Preclinical toxicity analyses of lentiviral vectors expressing the HIV-1 LTR-specific designer-recombinase Brec1.

Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies.

Time-resolved role of P2X4 and P2X7 during CD8+ T cell activation.

CD8+ T cells are a crucial part of the adaptive immune system, responsible for combating intracellular pathogens and tumor cells. The initial activation of T cells involves the formation of highly dynamic Ca2+ microdomains. Recently, purinergic signaling was shown to be involved in the formation of the initial Ca2+ microdomains in CD4+ T cells. In this study, the role of purinergic cation channels, particularly P2X4 and P2X7, in CD8+ T cell signaling from initial events to downstream responses was investigated, focusing on various aspects of T cell activation, including Ca2+ microdomains, global Ca2+ responses, NFAT-1 translocation, cytokine expression, and proliferation. While Ca2+ microdomain formation was significantly reduced in the first milliseconds to seconds in CD8+ T cells lacking P2X4 and P2X7 channels, global Ca2+ responses over minutes were comparable between wild-type (WT) and knockout cells. However, the onset velocity was reduced in P2X4-deficient cells, and P2X4, as well as P2X7-deficient cells, exhibited a delayed response to reach a certain level of free cytosolic Ca2+ concentration ([Ca2+]i). NFAT-1 translocation, a crucial transcription factor in T cell activation, was also impaired in CD8+ T cells lacking P2X4 and P2X7. In addition, the expression of IFN-γ, a major pro-inflammatory cytokine produced by activated CD8+ T cells, and Nur77, a negative regulator of T cell activation, was significantly reduced 18h post-stimulation in the knockout cells. In line, the proliferation of T cells after 3 days was also impaired in the absence of P2X4 and P2X7 channels. In summary, the study demonstrates that purinergic signaling through P2X4 and P2X7 enhances initial Ca2+ events during CD8+ T cell activation and plays a crucial role in regulating downstream responses, including NFAT-1 translocation, cytokine expression, and proliferation on multiple timescales. These findings suggest that targeting purinergic signaling pathways may offer potential therapeutic interventions.

The TRPM2 ion channel regulates metabolic and thermogenic adaptations in adipose tissue of cold-exposed mice.

Introduction: During thermogenesis, adipose tissue (AT) becomes more active and enhances oxidative metabolism. The promotion of this process in white AT (WAT) is called "browning" and, together with the brown AT (BAT) activation, is considered as a promising approach to counteract obesity and metabolic diseases. Transient receptor potential cation channel, subfamily M, member 2 (TRPM2), is an ion channel that allows extracellular Ca2+ influx into the cytosol, and is gated by adenosine diphosphate ribose (ADPR), produced from NAD+ degradation. The aim of this study was to investigate the relevance of TRPM2 in the regulation of energy metabolism in BAT, WAT, and liver during thermogenesis. Methods: Wild type (WT) and Trpm2-/- mice were exposed to 6°C and BAT, WAT and liver were collected to evaluate mRNA, protein levels and ADPR content. Furthermore, O2 consumption, CO2 production and energy expenditure were measured in these mice upon thermogenic stimulation. Finally, the effect of the pharmacological inhibition of TRPM2 was assessed in primary adipocytes, evaluating the response upon stimulation with the ß-adrenergic receptor agonist CL316,243. Results: Trpm2-/- mice displayed lower expression of browning markers in AT and lower energy expenditure in response to thermogenic stimulus, compared to WT animals. Trpm2 gene overexpression was observed in WAT, BAT and liver upon cold exposure. In addition, ADPR levels and mono/poly-ADPR hydrolases expression were higher in mice exposed to cold, compared to control mice, likely mediating ADPR generation. Discussion: Our data indicate TRPM2 as a fundamental player in BAT activation and WAT browning. TRPM2 agonists may represent new pharmacological strategies to fight obesity.

Pregnancy-induced transfer of pathogen-specific T cells from mother to fetus in mice.

Neonatal health is determined by the transfer of maternal antibodies from the mother to the fetus. Besides antibodies, maternal cells cross the placental barrier and seed into fetal organs. Contrary to maternal antibodies, maternal microchimeric cells (MMc) show a high longevity, as they can persist in the offspring until adulthood. Recent evidence highlights that MMc leukocytes promote neonatal immunity against early-life infections in mice and humans. As shown in mice, this promotion of immunity was attributable to an improved fetal immune development. Besides this indirect effect, MMc may be pathogen-specific and thus, directly clear pathogen threats in the offspring postnatally. By using ovalbumin recombinant Listeria monocytogenes (LmOVA), we here provide evidence that OVA-specific T cells are transferred from the mother to the fetus, which is associated with increased activation of T cells and a milder course of postnatal infection in the offspring. Our data highlight that maternally-derived passive immunity of the neonate is not limited to antibodies, as MMc have the potential to transfer immune memory between generations.

Short-term dietary changes can result in mucosal and systemic immune depression.

Omnivorous animals, including mice and humans, tend to prefer energy-dense nutrients rich in fat over plant-based diets, especially for short periods of time, but the health consequences of this short-term consumption of energy-dense nutrients are unclear. Here, we show that short-term reiterative switching to 'feast diets', mimicking our social eating behavior, breaches the potential buffering effect of the intestinal microbiota and reorganizes the immunological architecture of mucosa-associated lymphoid tissues. The first dietary switch was sufficient to induce transient mucosal immune depression and suppress systemic immunity, leading to higher susceptibility to Salmonella enterica serovar Typhimurium and Listeria monocytogenes infections. The ability to respond to antigenic challenges with a model antigen was also impaired. These observations could be explained by a reduction of CD4+ T cell metabolic fitness and cytokine production due to impaired mTOR activity in response to reduced microbial provision of fiber metabolites. Reintroducing dietary fiber rewired T cell metabolism and restored mucosal and systemic CD4+ T cell functions and immunity. Finally, dietary intervention with human volunteers confirmed the effect of short-term dietary switches on human CD4+ T cell functionality. Therefore, short-term nutritional changes cause a transient depression of mucosal and systemic immunity, creating a window of opportunity for pathogenic infection.

IRF4 is required for migration of CD4+ T cells to the intestine but not for Th2 and Th17 cell maintenance.

The transcription factor Interferon Regulatory Factor 4 (IRF4) is central in control of T cell activation and differentiation. Deficiency of IRF4 results in severe immune deficiency and affects maturation and function of most if not all T cell subsets. Here we use mouse infection models for Citrobacter rodentium and Strongyloides ratti to analyze the function of IRF4 in T helper (Th) 17 and Th2 cell responses, respectively. IRF4 deficient mice were impaired in the control of both pathogens, failed to mount Th17 and Th2 cell responses and showed impaired recruitment of T helper cells to the intestine, the infection site of both pathogens. Compromised intestinal migration was associated with reduced expression of the intestinal homing receptors α4ß7 integrin, CCR9 and GPR15. Identification of IRF4 binding sites in the gene loci of these receptors suggests a direct control of their expression by IRF4. Competitive T cell transfer assays further demonstrated that loss of one functional Irf4 allele already affected intestinal accumulation and Th2 and Th17 cell generation, indicating that lower IRF4 levels are of disadvantage for Th2 and Th17 cell differentiation as well as their migration to the intestine. Conversion of peripheral CD4+ T cells from an Irf4 wildtype to an Irf4 heterozygous or from an Irf4 heterozygous to a homozygous mutant genotype after C. rodentium or S. ratti infection did not reduce their capacity to produce Th17 or Th2 cytokines and only partially affected their persistence in the intestine, revealing that IRF4 is not essential for maintenance of the Th2 and Th17 phenotype and for survival of these T helper cells in the intestine. In conclusion, we demonstrate that the expression levels of IRF4 determine Th2 and Th17 cell differentiation and their intestinal accumulation but that IRF4 expression is not crucial for Th2 and Th17 cell survival.

Cell-Autonomous Constitutive gp130 Signaling in T Cells Amplifies TH17 Cell Responses and Causes Severe Lung Inflammation.

IL-6 plays a fundamental role in T cell differentiation and is strictly controlled by surface expression and shedding of IL-6R. IL-6 also acts on other cells that might affect T cell maturation. To study the impact of cell-autonomous and uncontrolled IL-6 signaling in T cells, we generated mice with a constitutively active IL-6R gp130 chain (Lgp130) expressed either in all T cells (Lgp130 × CD4Cre mice) or inducible in CD4+ T cells (Lgp130 × CD4CreERT2 mice). Lgp130 × CD4Cre mice accumulated activated T cells, including TH17 cells, in the lung, resulting in severe inflammation. Tamoxifen treatment of Lgp130 × CD4CreERT2 mice caused Lgp130 expression in 40-50% of CD4+ T cells, but mice developed lung disease only after several months. Lgp130+ CD4+ T cells were also enriched for TH17 cells; however, there was concomitant expansion of Lgp130- regulatory T cells, which likely restricted pathologic Lgp130+ T cells. In vitro, constitutive gp130 signaling in T cells enhanced but was not sufficient for TH17 cell differentiation. Augmented TH17 cell development of Lgp130+ T cells was also observed in Lgp130 × CD4CreERT2 mice infected with Staphylococcus aureus, but gp130 activation did not interfere with formation of TH1 cells against Listeria monocytogenes. Lgp130+ CD4+ T cells acquired a memory T cell phenotype and persisted in high numbers as a polyclonal T cell population in lymphoid and peripheral tissues, but we did not observe T cell lymphoma formation. In conclusion, cell-autonomous gp130 signaling alters T cell differentiation. Although gp130 signaling is not sufficient for TH17 cell differentiation, it still promotes accumulation of activated T cells in the lung that cause tissue inflammation.

Kidney-resident innate-like memory γδ T cells control chronic Staphylococcus aureus infection of mice.

γδ T cells are involved in the control of Staphylococcus aureus infection, but their importance in protection compared to other T cells is unclear. We used a mouse model of systemic S. aureus infection associated with high bacterial load and persistence in the kidney. Infection caused fulminant accumulation of γδ T cells in the kidney. Renal γδ T cells acquired tissue residency and were maintained in high numbers during chronic infection. At day 7, up to 50% of renal γδ T cells produced IL-17A in situ and a large fraction of renal γδ T cells remained IL-17A+ during chronic infection. Controlled depletion revealed that γδ T cells restricted renal S. aureus replication in the acute infection and provided protection during chronic renal infection and upon reinfection. Our results demonstrate that kidney-resident γδ T cells are nonredundant in limiting local S. aureus growth during chronic infection and provide enhanced protection against reinfection.

Role of Liver CD38 in the Regulation of Metabolic Pathways during Cold-Induced Thermogenesis in Mice.

Boosting NAD+ levels are considered a promising means to promote healthy aging and ameliorate dysfunctional metabolism. The expression of CD38, the major NAD+-consuming enzyme, is downregulated during thermogenesis in both brown and white adipose tissues (BAT and WAT). Moreover, BAT activation and WAT "browning" were enhanced in Cd38-/- mice. In this study, the role of CD38 in the liver during thermogenesis was investigated, with the liver being the central organ controlling systemic energy metabolism. Wild-type mice and Cd38-/- mice were exposed to cold temperatures, and levels of metabolites and enzymes were measured in the livers and plasma. During cold exposure, CD38 expression was downregulated in the liver, as in BAT and WAT, with a concomitant increase in NAD(H) and a marked decrease in NADPH levels. Glucose-6-phosphate dehydrogenase and the malic enzyme, along with enzymes in the glycolytic pathway, were downregulated, which is in line with glucose-6-P being re-directed towards glucose release. In Cd38-/- mice, the cross-regulation between glycolysis and glucose release was lost, although this did not impair the glucose release from glycogen. Glycerol levels were decreased in the liver from Cd38-/- animals upon cold exposure, suggesting that glyceroneogenesis, as gluconeogenesis, was not properly activated in the absence of CD38. SIRT3 activity, regulating mitochondrial metabolism, was enhanced by cold exposure, whereas its activity was already high at a warm temperature in Cd38-/- mice and was not further increased by the cold. Notably, FGF21 and bile acid release was enhanced in the liver of Cd38-/- mice, which might contribute to enhanced BAT activation in Cd38-/- mice. These results demonstrate that CD38 inhibition can be suggested as a strategy to boost NAD+ and would not negatively affect hepatic functions during thermogenesis.

The Properties of Proinflammatory Ly6Chi Monocytes Are Differentially Shaped by Parasitic and Bacterial Liver Infections.

In the past, proinflammatory CD11b+Ly6Chi monocytes were predominantly considered as a uniform population. However, recent investigations suggests that this population is far more diverse than previously thought. For example, in mouse models of Entamoeba (E.) histolytica and Listeria (L.) monocytogenes liver infections, it was shown that their absence had opposite effects. In the former model, it ameliorated parasite-dependent liver injury, whereas in the listeria model it exacerbated liver pathology. Here, we analyzed Ly6Chi monocytes from the liver of both infection models at transcriptome, protein, and functional levels. Paralleled by E. histolytica- and L. monocytogenes-specific differences in recruitment-relevant chemokines, both infections induced accumulation of Ly6C+ monocytes at infection sites. Transcriptomic analysis revealed a high similarity between monocytes from naïve and parasite-infected mice and a clear proinflammatory phenotype of listeria-induced monocytes. This was further reflected by the upregulation of M2-related transcription factors (e.g., Mafb, Nr4a1, Fos) and higher CD14 expression by Ly6Chi monocytes in the E. histolytica infection model. In contrast, monocytes from the listeria infection model expressed M1-related transcription factors (e.g., Irf2, Mndal, Ifi204) and showed higher expression of CD38, CD74, and CD86, as well as higher ROS production. Taken together, proinflammatory Ly6Chi monocytes vary considerably depending on the causative pathogen. By using markers identified in the study, Ly6Chi monocytes can be further subdivided into different populations.

Tissue-resident memory T cells in the kidney.

The identification of tissue-resident memory T cells (TRM cells) has significantly improved our understanding of immunity. In the last decade, studies have demonstrated that TRM cells are induced after an acute T-cell response, remain in peripheral organs for several years, and contribute to both an efficient host defense and autoimmune disease. TRM cells are found in the kidneys of healthy individuals and patients with various kidney diseases. A better understanding of these cells and their therapeutic targeting might provide new treatment options for infections, autoimmune diseases, graft rejection, and cancer. In this review, we address the definition, phenotype, and developmental mechanisms of TRM cells. Then, we further discuss the current understanding of TRM cells in kidney diseases, such as infection, autoimmune disease, cancer, and graft rejection after transplantation.

Th17 cell plasticity towards a T-bet-dependent Th1 phenotype is required for bacterial control in Staphylococcus aureus infection.

Staphylococcus aureus is frequently detected in patients with sepsis and thus represents a major health burden worldwide. CD4+ T helper cells are involved in the immune response to S. aureus by supporting antibody production and phagocytosis. In particular, Th1 and Th17 cells secreting IFN-γ and IL-17A, are involved in the control of systemic S. aureus infections in humans and mice. To investigate the role of T cells in severe S. aureus infections, we established a mouse sepsis model in which the kidney was identified to be the organ with the highest bacterial load and abundance of Th17 cells. In this model, IL-17A but not IFN-γ was required for bacterial control. Using Il17aCre × R26YFP mice we could show that Th17 fate cells produce Th17 and Th1 cytokines, indicating a high degree of Th17 cell plasticity. Single cell RNA-sequencing of renal Th17 fate cells uncovered their heterogeneity and identified a cluster with a Th1 expression profile within the Th17 cell population, which was absent in mice with T-bet/Tbx21-deficiency in Th17 cells (Il17aCre x R26eYFP x Tbx21-flox). Blocking Th17 to Th1 transdifferentiation in Th17 fate cells in these mice resulted in increased S. aureus tissue loads. In summary, we highlight the impact of Th17 cells in controlling systemic S. aureus infections and show that T-bet expression by Th17 cells is required for bacterial clearance. While targeting the Th17 cell immune response is an important therapeutic option in autoimmunity, silencing Th17 cells might have detrimental effects in bacterial infections.

P2X4 and P2X7 are essential players in basal T cell activity and Ca2+ signaling milliseconds after T cell activation.

Initial T cell activation is triggered by the formation of highly dynamic, spatiotemporally restricted Ca2+ microdomains. Purinergic signaling is known to be involved in Ca2+ influx in T cells at later stages compared to the initial microdomain formation. Using a high-resolution Ca2+ live-cell imaging system, we show that the two purinergic cation channels P2X4 and P2X7 not only are involved in the global Ca2+ signals but also promote initial Ca2+ microdomains tens of milliseconds after T cell stimulation. These Ca2+ microdomains were significantly decreased in T cells from P2rx4-/- and P2rx7-/- mice or by pharmacological inhibition or blocking. Furthermore, we show a pannexin-1-dependent activation of P2X4 in the absence of T cell receptor/CD3 stimulation. Subsequently, upon T cell receptor/CD3 stimulation, ATP release is increased and autocrine activation of both P2X4 and P2X7 then amplifies initial Ca2+ microdomains already in the first second of T cell activation.

Trans-Ned 19-Mediated Antagonism of Nicotinic Acid Adenine Nucleotide-Mediated Calcium Signaling Regulates Th17 Cell Plasticity in Mice.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing agent and its inhibition proved to inhibit T-cell activation. However, the impact of the NAADP signaling on CD4+ T-cell differentiation and plasticity and on the inflammation in tissues other than the central nervous system remains unclear. In this study, we used an antagonist of NAADP signaling, trans-Ned 19, to study the role of NAADP in CD4+ T-cell differentiation and effector function. Partial blockade of NAADP signaling in naïve CD4+ T cells in vitro promoted the differentiation of Th17 cells. Interestingly, trans-Ned 19 also promoted the production of IL-10, co-expression of LAG-3 and CD49b and increased the suppressive capacity of Th17 cells. Moreover, using an IL-17A fate mapping mouse model, we showed that NAADP inhibition promotes conversion of Th17 cells into regulatory T cells in vitro and in vivo. In line with the results, we found that inhibiting NAADP ameliorates disease in a mouse model of intestinal inflammation. Thus, these results reveal a novel function of NAADP in controlling the differentiation and plasticity of CD4+ T cells.

Dual NADPH oxidases DUOX1 and DUOX2 synthesize NAADP and are necessary for Ca2+ signaling during T cell activation.

The formation of Ca2+ microdomains during T cell activation is initiated by the production of nicotinic acid adenine dinucleotide phosphate (NAADP) from its reduced form NAADPH. The reverse reaction­NAADP to NAADPH­is catalyzed by glucose 6-phosphate dehydrogenase (G6PD). Here, we identified NADPH oxidases NOX and DUOX as NAADP-forming enzymes that convert NAADPH to NAADP under physiological conditions in vitro. T cells express NOX1, NOX2, and, to a minor extent, DUOX1 and DUOX2. Local and global Ca2+ signaling were decreased in mouse T cells with double knockout of Duoxa1 and Duoxa2 but not with knockout of Nox1 or Nox2. Ca2+ microdomains in the first 15 s upon T cell activation were significantly decreased in Duox2−/− but not in Duox1−/− T cells, whereas both DUOX1 and DUOX2 were required for global Ca2+ signaling between 4 and 12 min after stimulation. Our findings suggest that a DUOX2- and G6PD-catalyzed redox cycle rapidly produces and degrades NAADP through NAADPH as an inactive intermediate.

Vertically transferred maternal immune cells promote neonatal immunity against early life infections.

During mammalian pregnancy, immune cells are vertically transferred from mother to fetus. The functional role of these maternal microchimeric cells (MMc) in the offspring is mostly unknown. Here we show a mouse model in which MMc numbers are either normal or low, which enables functional assessment of MMc. We report a functional role of MMc in promoting fetal immune development. MMc induces preferential differentiation of hematopoietic stem cells in fetal bone marrow towards monocytes within the myeloid compartment. Neonatal mice with higher numbers of MMc and monocytes show enhanced resilience against cytomegalovirus infection. Similarly, higher numbers of MMc in human cord blood are linked to a lower number of respiratory infections during the first year of life. Our data highlight the importance of MMc in promoting fetal immune development, potentially averting the threats caused by early life exposure to pathogens.

Neutralization of IL-6 inhibits formation of autoreactive TH17 cells but does not prevent loss of renal function in experimental autoimmune glomerulonephritis.

In anti-glomerular basement membrane glomerulonephritis (anti-GBM GN), antibodies and T cells directed against the Goodpasture antigen, the non-collagenous domain of the α3-chain of type IV collagen (α3(IV)NC1), provoke renal inflammation resulting in rapidly progressing crescentic GN. Interleukin 6 (IL-6) is a pleiotropic cytokine with both pro- and anti-inflammatory activities, and IL-6 blockade is successfully used for treatment of diseases associated with acute and chronic inflammation. However, the role of IL-6 in anti-GBM GN is unclear. Here, we use the mouse model of experimental autoimmune glomerulonephritis (EAG) to study the role of IL-6 in anti-GBM GN. DBA/1J mice were immunized with α3(IV)NC1 and developed fatal crescentic GN. Treatment of mice with neutralizing anti-IL-6 antibodies impaired the generation of α3(VI)NC1-specific TH1 and TH17 cells. However, despite lasting reduction of the TH17 cell response, antibody treatment did not prevent crescentic GN. Antibody treatment was also ineffective in a therapeutic setting with pre-existing autoantibodies and T cells. In conclusion, our results indicate that although the blockade of IL-6 impairs the development of autoimmunity against α3(VI)NC1, this treatment does not ameliorate crescentic GN both in a preemptive and a therapeutic approach.

IL-6 signaling in macrophages is required for immunotherapy-driven regression of tumors.

BACKGROUND: High serum interleukin (IL-6) levels may cause resistance to immunotherapy by modulation of myeloid cells in the tumor microenvironment. IL-6 signaling blockade is tested in cancer, but as this inflammatory cytokine has pleiotropic effects, this treatment is not always effective. METHODS: IL-6 and IL-6R blockade was applied in an IL-6-mediated immunotherapy-resistant TC-1 tumor model (TC-1.IL-6) and immunotherapy-sensitive TC-1. CONTROL: Effects on therapeutic vaccination-induced tumor regression, recurrence and survival as well on T cells and myeloid cells in the tumor microenvironment were studied. The effects of IL-6 signaling in macrophages under therapy conditions were studied in Il6rafl/fl×LysMcre+ mice. RESULTS: Our therapeutic vaccination protocol elicits a strong tumor-specific CD8+ T-cell response, leading to enhanced intratumoral T-cell infiltration and recruitment of tumoricidal macrophages. Blockade of IL-6 signaling exacerbated tumor outgrowth, reflected by fewer complete regressions and more recurrences after therapeutic vaccination, especially in TC-1.IL-6 tumor-bearing mice. Early IL-6 signaling blockade partly inhibited the development of the vaccine-induced CD8+ T-cell response. However, the main mechanism was the malfunction of macrophages during therapy-induced tumor regression. Therapy efficacy was impaired in Il6rafl/fl×LysMcre+ but not cre-negative control mice, while no differences in the vaccine-induced CD8+ T-cell response were found between these mice. IL-6 signaling blockade resulted in decreased expression of suppressor of cytokine signaling 3, essential for effective M1-type function in macrophages, and increased expression of the phagocytic checkpoint molecule signal-regulatory protein alpha by macrophages. CONCLUSION: IL-6 signaling is critical for macrophage function under circumstances of immunotherapy-induced tumor tissue destruction, in line with the acute inflammatory functions of IL-6 signaling described in infections.

Interferon regulatory factor 4 controls effector functions of CD8+ memory T cells.

The transcription factor IRF4 is required for CD8+ T cell activation, proliferation, and differentiation to effector cells and thus is essential for robust CD8+ T cell responses. The function of IRF4 in memory CD8+ T cells yet needs to be explored. To investigate the role of IRF4 for maintaining differentiation state and survival of CD8+ memory T cells, we used a mouse model with tamoxifen-inducible Irf4 knockout to preclude effects due to inefficient memory cell differentiation in absence of IRF4. We infected mice with ovalbumin-recombinant listeria and induced Irf4 knockout after clearance of the pathogen. Loss of IRF4 resulted in phenotypical changes of CD8+ memory T cells but did not cause a reduction of the total memory T cell population. However, upon reencounter of the pathogen, CD8+ memory T cells showed impaired expansion and acquisition of effector functions. When compared to CD8+ effector memory T cells, CD8+ tissue-resident memory T cells (TRM cells) expressed higher IRF4 levels. Mice with constitutive Irf4 knockout had diminished CD8+ TRM-cell populations, and tamoxifen-induced Irf4 deletion caused a reduction of this cell population. In conclusion, our results demonstrate that IRF4 is required for effective reactivation but not for general survival of CD8+ memory T cells. Formation and maintenance of CD8+ TRM cells, in contrast, appear to depend on IRF4.