Memory CD8+ T cells generated during acute infections exhibit enhanced effector functions upon reactivation. However, persistent antigen exposure, such as in cancer, can impair their functionality. In this study, we compared memory CD8+ T cells generated following tumor rejection (Tum-CD8+) with those arising from an acute viral infection (Vir-CD8+). Using vaccinia virus and EL4 tumor models expressing the same antigen, we found that Tum-CD8+ cells displayed a distinct phenotype, including sustained expression of inhibitory receptors (PD-1, TIM-3), altered integrin expression, and reduced production of IFNγ and TNF. Despite retaining cytotoxic activity, their protective capacity was compromised, even after viral recall. Transcriptomic and functional analyses revealed that transient tumor exposure imprints a stable, exhaustion-like program on memory CD8+ T cells. These findings highlight how suboptimal priming conditions during tumor challenge durably shape memory T cell responses.

IRF2 plays an indirect role in inflammasome activation by regulating Caspase-4 and Gasdermin D (GSDMD) levels. However, the in vivo relevance of this regulatory circuit is unknown. We generate IRF2 KO mice and demonstrate that they are equally susceptible to Francisella novicida infection as GSDMD KO mice. Interestingly, the phenotypes of IRF2 KO and GSDMD KO mice diverge with respect to IFN-γ. Specifically, IRF2 KO mice exhibit a profound defect in IFN-γ production, which we attribute to an intrinsic role of IRF2 in regulating both the number and maturation of NK cells. IRF2 KO NK cells fail to express the antibacterial effectors IL-18R and Granzyme A, thereby impairing bacterial clearance. IFN-γ therapy partially restores immune responses in IRF2 KO mice and resistance to infection. These findings confirm IRF2 as a dual regulator of inflammasome activity and NK cell function, highlighting its pivotal role in innate immunity. Moreover, they underscore the potential of IFN-γ therapy as a promising treatment for severe infections in patients with primary immunodeficiencies affecting multiple immune pathways.

Post-acute sequelae of COVID-19 (PASC) is a complex and multifaceted clinical challenge requiring to emphasize its underlying pathophysiological mechanisms. This study assessed hundreds of virological, serological, immunological, and tissue damage biomarkers in two patient cohorts who experienced mild (n=270) or severe (n=188) COVID-19, 6 to 9 months post-initial infection, and in which 40% and 57.4% of patients, respectively, developed PASC. Blood analysis showed that mains differences observed in humoral, viral, and biological biomarkers were associated with the initial COVID-19 severity, rather than being specifically linked to PASC. However, patients with PASC displayed altered CD4+ and CD8+ memory T-cell subsets, with higher cytokine-secreting cells and increased terminally differentiated CD45RA+ effector memory T cells (TEMRA). Elevated SARS-CoV-2-specific T cells responsive to nucleocapsid/membrane proteins with a TEMRA phenotype were also observed. A random forest model identified these features and initial symptom duration as top variables discriminating PASC, achieving over 80% classification accuracy.

Post-acute sequelae of COVID-19 (PASC) are a complex clinical condition that requires a better understanding of its underlying biological mechanisms. In this study, we assessed hundreds of virological, serological, immunological, and tissue damage biomarkers in two cohorts of patients who had experienced either mild (n = 270) or severe (n = 188) COVID-19, 6 to 9 months post-initial infection, and in which 40% and 57.4% of patients, respectively, developed PASC. Blood analysis showed that the main differences observed in humoral, viral, and biological biomarkers were associated with the initial COVID-19 severity, rather than being specifically linked to PASC. However, patients with PASC displayed altered CD4+ and CD8+ memory T cell subsets, with higher cytokine-secreting cells and increased terminally differentiated CD45RA+ effector memory T cells (TEMRA). Elevated SARS-CoV-2-specific T cells responsive to nucleocapsid/membrane proteins with a TEMRA phenotype were also observed. A random forest model identified these features and initial symptom duration as top variables discriminating PASC, achieving over 80% classification accuracy.

Memory CD8 T cells typically exhibit improved effector functions compared to their naive counterparts. However, under certain activation conditions, such as chronic viral infections or cancer, these cells may develop functional defects. In this study, we compared the functional quality of memory CD8 T cells generated following tu-mor rejection with those arising from an acute viral infection. We found that tumor-induced (Tum-CD8) memory cells exhibited a distinct phenotype and transcriptomic profile compared with viral-induced (Vir-CD8) memory cells. These memory cells are characterized by the expression of inhibitory receptors and displayed altered functions including reduced IFNg and TNFa production as well as changes in integrin expression. Additionally, the protective capacity of Tum-CD8 memory cells was flawed relative to that of Vir-CD8 memory cells. Importantly, the functional defects of Tum-CD8 persisted upon viral recall. Together, these findings indicate that transient tumoral stimulation can imprint a stable partial exhaustion-like program on memory CD8 T cells. ### Competing Interest Statement The authors have declared no competing interest.

To investigate the impact of paracrine IL -2 signals on memory precursor (MP) cell differentiation, we activated CD8 T cell in vitro in the presence or absence of exogenous IL -2 (ex -IL -2). We assessed memory differentiation by transferring these cells into virus -infected mice. Both conditions generated CD8 T cells that participate in the ongoing response and gave rise to similar memory cells. Nevertheless, when transferred into a naive host, T cells activated with ex -IL -2 generated a higher frequency of memory cells displaying increased functional memory traits. Single -cell RNA-seq analysis indicated that without ex -IL -2, cells rapidly acquire an MP signature, while in its presence they adopted an effector signature. This was confirmed at the protein level and in a functional assay. Overall, ex -IL -2 delays the transition into MP cells, allowing the acquisition of effector functions that become imprinted in their progeny. These findings may help to optimize the generation of therapeutic T cells.

Ly49+ CD8 T cells are memory-phenotype (MP) cells expressing Ly49 family inhibitory receptors, that control autoimmune diseases development through the regulation of follicular CD4 T cells. During viral infection, their number increases, suggesting an involvement in antiviral responses. These cells do not derive from naive cells and are thought to develop in the thymus. This study identified two subsets of Ly49+ CD8 T cells, based on CD8beta expression, in mice and humans. Lineage tracing and reliance on the transcription factor Zeb1 indicate a thymic origin via agonist selection. scRNAseq analysis revealed that a small fraction of CD8alphabeta-Ly49+ cells acquired an effector profile during vaccinia virus infection. Moreover, the majority of Ly49+ CD8 T cells seems to respond to cytokine-driven bystander signals. In vitro, these signals promoted the expression of effector molecules such as IFNgamma and granzyme B, as well as the homing chemokine receptor CXCR5, potentially driving their recruitment to germinal centers. ### Competing Interest Statement The authors have declared no competing interest.

OBJECTIVES:In the post-SARS-CoV-2 pandemic era, "breakthrough infections" are still documented, due to variants of concerns (VoCs) emergence and waning humoral immunity. Despite widespread utilization, the definition of the anti-Spike (S) immunoglobulin-G (IgG) threshold to define protection has unveiled several limitations. Here, we explore the advantages of incorporating T-cell response assessment to enhance the definition of immune memory profile. METHODS:SARS-CoV-2 interferon-gamma release assay test (IGRA) was performed on samples collected longitudinally from immunocompetent healthcare workers throughout their immunization by infection and/or vaccination, anti-receptor-binding domain IgG levels were assessed in parallel. The risk of symptomatic infection according to cellular/humoral immune capacities during Omicron BA.1 wave was then estimated. RESULTS:Close to 40% of our samples were exclusively IGRA-positive, largely due to time elapsed since their last immunization. This suggests that individuals have sustained long-lasting cellular immunity, while they would have been classified as lacking protective immunity based solely on IgG threshold. Moreover, the Cox regression model highlighted that Omicron BA.1 circulation raises the risk of symptomatic infection while increased anti-receptor-binding domain IgG and IGRA levels tended to reduce it. CONCLUSION:The discrepancy between humoral and cellular responses highlights the significance of assessing the overall adaptive immune response. This integrated approach allows the identification of vulnerable subjects and can be of interest to guide antiviral prophylaxis at an individual level.

Dengue virus (DENV) infection continues to be a public health challenge, lacking a specific cure. Vaccination remains the primary strategy against dengue; however, existing live-attenuated vaccines display variable efficacy across four serotypes, influenced by host serostatus and age, and predominantly inducing humoral responses. To address this limitation, this study investigates a multiepitope-based immunogen designed to induce robust cellular immunity across all DENV serotypes. The chimeric immunogen integrates H-2d specific MHC-I binding T-cell epitopes derived from conserved domains within the DENV envelope protein. Immuno-informatics analyses supported its stability, non-allergenic nature, and strong MHC-I binding affinity as an antigen. To assess the immunogenicity of the multiepitope, it was expressed in murine bone-marrow-derived dendritic cells (BMDCs) that were used to prime mice. In this experimental model, simultaneous exposure to T-cell epitopes from all four DENV serotypes initiated distinct IFNγ-CD8 T-cell responses for different serotypes. These results supported the potential of the multiepitope construct as a vaccine candidate. While the optimization of the immunogen design remains a continuous pursuit, this proof-of-concept study provides a starting point for evaluating its protective efficacy against dengue infection in vivo. Moreover, our results support the development of a multiepitope vaccine that could trigger a pan-serotype anti-dengue CD8 response.

Abstract Adoptive cell transfer between genetically identical hosts is a key tool in the study of the murine immune system. In most models, host and donor differ by a congenic marker, i.e. alleles of a gene coding for a surface protein expressed by immune cells and which allows to trace the donor cells using an epitope specific antibody (Ab). CD45, a glycoprotein expressed by all hematopoietic cells and encoded by the Ptprc gene, constitute one of the main congenic markers used. Its two isoforms, CD45.1 and CD45.2, can be discriminated by flow cytometry using the anti-CD45.1 (clone A20) and anti-CD45.2 (clone 104) Ab. As a consequence, C57BL/6J (B6; CD45.2) and B6.SJL-Ptprca Pepcb/BoyJ (B6.SJL; CD45.1) mice are widely used in adoptive cell transfer experiments, under the presumption that they differ only at the CD45 (Ptprc) locus. However, the B6.SJL congenic mouse was generated by crossing an SJL mouse with a B6 mouse, followed by serial back-crossing on B6 mice with the selection of CD45.1 expressing animals at each generation. Recent studies identified genetic variations between these congenic strains and highlighted, among other, a differential expression of cathepsin E (CTSE). B6.SJL mouse presents a number of functional differences in hematopoietic stem cell (HSC) engraftment-potential or immune cell numbers. We show that B6 and B6.SJL mice also differ in their CD8 T cells compartment and CD8 T cells responses to viral infection compared to B6 mouse. We identified Ctse as the most differentially expressed gene between CD8 T cells of B6 and B6.SJL demonstrate that the differences reported between these two mouse strains are not due to CTSE. In order to avoid biases associated with known or unknown polymorphisms found in congenic mice, we used CRISPR/Cas9 genome editing to introduce, in the Ptprc gene of B6 mouse (CD45.2 epitope), the point mutation(s) associated with its transformation into the CD45.1 epitope. We show that only one nucleotide mutation (A904G) leading to an amino acid change (K302E) is sufficient for the protein to be recognized by the anti-CD45.1 Ab. We show that this new B6-Ptprcem(K302E)Jmar/J mouse has similar immune compartment compared to B6 mouse and does not show any difference in HSC engraftment or CD8 antiviral responses. This new strain resolves the experimental biases reported between B6 and B6.SJL mouse lines and should thus represent the new gold standard for adoptive transfer experiments in B6.

Supplementary Text - PDF file 132K, Supplementary Material and methods, and Supplementary figure legends

ABSTRACT Increasing evidence suggests that the beta genus of human papillomavirus (β-HPV) cooperates with ultraviolet B (UVB) radiation in inducing non-melanoma skin cancer (NMSC). For instance, expression of E6 and E7 oncoproteins from cutaneous human papillomavirus type 38 (HPV38) in a transgenic mouse model (Tg38) leads to the development of squamous cell carcinoma (SCC) after chronic UVB exposure. Transcriptomic analysis of the Tg38 SCC samples revealed a deregulation of inflammasome pathway-related genes such as IL-18, which is consistent with the well-known capability of several oncogenic viruses to modulate the immune response, in order to efficiently produce new progeny. However, the mechanism at the gene expression level by which UVB and HPV38 synergize and modulate the innate immune response has not been elucidated yet. Here, we propose a new molecular mechanism for the regulation of the IL-18 promoter that involves direct binding of p53 to specific response elements. This mechanism seems to be impaired in the presence of the β-HPV38 oncoproteins. Instead, a newly described inhibitory complex formed by DNA methyltransferase 1 (DNMT1)/PKR/ΔNp73α is recruited to the region formerly occupied by p53 in primary keratinocytes. Together, these results corroborate our hypothesis that β-HPV38 plays an important role in the inhibition of the ultraviolet-induced inflammasome response. This downregulation is likely to impair the ability of keratinocytes to recruit the immune cell population at the UVB-exposed area, causing a defect in the clearance of cells harboring DNA damage and promoting a favorable environment for the progression of NMSC. IMPORTANCE Here, we demonstrate that the direct binding of p53 on the IL-18 promoter region regulates its gene expression. However, the presence of E6 and E7 from human papillomavirus type 38 impairs this mechanism via a new inhibitory complex formed by DNA methyltransferase 1 (DNMT1)/PKR/ΔNp73α, which binds to the region formerly occupied by p53 in primary keratinocytes.

Cellular senescence is induced by many stresses including telomere shortening, DNA damage, oxidative, or metabolic stresses. Senescent cells are stably cell cycle arrested and they secrete many factors including cytokines and chemokines. Accumulation of senescent cells promotes many age-related alterations and diseases. In this study, we investigated the role of the pro-senescent phospholipase A2 receptor 1 (PLA2R1) in regulating some age-related alterations in old mice and in mice subjected to a Western diet, whereas aged wild-type mice displayed a decreased ability to regulate their glycemia during glucose and insulin tolerance tests, aged Pla2r1 knockout (KO) mice efficiently regulated their glycemia and displayed fewer signs of aging. Loss of Pla2r1 was also found protective against the deleterious effects of a Western diet. Moreover, these Pla2r1 KO mice were partially protected from diet-induced senescent cell accumulation, steatosis, and fibrosis. Together these results support that Pla2r1 drives several age-related alterations, especially in the liver, arising during aging or through a Western diet.

Adoptive cell transfer between genetically identical hosts relies on the use of a congenic marker to distinguish the donor cells from the host cells. CD45, a glycoprotein expressed by all hematopoietic cells, is one of the main congenic markers used because its two isoforms, CD45.1 and CD45.2, can be discriminated by flow cytometry. As a consequence, C57BL/6J (B6; CD45.2) and B6.SJL-Ptprca Pepcb/BoyJ (B6.SJL; CD45.1) mice are widely used in adoptive cell transfer experiments, under the presumption that they differ only at the CD45 (Ptprc) locus. However, recent studies have identified genetic variations between these congenic strains and have notably highlighted a differential expression of cathepsin E (CTSE). The B6.SJL mouse presents a number of functional differences in hematopoietic stem cell engraftment potential and immune cell numbers compared with the B6 mouse. In this study, we showed that B6 and B6.SJL mice also differ in their CD8+ T cell compartment and CD8+ T cell responses to viral infection. We identified Ctse as the most differentially expressed gene between CD8+ T cells of B6 and B6.SJL and demonstrated that the differences reported between these two mouse strains are not due to CTSE. Finally, using CRISPR–Cas9 genome editing, we generated a CD45.1-expressing B6 mouse by inserting one nucleotide mutation (A904G) leading to an amino acid change (K302E) in the Ptprc gene of the B6 mouse. We showed that this new B6-Ptprcem(K302E)Jmar/J mouse resolves the experimental biases reported between the B6 and B6.SJL mouse lines and should thus represent the new gold standard for adoptive cell transfer experiments in B6. The authors used a gene-editing approach to generate a C57BL/6J mouse model expressing the CD45.1 epitope. The model, which overcomes some of the issues reported with the congenic mouse B6.SJL, could be useful for adoptive cell transfer experiments.

Supplementary Figures - PDF file 322K, S1. Spontaneous MMTVneu display high Her2/neu expression. S2. FoxP3 expression among CD4+ CD25+ T cells correlates with CD45RB negativity. S3. NEU15WT TApDC display unaltered uptake ability. S4. NEU15WT TApDC display unaltered TLR7 and TLR9 expression. S5. Fold changes in gene expression upon intratumoral TLR7L injection. S6. Intratumoral TLR7L or CpG injection induced distinct immune cell recruitment. S7. List of flurochrome-conjugated antibodies used for multi-parametric flow cytometry analysis

Staphylococcus aureus (S. aureus) is a pathogen associated with a wide variety of diseases, from minor to life-threatening infections. Antibiotic-resistant strains have emerged, leading to increasing concern about the control of S. aureus infections. The development of vaccines may be one way to overcome these resistant strains. However, S. aureus ability to internalize into cells – and thus to form a reservoir escaping humoral immunity – is a challenge for vaccine development. A role of T cells in the elimination of persistent S. aureus has been established in mice but it remains to be established if CD8+ T cells could display a cytotoxic activity against S. aureus infected cells. We examined in vitro the ability of CD8+ T cells to recognize and kill dendritic cells infected with S. aureus. We first evidenced that both primary mouse dendritic cells and DC2.4 cell line can be infected with S. aureus. We then generated a strain of S. aureus expressing a model CD8 epitope and transgenic F5 CD8+ T cells recognizing this model epitope were used as reporter T cells. In response to S. aureus-infected dendritic cells, F5 CD8+ T cells produced IFN-γ in an antigen-specific manner and displayed an increased ability to kill infected cells. Altogether, these results demonstrate that cells infected by S. aureus display bacteria-derived epitopes at their surface that are recognized by CD8+ T cells. This paves the way for the development of CD8+ T cell-based therapies against S. aureus.

The diversity of vaccination modalities and infection history are both variables that have an impact on the immune memory of individuals vaccinated against SARS-CoV-2. To gain more accurate knowledge of how these parameters imprint on immune memory, we conducted a long-term follow-up of SARS-CoV-2 spike protein–specific immune memory in unvaccinated and vaccinated COVID-19 convalescent individuals as well as in infection-naïve vaccinated individuals. Here, we report that individuals from the convalescent vaccinated (hybrid immunity) group have the highest concentrations of spike protein–specific antibodies at 6 months after vaccination. As compared with infection-naïve vaccinated individuals, they also display increased frequencies of an atypical mucosa-targeted memory B cell subset. These individuals also exhibited enhanced T H 1 polarization of their SARS-CoV-2 spike protein–specific follicular T helper cell pool. Together, our data suggest that prior SARS-CoV-2 infection increases the titers of SARS-CoV-2 spike protein–specific antibody responses elicited by subsequent vaccination and induces modifications in the composition of the spike protein–specific memory B cell pool that are compatible with enhanced functional protection at mucosal sites.

BackgroundEpicutaneous immunotherapy (EPIT) protocols have recently been developed to restore tolerance in patients with food allergy. The mechanisms by which EPIT protocols promote desensitization rely on a profound immune deviation of pathogenic T- and B-cell responses.ObjectiveTo date, little is known about the contribution of skin dendritic cells (skDCs) to T-cell remodeling and EPIT efficacy.MethodsWe capitalized on a preclinical model of food allergy to ovalbumin (OVA) to characterize the phenotype and functions of OVA+ skDCs throughout the course of EPIT.ResultsOur results showed that both Langerhans cells and dermal conventional cDC1 and cDC2 subsets retained their ability to capture OVA in the skin and to migrate toward the skin-draining lymph nodes during EPIT. However, their activation/maturation status was significantly impaired, as evidenced by the gradual and selective reduction of CD86, CD40, and OVA protein expression in respective subsets. Phenotypic changes during EPIT were also characterized by a progressive diversification of single-cell gene signatures within each DC subset. Interestingly, we observed that OVA+ Langerhans cells progressively lost their capacity to prime CD4+ TEFF cells, but gained regulatory T-cell stimulatory properties. In contrast, cDC1 were inefficient in priming CD4+ TEFF cells or in reactivating TMEM cells in vitro, whereas cDC2 retained moderate stimulatory properties, and progressively biased type 2 immunity toward type 1 and type 17 responses.ConclusionsOur results therefore emphasize that the acquisition of distinct phenotypic and functional specializations by skDCs during EPIT is at the cornerstone of the desensitization process.

In this work, we studied the generation of memory precursor cells following an acute infection by analyzing single-cell RNA-seq data that contained CD8 T cells collected during the post-infection expansion phase. We used different tools to reconstruct the developmental trajectory that CD8 T cells followed after activation. Cells that exhibited a memory precursor signature were identified and positioned on this trajectory. We found that memory precursors are generated continuously with increasing numbers being generated over time. Similarly, expression of genes associated with effector functions was also found to be raised in memory precursors at later time points. The ability of cells to enter quiescence to differentiate into memory cells was confirmed by BrdU pulse-chase experiment in vivo. Analysis of cell counts indicates that the vast majority of memory cells are generated at later time points from cells that have extensively divided.

Background; As the Covid-19 pandemic remains uncontrolled despite the introduction of multiple vaccines, it is essential to identify populations with the highest risk of being infected or re-infected.Methods: To address this question, Spike-specific humoral and cellular immunity were compared in several groups of subjects from different cohorts, 6 months post-infection or post-vaccination in naïve or previously infected individuals (convalescents).Findings: Vaccinated convalescent subjects have significantly higher Spike-specific antibody levels, serum neutralization capacity, and memory T cell frequency than naïve vaccinated or unvaccinated convalescent individuals. This advantage is independent of the vaccination regimen post infection (ChAdOx1, BNT162b2 one dose, BNT162b2 two doses). Moreover, it correlates with a lower frequency of breakthrough infections recorded in the convalescent vaccinated group (N=1/2572) compared to vaccinated without prior infection (N=112/10614), in a two-month real life monitoring during the fifth Covid-19 wave dominated by the Delta variant.Interpretation: Hybrid immunity conferred by vaccination following infection is greatly superior to all other forms of immunity. These results suggest that vaccinated individuals with no history of prior infection should be prioritized for booster doses 6 months post initial vaccination and that monitoring of spike-specific immunity is required for vaccines follow-up.Trial Registration Details: The study was registered on ClinicalTrials.gov (NCT04341142).Funding Information: This study was supported by ANRS-MIE (Emergen study), REACTing (Research and ACTion targeting emerging infectious diseases)-INSERM, the Fondation AnBer (http://fondationanber.fr/).and institutional grants from INSERM, CNRS, UCBL1, and ENS de Lyon.Declaration of Interests: Philippe Vanhems declared the following conflict of interest: grants and consulting fees by SANOFI, PFIZER, MSD, Astellas since 2018. Christelle Compagnon and Karen Brengel-Pesce are employees of Biomerieux. The other authors have no conflict of interest to declare.Ethics Approval Statement: The use and analysis of data from the occupational health medical file were authorized after a regulatory declaration to the National Commission for Information Technology and Civil Liberties (CNIL) according to the reference methodology (declaration MR004 n° 20-121 of April 30th 2020). Written informed consent was obtained from all participants; ethics approval was obtained from the national review board for biomedical research in April 2020 (Comité de Protection des Personnes Sud Méditerranée I, Marseille, France; ID RCB 2020-A00932-37).