A comprehensive genetic map of cytokine responses in Lyme borreliosis.

The incidence of Lyme borreliosis has risen, accompanied by persistent symptoms. The innate immune system and related cytokines are crucial in the host response and symptom development. We characterized cytokine production capacity before and after antibiotic treatment in 1,060 Lyme borreliosis patients. We observed a negative correlation between antibody production and IL-10 responses, as well as increased IL-1Ra responses in patients with disseminated disease. Genome-wide mapping the cytokine production allowed us to identify 34 cytokine quantitative trait loci (cQTLs), with 31 novel ones. We pinpointed the causal variant at the TLR1-6-10 locus and validated the regulation of IL-1Ra responses at transcritpome level using an independent cohort. We found that cQTLs contribute to Lyme borreliosis susceptibility and are relevant to other immune-mediated diseases. Our findings improve the understanding of cytokine responses in Lyme borreliosis and provide a genetic map of immune function as an expanded resource.

Early-life vitamin A treatment rescues neonatal infection-induced durably impaired tolerogenic properties of celiac lymph nodes.

Gut-draining mesenteric and celiac lymph nodes (mLNs and celLNs) critically contribute to peripheral tolerance toward food and microbial antigens by supporting the de novo induction of regulatory T cells (Tregs). These tolerogenic properties of mLNs and celLNs are stably imprinted within stromal cells (SCs) by microbial signals and vitamin A (VA), respectively. Here, we report that a single, transient gastrointestinal infection in the neonatal, but not adult, period durably abrogates the efficient Treg-inducing capacity of celLNs by altering the subset composition and gene expression profile of celLNSCs. These cells carry information about the early-life pathogen encounter until adulthood and durably instruct migratory dendritic cells entering the celLN with reduced tolerogenic properties. Mechanistically, transiently reduced VA levels cause long-lasting celLN functional impairment, which can be rescued by early-life treatment with VA. Together, our data highlight the therapeutic potential of VA to prevent sequelae post gastrointestinal infections in infants.

Impact of vitamin C on the development, differentiation and functional properties of T cells.

Vitamin C plays a multifaceted role in various biological processes and is well-known to facilitate pleiotropic activities in both innate and adaptive immune responses, where the antioxidant capacity of vitamin C is most likely highly relevant since immune responses mainly occur in reducing environments. Beyond its antioxidant properties, vitamin C can enhance the transcription potential of genes by promoting DNA demethylation through ten-eleven-translocation (Tet) methylcytosine dioxygenases, which have been recently demonstrated to be critical for the development and differentiation of T cells. In this minireview, we will provide a broader overview on the impact of vitamin C on signaling and regulatory activities in both innate and adaptive immune cells. Particularly, we will summarize recent findings on the decisive role of finely tuned vitamin C concentrations for T cell development, T helper cell differentiation, and optimal T cell-mediated immune responses.

The effector program of human CD8 T cells supports tissue remodeling.

CD8 T lymphocytes are classically viewed as cytotoxic T cells. Whether human CD8 T cells can, in parallel, induce a tissue regeneration program is poorly understood. Here, antigen-specific assay systems revealed that human CD8 T cells not only mediated cytotoxicity but also promoted tissue remodeling. Activated CD8 T cells could produce the epidermal growth factor receptor (EGFR)-ligand amphiregulin (AREG) and sensitize epithelial cells for enhanced regeneration potential. Blocking the EGFR or the effector cytokines IFN-γ and TNF could inhibit tissue remodeling. This regenerative program enhanced tumor spheroid and stem cell-mediated organoid growth. Using single-cell gene expression analysis, we identified an AREG+, tissue-resident CD8 T cell population in skin and adipose tissue from patients undergoing abdominal wall or abdominoplasty surgery. These tissue-resident CD8 T cells showed a strong TCR clonal relation to blood PD1+TIGIT+ CD8 T cells with tissue remodeling abilities. These findings may help to understand the complex CD8 biology in tumors and could become relevant for the design of therapeutic T cell products.

RORγt+ c-Maf+ Vγ4+ γδ T cells are generated in the adult thymus but do not reach the periphery.

T cell receptor (TCR) Vγ4-expressing γδ T cells comprise interferon γ (IFNγ)- and interleukin-17 (IL-17)-producing effector subsets, with a preference for IL-17 effector fate decisions during early ontogeny. The existence of adult-thymus-derived IL-17+ T cells (γδ17) remains controversial. Here, we use a mouse model in which T cells are generated exclusively in the adult thymus and employ single-cell chromatin state analysis to study their development. We identify adult-thymus-derived Vγ4 T cells that have all the molecular programs to become IL-17 producers. However, they have reduced IL-17 production capabilities and rarely reach the periphery. Moreover, this study provides high-resolution profiles of Vγ4 T cells in the adult thymus and lymph nodes and identifies Zeb1 as a potential γδ17 cell regulator. Together, this study provides valuable insights into the developmental traits of Vγ4 T cells during adulthood and supports the idea of age-specific signals required for thymic export and/or peripheral maturation of γδ17 cells.

Identification of the novel FOXP3-dependent Treg cell transcription factor MEOX1 by high-dimensional analysis of human CD4+ T cells.

CD4+ T cells play a central role in the adaptive immune response through their capacity to activate, support and control other immune cells. Although these cells have become the focus of intense research, a comprehensive understanding of the underlying regulatory networks that orchestrate CD4+ T cell function and activation is still incomplete. Here, we analyzed a large transcriptomic dataset consisting of 48 different human CD4+ T cell conditions. By performing reverse network engineering, we identified six common denominators of CD4+ T cell functionality (CREB1, E2F3, AHR, STAT1, NFAT5 and NFATC3). Moreover, we also analyzed condition-specific genes which led us to the identification of the transcription factor MEOX1 in Treg cells. Expression of MEOX1 was comparable to FOXP3 in Treg cells and can be upregulated by IL-2. Epigenetic analyses revealed a permissive epigenetic landscape for MEOX1 solely in Treg cells. Knockdown of MEOX1 in Treg cells revealed a profound impact on downstream gene expression programs and Treg cell suppressive capacity. These findings in the context of CD4+ T cells contribute to a better understanding of the transcriptional networks and biological mechanisms controlling CD4+ T cell functionality, which opens new avenues for future therapeutic strategies.

Zfp362 potentiates murine colonic inflammation by constraining Treg cell function rather than promoting Th17 cell differentiation.

Mucosal barrier integrity and pathogen clearance is a complex process influenced by both Th17 and Treg cells. Previously, we had described the DNA methylation profile of Th17 cells and identified Zinc finger protein (Zfp)362 to be uniquely demethylated. Here, we generated Zfp362-/- mice to unravel the role of Zfp362 for Th17 cell biology. Zfp362-/- mice appeared clinically normal, showed no phenotypic alterations in the T-cell compartment, and upon colonization with segmented filamentous bacteria, no effect of Zfp362 deficiency on Th17 cell differentiation was observed. By contrast, Zfp362 deletion resulted in increased frequencies of colonic Foxp3+ Treg cells and IL-10+ and RORγt+ Treg cell subsets in mesenteric lymph nodes. Adoptive transfer of naïve CD4+ T cells from Zfp362-/- mice into Rag2-/- mice resulted in a significantly lower weight loss when compared with controls receiving cells from Zfp362+/+ littermates. However, this attenuated weight loss did not correlate with alterations of Th17 cells but instead was associated with an increase of effector Treg cells in mesenteric lymph nodes. Together, these results suggest that Zfp362 plays an important role in promoting colonic inflammation; however, this function is derived from constraining the effector function of Treg cells rather than directly promoting Th17 cell differentiation.

IFNAR signaling of neuroectodermal cells is essential for the survival of C57BL/6 mice infected with Theiler's murine encephalomyelitis virus.

BACKGROUND: Theiler's murine encephalomyelitis virus (TMEV) is a single-stranded RNA virus that causes encephalitis followed by chronic demyelination in SJL mice and spontaneous seizures in C57BL/6 mice. Since earlier studies indicated a critical role of type I interferon (IFN-I) signaling in the control of viral replication in the central nervous system (CNS), mouse strain-specific differences in pathways induced by the IFN-I receptor (IFNAR) might determine the outcome of TMEV infection. METHODS: Data of RNA-seq analysis and immunohistochemistry were used to compare the gene and protein expression of IFN-I signaling pathway members between mock- and TMEV-infected SJL and C57BL/6 mice at 4, 7 and 14 days post-infection (dpi). To address the impact of IFNAR signaling in selected brain-resident cell types, conditional knockout mice with an IFNAR deficiency in cells of the neuroectodermal lineage (NesCre±IFNARfl/fl), neurons (Syn1Cre±IFNARfl/fl), astrocytes (GFAPCre±IFNARfl/fl), and microglia (Sall1CreER±IFNARfl/fl) on a C57BL/6 background were tested. PCR and an immunoassay were used to quantify TMEV RNA and cytokine and chemokine expression in their brain at 4 dpi. RESULTS: RNA-seq analysis revealed upregulation of most ISGs in SJL and C57BL/6 mice, but Ifi202b mRNA transcripts were only increased in SJL and Trim12a only in C57BL/6 mice. Immunohistochemistry showed minor differences in ISG expression (ISG15, OAS, PKR) between both mouse strains. While all immunocompetent Cre-negative control mice and the majority of mice with IFNAR deficiency in neurons or microglia survived until 14 dpi, lack of IFNAR expression in all cells (IFNAR-/-), neuroectodermal cells, or astrocytes induced lethal disease in most of the analyzed mice, which was associated with unrestricted viral replication. NesCre±IFNARfl/fl mice showed more Ifnb1, Tnfa, Il6, Il10, Il12b and Ifng mRNA transcripts than Cre-/-IFNARfl/fl mice. IFNAR-/- mice also demonstrated increased IFN-α, IFN-ß, IL1-ß, IL-6, and CXCL-1 protein levels, which highly correlated with viral load. CONCLUSIONS: Ifi202b and Trim12a expression levels likely contribute to mouse strain-specific susceptibility to TMEV-induced CNS lesions. Restriction of viral replication is strongly dependent on IFNAR signaling of neuroectodermal cells, which also controls the expression of key pro- and anti-inflammatory cytokines during viral brain infection.

Altered and allele-specific open chromatin landscape reveals epigenetic and genetic regulators of innate immunity in COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes severe COVID-19 in some patients and mild COVID-19 in others. Dysfunctional innate immune responses have been identified to contribute to COVID-19 severity, but the key regulators are still unknown. Here, we present an integrative single-cell multi-omics analysis of peripheral blood mononuclear cells from hospitalized and convalescent COVID-19 patients. In classical monocytes, we identified genes that were potentially regulated by differential chromatin accessibility. Then, sub-clustering and motif-enrichment analyses revealed disease condition-specific regulation by transcription factors and their targets, including an interaction between C/EBPs and a long-noncoding RNA LUCAT1, which we validated through loss-of-function experiments. Finally, we investigated genetic risk variants that exhibit allele-specific open chromatin (ASoC) in COVID-19 patients and identified a SNP rs6800484-C, which is associated with lower expression of CCR2 and may contribute to higher viral loads and higher risk of COVID-19 hospitalization. Altogether, our study highlights the diverse genetic and epigenetic regulators that contribute to COVID-19.

Stepwise acquisition of unique epigenetic signatures during differentiation of tissue Treg cells.

Regulatory T cells in non-lymphoid tissues are not only critical for maintaining self-tolerance, but are also important for promoting organ homeostasis and tissue repair. It is proposed that the generation of tissue Treg cells is a stepwise, multi-site process, accompanied by extensive epigenome remodeling, finally leading to the acquisition of unique tissue-specific epigenetic signatures. This process is initiated in the thymus, where Treg cells acquire core phenotypic and functional properties, followed by a priming step in secondary lymphoid organs that permits Treg cells to exit the lymphoid organs and seed into non-lymphoid tissues. There, a final specialization process takes place in response to unique microenvironmental cues in the respective tissue. In this review, we will summarize recent findings on this multi-site tissue Treg cell differentiation and highlight the importance of epigenetic remodeling during these stepwise events.

Postnatal expansion of mesenteric lymph node stromal cells towards reticular and CD34+ stromal cell subsets.

Gut-draining mesenteric lymph nodes (LN) provide the framework to shape intestinal adaptive immune responses. Based on the transcriptional signatures established by our previous work, the composition and immunomodulatory function of LN stromal cells (SC) vary according to location. Here, we describe the single-cell composition and development of the SC compartment within mesenteric LNs derived from postnatal to aged mice. We identify CD34+ SC and fibroblastic reticular stromal cell (FRC) progenitors as putative progenitors, both supplying the typical rapid postnatal mesenteric LN expansion. We further establish the location-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs and identify a microbiota-independent core epigenomic signature, showing characteristic differences between SCs from mesenteric and skin-draining peripheral LNs. The epigenomic landscape of SCs points to dynamic expression of Irf3 along the differentiation trajectories of FRCs. Accordingly, a mesenchymal stem cell line acquires a Cxcl9+ FRC molecular phenotype upon lentiviral overexpression of Irf3, and the relevance of Irf3 for SC biology is further underscored by the diminished proportion of Ccl19+ and Cxcl9+ FRCs in LNs of Irf3-/- mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of mesenteric LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs.

Distinct immunological and molecular signatures underpinning influenza vaccine responsiveness in the elderly.

Seasonal influenza outbreaks, especially in high-risk groups such as the elderly, represent an important public health problem. Prevailing inadequate efficacy of seasonal vaccines is a crucial bottleneck. Understanding the immunological and molecular mechanisms underpinning differential influenza vaccine responsiveness is essential to improve vaccination strategies. Here we show comprehensive characterization of the immune response of randomly selected elderly participants (≥ 65 years), immunized with the adjuvanted influenza vaccine Fluad. In-depth analyses by serology, multi-parametric flow cytometry, multiplex and transcriptome analysis, coupled to bioinformatics and mathematical modelling, reveal distinguishing immunological and molecular features between responders and non-responders defined by vaccine-induced seroconversion. Non-responders are specifically characterized by multiple suppressive immune mechanisms. The generated comprehensive high dimensional dataset enables the identification of putative mechanisms and nodes responsible for vaccine non-responsiveness independently of confounding age-related effects, with the potential to facilitate development of tailored vaccination strategies for the elderly.

Fibroblastic reticular cells mitigate acute GvHD via MHCII-dependent maintenance of regulatory T cells.

Acute graft versus host disease (aGvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT) inflicted by alloreactive T cells primed in secondary lymphoid organs (SLOs) and subsequent damage to aGvHD target tissues. In recent years, Treg transfer and/or expansion has emerged as a promising therapy to modulate aGvHD. However, cellular niches essential for fostering Tregs to prevent aGvHD have not been explored. Here, we tested whether and to what extent MHC class II (MHCII) expressed on Ccl19+ fibroblastic reticular cells (FRCs) shape the donor CD4+ T cell response during aGvHD. Animals lacking MHCII expression on Ccl19-Cre-expressing FRCs (MHCIIΔCcl19) showed aberrant CD4+ T cell activation in the effector phase, resulting in exacerbated aGvHD that was associated with significantly reduced expansion of Foxp3+ Tregs and invariant NK T (iNKT) cells. Skewed Treg maintenance in MHCIIΔCcl19 mice resulted in loss of protection from aGvHD provided by adoptively transferred donor Tregs. In contrast, although FRCs upregulated costimulatory surface receptors, and although they degraded and processed exogenous antigens after myeloablative irradiation, FRCs were dispensable to activate alloreactive CD4+ T cells in 2 mouse models of aGvHD. In summary, these data reveal an immunoprotective, MHCII-mediated function of FRC niches in secondary lymphoid organs (SLOs) after allo-HCT and highlight a framework of cellular and molecular interactions that regulate CD4+ T cell alloimmunity.

Inflammatory perturbations in early life long-lastingly shape the transcriptome and TCR repertoire of the first wave of regulatory T cells.

The first wave of Foxp3+ regulatory T cells (Tregs) generated in neonates is critical for the life-long prevention of autoimmunity. Although it is widely accepted that neonates are highly susceptible to infections, the impact of neonatal infections on this first wave of Tregs is completely unknown. Here, we challenged newborn Treg fate-mapping mice (Foxp3eGFPCreERT2xROSA26STOP-eYFP) with the Toll-like receptor (TLR) agonists LPS and poly I:C to mimic inflammatory perturbations upon neonatal bacterial or viral infections, respectively, and subsequently administrated tamoxifen during the first 8 days of life to selectively label the first wave of Tregs. Neonatally-tagged Tregs preferentially accumulated in non-lymphoid tissues (NLTs) when compared to secondary lymphoid organs (SLOs) irrespective of the treatment. One week post challenge, no differences in the frequency and phenotypes of neonatally-tagged Tregs were observed between challenged mice and untreated controls. However, upon aging, a decreased frequency of neonatally-tagged Tregs in both NLTs and SLOs was detected in challenged mice when compared to untreated controls. This decrease became significant 12 weeks post challenge, with no signs of altered Foxp3 stability. Remarkably, this late decrease in the frequency of neonatally-tagged Tregs only occurred when newborns were challenged, as treating 8-days-old mice with TLR agonists did not result in long-lasting alterations of the first wave of Tregs. Combined single-cell T cell receptor (TCR)-seq and RNA-seq revealed that neonatal inflammatory perturbations drastically diminished TCR diversity and long-lastingly altered the transcriptome of neonatally-tagged Tregs, exemplified by lower expression of Tigit, Foxp3, and Il2ra. Together, our data demonstrate that a single, transient encounter with a pathogen in early life can have long-lasting consequences for the first wave of Tregs, which might affect immunological tolerance, prevention of autoimmunity, and other non-canonical functions of tissue-resident Tregs in adulthood.

Profiling of epigenetic marker regions in murine ILCs under homeostatic and inflammatory conditions.

Epigenetic modifications such as DNA methylation play an essential role in imprinting specific transcriptional patterns in cells. We performed genome-wide DNA methylation profiling of murine lymph node-derived ILCs, which led to the identification of differentially methylated regions (DMRs) and the definition of epigenetic marker regions in ILCs. Marker regions were located in genes with a described function for ILCs, such as Tbx21, Gata3, or Il23r, but also in genes that have not been related to ILC biology. Methylation levels of the marker regions and expression of the associated genes were strongly correlated, indicating their functional relevance. Comparison with T helper cell methylomes revealed clear lineage differences, despite partial similarities in the methylation of specific ILC marker regions. IL-33-mediated challenge affected methylation of ILC2 epigenetic marker regions in the liver, while remaining relatively stable in the lung. In our study, we identified a set of epigenetic markers that can serve as a tool to study phenotypic and functional properties of ILCs.

The dark side of Tregs during aging.

In the last century, we have seen a dramatic rise in the number of older persons globally, a trend known as the grey (or silver) tsunami. People live markedly longer than their predecessors worldwide, due to remarkable changes in their lifestyle and in progresses made by modern medicine. However, the older we become, the more susceptible we are to a series of age-related pathologies, including infections, cancers, autoimmune diseases, and multi-morbidities. Therefore, a key challenge for our modern societies is how to cope with this fragile portion of the population, so that everybody could have the opportunity to live a long and healthy life. From a holistic point of view, aging results from the progressive decline of various systems. Among them, the distinctive age-dependent changes in the immune system contribute to the enhanced frailty of the elderly. One of these affects a population of lymphocytes, known as regulatory T cells (Tregs), as accumulating evidence suggest that there is a significant increase in the frequency of these cells in secondary lymphoid organs (SLOs) of aged animals. Although there are still discrepancies in the literature about modifications to their functional properties during aging, mounting evidence suggests a detrimental role for Tregs in the elderly in the context of bacterial and viral infections by suppressing immune responses against non-self-antigens. Interestingly, Tregs seem to also contribute to the reduced effectiveness of immunizations against many pathogens by limiting the production of vaccine-induced protective antibodies. In this review, we will analyze the current state of understandings about the role of Tregs in acute and chronic infections as well as in vaccination response in both humans and mice. Lastly, we provide an overview of current strategies for Treg modulation with potential future applications to improve the effectiveness of vaccines in older individuals.

Lymphatic migration of unconventional T cells promotes site-specific immunity in distinct lymph nodes.

Lymphatic transport of molecules and migration of myeloid cells to lymph nodes (LNs) continuously inform lymphocytes on changes in drained tissues. Here, using LN transplantation, single-cell RNA-seq, spectral flow cytometry, and a transgenic mouse model for photolabeling, we showed that tissue-derived unconventional T cells (UTCs) migrate via the lymphatic route to locally draining LNs. As each tissue harbored a distinct spectrum of UTCs with locally adapted differentiation states and distinct T cell receptor repertoires, every draining LN was thus populated by a distinctive tissue-determined mix of these lymphocytes. By making use of single UTC lineage-deficient mouse models, we found that UTCs functionally cooperated in interconnected units and generated and shaped characteristic innate and adaptive immune responses that differed between LNs that drained distinct tissues. Lymphatic migration of UTCs is, therefore, a key determinant of site-specific immunity initiated in distinct LNs with potential implications for vaccination strategies and immunotherapeutic approaches.

Impact of gut microenvironment on epigenetic signatures of intestinal T helper cell subsets.

The intestine, which is constantly exposed to an extensive range of antigens and immune stimuli, harbours a large number of highly diverse CD4+ T helper (Th) cells. Maintaining a balance between pro-inflammatory and tolerogenic intestinal Th cell subsets is crucial for the homeostasis and functionality of the gastrointestinal tract. Recent evidence suggests that epigenetic mechanisms play a vital role for Th cell differentiation and specialization, and dysregulation of these epigenetic regulations can result in an imbalance of Th cell subsets, causing the onset and progression of intestinal inflammatory diseases. Furthermore, epigenetic alterations might be orchestrated through microbiota-derived metabolites, which can function as epigenetic modifiers by catalyzing major enzymes involved in epigenetic modifications. In this review, we focus on the epigenetic control of Th cell differentiation and functional specialization in intestinal tissues in health and disease, and also discuss the role of microbiota in shaping intestinal Th cell epigenomes.

Lymph node stromal cells support the maturation of pre-DCs into cDC-like cells via colony-stimulating factor 1.

Conventional dendritic cells (cDCs) arise from committed precursor dendritic cells (pre-DCs) in the bone marrow that continuously seed the periphery. Pre-DCs and other upstream progenitors proliferate and mature in response to Fms-related receptor tyrosine kinase 3 ligand, which is considered the key cytokine for cDC development. However, other cytokines such as stem cell factor and colony-stimulating factor 1 (CSF1) were also shown to induce pre-DC maturation into DC-like cells. Yet, it is still only incompletely understood which cells contribute to cDC development once pre-DCs arrive in peripheral tissues. Here, we analysed the impact of lymph node (LN) fibroblastic stromal cells (FSCs) on the maturation of pre-DCs into cDC-like cells. We could demonstrate that ex vivo isolated LN FSCs co-cultured with pre-DCs induce precursor maturation into DC-like cells, which were capable of efficiently promoting the proliferation of naïve CD4+ T cells. Interestingly, FSCs isolated from distinct LNs induced DC-like cells with highly comparable transcriptomes, characterized by the expression of signature genes of both ex vivo isolated DCs and macrophages. Finally, by performing supplementation and receptor blocking studies, we could demonstrate that CSF1 is a driving factor for LN FSC-mediated pre-DC maturation into DC-like cells. In summary, we could identify CSF1 as a stromal cell-derived factor that has the potential to support the maturation of pre-DCs into cDC-like cells within secondary lymphoid organs.

Enhancement of Antiviral T-Cell Responses by Vitamin C Suggests New Strategies to Improve Manufacturing of Virus-Specific T Cells for Adoptive Immunotherapy.

Allogeneic and autologous transplantation of hematopoietic stem cells (HSCT) are being routinely used to treat patients with leukemia and lymphoma. Due to the required immunosuppression after stem cell transplantation, infection and reactivation by viruses are life-threatening complications. In recent years, adoptive transfer using virus-specific T cells (VSTs) has emerged as alternative to conventional therapies. Since vitamins are described to influence the immune system and its cellular components, the aim of this study was to examine whether vitamins modulate VST function and thereby enable an improvement of therapy. For that, we investigated the impact of vitamin C and D on the functionality of cytomegalovirus (CMV)-specific T cells isolated from CMV-seropositive healthy donors. We were able to show that vitamin C increases the expansion and activation state of CMV-specific T cells, and an increased influence of vitamin C was observed on cells isolated from male donors and donors above 40 years of age. A higher frequency of the terminally differentiated effector memory CD8+ T-cell population in these donors indicates a connection between these cells and the enhanced response to vitamin C. Thus, here we provide insights into the impact of vitamin C on cytotoxic T cells as well as possible additional selection criteria and strategies to improve VST functionality.