviral proteins that have been proposed to be involved in cellular transformation and cancer development. We found that, in chemo-resistant U87 RETO glioblastoma cells, cytotoxic stress induced by etoposide promotes accumulation and large-scale fission of mitochondria, associated with the detection of HERV-WE 1 (syncytin-1) and HERV-FRD 1 (syncytin-2) in these organelles. In addition, mitochondrial preparations also contained the corresponding receptors, i.e. ASCT2 and MFSD2. We clearly demonstrated that mitochondria associated with HERV-proteins were shuttled between adjacent cancer cells not only via tunneling tubes, but also by direct cellular uptake across the cell membrane. Furthermore, anti-syncytin-1 and anti-syncytin-2 antibodies were able to specifically block this direct cellular uptake of mitochondria even more than antibodies targeting the cognate receptors. Here, we suggest that the association of mitochondria with syncytin-1/syncytin-2 together with their respective receptors could represent a novel mechanism of cell-to-cell transfer. In chemotherapy-refractory cancer cells, this might open up attractive avenues to novel mitochondria-targeting therapies., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest or financial ties to disclose.

Immunosenescence is a hallmark of the aging immune system and is considered the main cause of a reduced vaccine efficacy in the elderly. Although γδ T cells can become activated by recombinant influenza hemagglutinin, their age-related immunocompetence during a virus-induced immune response has so far not been investigated. In this study we evaluate the kinetics of γδ T cells after vaccination with the trivalent 2011/2012 northern hemisphere seasonal influenza vaccine. We applied multi-parametric flow cytometry to a cohort of 21 young (19-30 years) and 23 elderly (53-67 years) healthy individuals. Activated and proliferating γδ T cells, as identified by CD38 and Ki67 expression, were quantified on the days 0, 3, 7, 10, 14, 17, and 21. We observed a significantly lower number of activated and proliferating γδ T cells at baseline and following vaccination in elderly as compared to young individuals. The kinetics changes of activated γδ T cells were much stronger in the young, while corresponding changes in the elderly occurred slower. In addition, we observed an association between day 21 HAI titers of influenza A and the frequencies of Ki67+ γδ T cells at day 7 in the young. In conclusion, aging induces alterations of the γδ T cell response that might have negative implications for vaccination efficacy.

FOXP3+ regulatory T cells (Tregs) maintain tolerance against self-antigens and innocuous environmental antigens. However, it is still unknown whether Treg-mediated tolerance is antigen specific and how Treg specificity contributes to the selective loss of tolerance, as observed in human immunopathologies such as allergies. Here, we used antigen-reactive T cell enrichment to identify antigen-specific human Tregs. We demonstrate dominant Treg-mediated tolerance against particulate aeroallergens, such as pollen, house dust mites, and fungal spores. Surprisingly, we found no evidence of functional impairment of Treg responses in allergic donors. Rather, major allergenic proteins, known to rapidly dissociate from inhaled allergenic particles, have a generally reduced capability to generate Treg responses. Most strikingly, in individual allergic donors, Th2 cells and Tregs always target disparate proteins. Thus, our data highlight the importance of Treg antigen-specificity for tolerance in humans and identify antigen-specific escape from Treg control as an important mechanism enabling antigen-specific loss of tolerance in human allergy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Reactivation of Polyomavirus BKV is a severe complication in kidney transplant patients. Current treatment requires close monitoring, and modification of immunosuppressive drugs. As an important additional tool, the monitoring of BKV immunity has been based on detection of cytokine-secreting T cells upon BKV-antigen challenge. However, low frequent BKV-specific T cells are often barely detectable and their roles in BKV clearance remain unclear. Here, we analyzed the effects of immunosuppressive agents on BKV-specific T cells in vitro. Significant reductions in expression of several markers, and reduced killing functions upon treatment with calcineurin but not mTOR inhibitors were detected. However, effects of these drugs on expression of surface markers and GranzymeB were substantially less striking than effects on cytokine expression. Consequently, we applied a novel detection strategy for BKV-specific T cells in immunosuppressed kidney transplant patients using these more robust markers, and showed significantly improved sensitivity compared with the conventional IFNγ-based method. Using this strategy and 17-color flow cytometry, we found BKV-specific helper and cytolytic CD4+ T-cell subsets that differed in their memory phenotype, which corresponded with BKV clearance in kidney transplant patients. Thus, our results offer an improved detection strategy for BKV-specific T cells in kidney transplant patients, and shed light on the contributions of these cells to BKV clearance.

Immunosenescence results from a continuous deterioration of immune responses resulting in a decreased response to vaccines. A well-described age-related alteration of the immune system is the decrease of de novo generation of T and B cells. In addition, the accumulation of memory cells and loss of diversity in antigen specificities resulting from a lifetime of exposure to pathogens has also been described. However, the effect of aging on subsets of γδTCR(+) T cells and Tregs has been poorly described, and the efficacy of the recall response to common persistent infections in the elderly remains obscure. Here, we investigated alterations in the subpopulations of the B and T cells among 24 healthy young (aged 19-30) and 26 healthy elderly (aged 53-67) individuals. The analysis was performed by flow cytometry using freshly collected peripheral blood. γδTCR(+) T cells were overall decreased, while CD4(+)CD8(-) cells among γδTCR(+) T cells were increased in the elderly. Helios(+)Foxp3(+) and Helios(-)Foxp3(+) Treg cells were unaffected with age. Recent thymic emigrants, based on CD31 expression, were decreased among the Helios(+)Foxp3(+), but not the Helios(-)Foxp3(+) cell populations. We observed a decrease in Adenovirus-specific CD4(+) and CD8(+) T cells and an increase in CMV-specific CD4(+) T cells in the elderly. Similarly, INFγ(+)TNFα(+) double-positive cells were decreased among activated T cells after Adenovirus stimulation but increased after CMV stimulation. The data presented here indicate that γδTCR(+) T cells might stabilize B cells, and functional senescence might dominate at higher ages than those studied here.

Immunosenescence is a hallmark of the aging immune system, leading to increased susceptibility to infections in the aged population and decreased ability to eradicate infectious pathogens. These effects, in turn, result in an increased burden on the healthcare system due to elevated frequency and duration of hospital visits. Growing evidence suggests that cells of the innate immune system are central modulators for the initiation and maintenance of an adequate pathogen-specific response through the adaptive immune system. While there are many reports on age-dependent alterations and dysfunctions of the adaptive immune system, the underlying mechanisms and effects of natural aging on the composition of the innate immune system remain unknown. Here, we present the results obtained from the comprehensive immunophenotyping of innate leukocyte populations, examined for age-related alterations within different sub-populations assessed using multi-parametric flow cytometry. We compared peripheral blood mononuclear cells from 24 young (aged 19-30 years) and 26 elderly (aged 53-67 years) donors. For classical CD16(+)CD56(dim) NK cells, the fraction of CD62L(+)CD57(+) was diminished in the elderly donors compared with young individuals, while the other investigated NK subsets remained unaffected by age. Both transitional monocytes and non-classical CD14(+-)CD16(++) monocytes were increased in the elderly compared with the young. The populations of pDCs and mDC2 were decreased among the elderly. These data demonstrate that the dynamics of the mDC subsets might counteract decreased virus surveillance. Furthermore, these data show that the maturation of NK cells might gradually slow down.

Motivation: Recombined T- and B-cell receptor repertoires are increasingly being studied using next generation sequencing (NGS) in order to interrogate the repertoire composition as well as changes in the distribution of receptor clones under different physiological and disease states. This type of analysis requires efficient and unambiguous clonotype assignment to a large number of NGS read sequences, including the identification of the incorporated V and J gene segments and the CDR3 sequence. Current tools have deficits with respect to performance, accuracy and documentation of their underlying algorithms and usage., Results: We present IMSEQ, a method to derive clonotype repertoires from NGS data with sophisticated routines for handling errors stemming from PCR and sequencing artefacts. The application can handle different kinds of input data originating from single- or paired-end sequencing in different configurations and is generic regarding the species and gene of interest. We have carefully evaluated our method with simulated and real world data and show that IMSEQ is superior to other tools with respect to its clonotyping as well as standalone error correction and runtime performance., Availability and Implementation: IMSEQ was implemented in C++ using the SeqAn library for efficient sequence analysis. It is freely available under the GPLv2 open source license and can be downloaded at www.imtools.org., Supplementary Information: Supplementary data are available at Bioinformatics online., Contact: lkuchenb@inf.fu-berlin.de or peter.robinson@charite.de., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)