S6K-STING interaction regulates cytosolic DNA-mediated activation of the transcription factor IRF3.

Cytosolic DNA-mediated activation of the transcription factor IRF3 is a key event in host antiviral responses. Here we found that infection with DNA viruses induced interaction of the metabolic checkpoint kinase mTOR downstream effector and kinase S6K1 and the signaling adaptor STING in a manner dependent on the DNA sensor cGAS. We further demonstrated that the kinase domain, but not the kinase function, of S6K1 was required for the S6K1-STING interaction and that the TBK1 critically promoted this process. The formation of a tripartite S6K1-STING-TBK1 complex was necessary for the activation of IRF3, and disruption of this signaling axis impaired the early-phase expression of IRF3 target genes and the induction of T cell responses and mucosal antiviral immunity. Thus, our results have uncovered a fundamental regulatory mechanism for the activation of IRF3 in the cytosolic DNA pathway.

Early specification of CD8+ T lymphocyte fates during adaptive immunity revealed by single-cell gene-expression analyses.

T lymphocytes responding to microbial infection give rise to effector cells that mediate acute host defense and memory cells that provide long-lived immunity, but the fundamental question of when and how these cells arise remains unresolved. Here we combined single-cell gene-expression analyses with 'machine-learning' approaches to trace the transcriptional 'roadmap' of individual CD8(+) T lymphocytes throughout the course of an immune response in vivo. Gene-expression signatures predictive of eventual fates could be discerned as early as the first T lymphocyte division and may have been influenced by asymmetric partitioning of the receptor for interleukin 2 (IL-2Rα) during mitosis. Our findings emphasize the importance of single-cell analyses in understanding fate determination and provide new insights into the specification of divergent lymphocyte fates early during an immune response to microbial infection.

CD8+ T-Lymphocyte-Driven Limbic Encephalitis Results in Temporal Lobe Epilepsy.

OBJECTIVE: Limbic encephalitis (LE) comprises a spectrum of inflammatory changes in affected brain structures including the presence of autoantibodies and lymphoid cells. However, the potential of distinct lymphocyte subsets alone to elicit key clinicopathological sequelae of LE potentially inducing temporal lobe epilepsy (TLE) with chronic spontaneous seizures and hippocampal sclerosis (HS) is unresolved. METHODS: Here, we scrutinized pathogenic consequences emerging from CD8+ T cells targeting hippocampal neurons by recombinant adeno-associated virus-mediated expression of the model-autoantigen ovalbumin (OVA) in CA1 neurons of OT-I/RAG1-/- mice (termed "OVA-CD8+ LE model"). RESULTS: Viral-mediated antigen transfer caused dense CD8+ T cell infiltrates confined to the hippocampal formation starting on day 5 after virus transduction. Flow cytometry indicated priming of CD8+ T cells in brain-draining lymph nodes preceding hippocampal invasion. At the acute model stage, the inflammatory process was accompanied by frequent seizure activity and impairment of hippocampal memory skills. Magnetic resonance imaging scans at day 7 of the OVA-CD8+ LE model revealed hippocampal edema and blood-brain barrier disruption that converted into atrophy until day 40. CD8+ T cells specifically targeted OVA-expressing, SIINFEKL-H-2Kb -positive CA1 neurons and caused segmental apoptotic neurodegeneration, astrogliosis, and microglial activation. At the chronic model stage, mice exhibited spontaneous recurrent seizures and persisting memory deficits, and the sclerotic hippocampus was populated with CD8+ T cells escorted by NK cells. INTERPRETATION: These data indicate that a CD8+ T-cell-initiated attack of distinct hippocampal neurons is sufficient to induce LE converting into TLE-HS. Intriguingly, the role of CD8+ T cells exceeds neurotoxic effects and points to their major pathogenic role in TLE following LE. ANN NEUROL 2021;89:666-685.

Polyglutamine-Related Aggregates Can Serve as a Potent Antigen Source for Cross-Presentation by Dendritic Cells.

Protective MHC class I-dependent immune responses require an overlap between repertoires of proteins directly presented on target cells and cross-presented by professional APC, specifically dendritic cells. How stable proteins that rely on defective ribosomal proteins for direct presentation are captured for cell-to-cell transfer remains enigmatic. In this study, we address this issue using a combination of in vitro (C57BL/6-derived mouse cell lines) and in vivo (C57BL/6 mouse strains) approaches involving stable and unstable versions of OVA model Ags displaying defective ribosomal protein-dependent and -independent Ag presentation, respectively. Apoptosis, but not necrosis, of donor cells was found associated with robust global protein aggregate formation and captured stable proteins permissive for cross-presentation. Potency of aggregates to serve as Ag source was directly demonstrated using polyglutamine-equipped model substrates. Collectively, our data implicate global protein aggregation in apoptotic cells as a mechanism that ensures the overlap between MHC class I epitopes presented directly or cross-presented by APC and demonstrate the unusual ability of dendritic cells to process stable protein aggregates.

Cutting Edge: Tissue Antigen Expression Levels Fine-Tune T Cell Differentiation Decisions In Vivo.

Immune homeostasis in peripheral tissues is, to a large degree, maintained by the differentiation and action of regulatory T cells (Treg) specific for tissue Ags. Using a novel mouse model, we have studied the differentiation of naive CD4+ T cells into Foxp3+ Treg in response to a cutaneous Ag (OVA). We found that expression of OVA resulted in fatal autoimmunity and in prevention of peripheral Treg generation. Inhibiting mTOR activity with rapamycin rescued the generation of Foxp3+ T cells. When we varied the level of Ag expression to modulate TCR signaling, we found that low Ag concentrations promoted the generation of Foxp3+ T cells, whereas high levels expanded effector T cells and caused severe autoimmunity. Our findings indicate that the expression level of tissue Ag is a key determinant of the balance between tissue-reactive effector and peripheral Foxp3+ T cells, which determines the choice between tolerance and autoimmunity.

Appropriate aglycone modification significantly expands the glycan substrate acceptability of α1,6-fucosyltransferase (FUT8).

The α1,6-fucosyltransferase, FUT8, is the sole enzyme catalyzing the core-fucosylation of N-glycoproteins in mammalian systems. Previous studies using free N-glycans as acceptor substrates indicated that a terminal ß1,2-GlcNAc moiety on the Man-α1,3-Man arm of N-glycan substrates is required for efficient FUT8-catalyzed core-fucosylation. In contrast, we recently demonstrated that, in a proper protein context, FUT8 could also fucosylate Man5GlcNAc2 without a GlcNAc at the non-reducing end. We describe here a further study of the substrate specificity of FUT8 using a range of N-glycans containing different aglycones. We found that FUT8 could fucosylate most of high-mannose and complex-type N-glycans, including highly branched N-glycans from chicken ovalbumin, when the aglycone moiety is modified with a 9-fluorenylmethyloxycarbonyl (Fmoc) moiety or in a suitable peptide/protein context, even if they lack the terminal GlcNAc moiety on the Man-α1,3-Man arm. FUT8 could also fucosylate paucimannose structures when they are on glycoprotein substrates. Such core-fucosylated paucimannosylation is a prominent feature of lysosomal proteins of human neutrophils and several types of cancers. We also found that sialylation of N-glycans significantly reduced their activity as a substrate of FUT8. Kinetic analysis demonstrated that Fmoc aglycone modification could either improve the turnover rate or decrease the KM value depending on the nature of the substrates, thus significantly enhancing the overall efficiency of FUT8 catalyzed fucosylation. Our results indicate that an appropriate aglycone context of N-glycans could significantly broaden the acceptor substrate specificity of FUT8 beyond what has previously been thought.

The Serpin-like Loop Insertion of Ovalbumin Increases the Stability and Decreases the OVA 323-339 Epitope Processing Efficiency.

Chicken ovalbumin (cOVA) has been studied for decades primarily due to the robust genetic and molecular resources that are available for experimental investigations. cOVA is a member of the serpin superfamily of proteins that function as protease inhibitors, although cOVA does not exhibit this activity. As a serpin, cOVA possesses a protease-sensitive reactive center loop that lies adjacent to the OVA 323-339 CD4+ T-cell epitope. We took advantage of the previously described single-substitution variant, OVA R339T, which can undergo the dramatic structural transition observed in serpins, to study how changes in loop size and protein stability influence the processing and presentation of the OVA 323-339 epitope. We observed that the OVA R339T loop insertion increases the stability and protease resistance, resulting in the reduced presentation of the OVA 323-339 epitope in vitro. These findings have implications for the design of more effective vaccines for the treatment of infectious diseases and cancer as well as the development of more robust CD4+ T-cell epitope prediction tools.

IL-17A/F enable cholangiocytes to restrict T cell-driven experimental cholangitis by upregulating PD-L1 expression.

BACKGROUND & AIMS: IL-17A-producing T cells are present in autoimmune cholestatic liver diseases; however, little is known about the contribution of IL-17 to periductal immune responses. Herein, we investigated the role of IL-17 produced by antigen-specific CD8+ T cells in a mouse model of cholangitis and in vitro in human cholangiocyte organoids. METHODS: K14-OVAp mice express a major histocompatibility complex I-restricted ovalbumin (OVA) peptide sequence (SIINFEKL) on cholangiocytes. Cholangitis was induced by the adoptive transfer of transgenic OVA-specific ovalbumin transgene (OT)-1 CD8+ T cells that either had OT-1wt or lacked IL-17A/F (OT-1IL17ko). The response of mouse and human cholangiocytes/organoids to IL-17A was assessed in vitro. RESULTS: Transfer of OVA-specific OT-1IL17ko cells significantly aggravated periductal inflammation in K14-OVAp recipient mice compared with transfer of OT-1wt T cells. OT-1IL17ko T cells were highly activated in the liver and displayed increased cytotoxicity and proliferation. IL-17A/F produced by transferred OT-1wt CD8+ T cells induced upregulation of the inhibitory molecule programmed cell death ligand 1 (PD-L1) on cholangiocytes, restricting cholangitis by limiting cytotoxicity and proliferation of transferred cells. In contrast, OT-1IL17ko T cells failed to induce PD-L1 on cholangiocytes, resulting in uncontrolled expansion of cytotoxic CD8+ T cells and aggravated cholangitis. Blockade of PD-L1 after transfer of OT-1wt T cells with anti-PD-L1 antibody also resulted in aggravated cholangitis. Using human cholangiocyte organoids, we were able to confirm that IL-17A induces PD-L1 expression in cholangiocytes. CONCLUSIONS: We demonstrate that by upregulating PD-L1 on cholangiocytes, IL-17 has an important role in restricting cholangitis and protecting against CD8+ T cell-mediated inflammatory bile duct injury. Caution should be exercised when targeting IL-17 for the treatment of cholangitis. LAY SUMMARY: IL-17 is assumed to be a driver of inflammation in several autoimmune diseases, such as psoriasis. IL-17 is also present in inflammatory diseases of the bile duct, but its role in these conditions is not clear, as the effects of IL-17 depend on the context of its expression. Herein, we investigated the role of IL-17 in an experimental autoimmune cholangitis mouse model, and we identified an important protective effect of IL-17 on cholangiocytes, enabling them to downregulate bile duct inflammation via checkpoint inhibitor PD-L1.

Cholinergic neuroplasticity in asthma driven by TrkB signaling.

Parasympathetic neurons in the airways control bronchomotor tone. Increased activity of cholinergic neurons are mediators of airway hyperresponsiveness (AHR) in asthma, however, mechanisms are not elucidated. We describe remodeling of the cholinergic neuronal network in asthmatic airways driven by brain-derived neurotrophic factor (BDNF) and Tropomyosin receptor kinase B (TrkB). Human bronchial biopsies were stained for cholinergic marker vesicular acetylcholine transporter (VAChT). Human lung gene expression and single nucleotide polymorphisms (SNP) in neuroplasticity-related genes were compared between asthma and healthy patients. Wild-type (WT) and mutated TrkB knock-in mice (Ntrk2tm1Ddg/J) with impaired BDNF signaling were chronically exposed to ovalbumin (OVA). Neuronal VAChT staining and airway narrowing in response to electrical field stimulation in precision cut lung slices (PCLS) were assessed. Increased cholinergic fibers in asthmatic airway biopsies was found, paralleled by increased TrkB gene expression in human lung tissue, and SNPs in the NTRK2 [TrkB] and BDNF genes linked to asthma. Chronic allergen exposure in mice resulted in increased density of cholinergic nerves, which was prevented by inhibiting TrkB. Increased nerve density resulted in AHR in vivo and in increased nerve-dependent airway reactivity in lung slices mediated via TrkB. These findings show cholinergic neuroplasticity in asthma driven by TrkB signaling and suggest that the BDNF-TrkB pathway may be a potential target.

Vesicular Stomatitis Virus Encoding a Destabilized Tumor Antigen Improves Activation of Anti-tumor T Cell Responses.

Enhancing the immunogenicity of tumor-associated antigens would represent a major advance for anti-tumor vaccination strategies. Here, we investigated structure-directed antigen destabilization as a strategy to improve the degradation, immunogenic epitope presentation, and T cell activation against a vesicular stomatitis virus (VSV)-encoded tumor antigen. We used the crystal structure of the model antigen ovalbumin to identify charge-disrupting amino acid mutations that were predicted to decrease the stability of the protein. One mutation, OVA-C12R, significantly reduced the half-life of the protein and was preferentially degraded in a 26-S proteasomal-dependent manner. The destabilized ovalbumin protein exhibited enhanced presentation of the major histocompatibility complex (MHC) class I immunogenic epitope, SIINFEKL, on the surface of B16F10 cells or murine bone marrow-derived dendritic cells (BMDCs) in vitro. Enhanced presentation correlated with better recognition by cognate CD8 OT-I T cells as measured by activation, proliferation, and effector cytokine production. Finally, VSV encoding the degradation-prone antigen was better able to prime an antigen ovalbumin-specific CD8 T cell response in vivo without altering the anti-viral CD8 T cell response. Our studies highlight that not only is the choice of antigen in cancer vaccines of importance, but that emphasis should be placed on modifying antigen quality to ensure optimal priming of anti-tumor responses.

TLR signals license CD8 T cells to destroy oligodendrocytes expressing an antigen shared with a Listeria pathogen.

Increasing evidence suggests a role of CD8 T cells in autoimmune demyelinating CNS disease, which, however, is still controversially discussed. Mice, which express ovalbumin (OVA) as cytosolic self-antigen in oligodendrocytes (ODC-OVA mice), respond to CNS infection induced by OVA-expressing attenuated Listeria with CD8 T cell-mediated inflammatory demyelination. This model is suitable to decipher the contribution of CD8 T cells and the pathogen in autoimmune CNS disease. Here, we show that both antigen and pathogen are required in the CNS for disease induction, though not in a physically linked fashion. Intracerebral challenge with combined toll like receptor (TLR) TLR2 and TLR9 as well as TLR7 and TLR9 agonists substituted for the bacterial stimulus, but not with individual TLR agonists (TLR2, TLR3,TLR5,TLR7, TLR9). Furthermore, MyD88 inactivation rendered ODC-OVA mice resistant to disease induction. Collectively, CD8 T cell-mediated destruction of oligodendrocytes is activated if (i) an antigen shared with an infectious agent is provided in the CNS microenvironment and (ii) innate immune signals inform the CNS microenvironment that pathogen removal warrants an immune attack by CD8 T cells, even at the expense of locally restricted demyelination.

The adjuvant effect of melanin is superior to incomplete Freund's adjuvant in subunit/peptide vaccines in mice.

Peptide vaccines represent an attractive alternative to conventional anti-tumor therapies, but have not yet achieved significant clinical efficacy with commonly used formulations. Combination of short antigenic peptides, synthetic melanin and TLR9 agonist (Toll-like receptor 9, CpG-28) was reported as highly efficient to trigger strong CD8 + T-cell responses. We compared this vaccine approach to the standard adjuvant formulation that combines the incomplete Freund's adjuvant (IFA) and CpG-28, using either an ovalbumin epitope (pOVA30) or a spontaneously occurring tumor neoepitope (mAdpgk).Melanin-based vaccine induced significantly higher cytotoxic T lymphocytes (CTL) responses than IFA-based vaccine in both pOVA30- and mAdpgk-targeted vaccines. The anti-tumor efficacy of melanin-based vaccine was further assessed in mice, grafted either with E.G7-OVA cells (E.G7 cells transfected with ovalbumin) or with MC38 cells that spontaneously express the mAdpgk neoepitope. Melanin-based vaccine induced a major inhibition of E.G7-OVA tumor growth when compared to IFA-based vaccine (p < 0.001), but tumors eventually relapsed from day 24. In the MC38 tumor model, no significant inhibition of tumor growth was observed. In both cases, tumor escape appeared related to the loss of antigen presentation by tumor cells (loss of ovalbumin expression in E.G7-OVA model; poor presentation of mAdpgk in MC38 model), although the CTL responses displayed an effector memory phenotype, a high cytolytic potential and low programmed cell death-1 (PD1) expression.In conclusion, synthetic melanin can be efficiently used as an adjuvant to enhance T-cells response against subunit vaccine antigens and compared favorably to the classic combination of IFA and TLR9 agonist in mice.

Modified Vaccinia Virus Ankara Can Induce Optimal CD8+ T Cell Responses to Directly Primed Antigens Depending on Vaccine Design.

A variety of strains of vaccinia virus (VACV) have been used as recombinant vaccine vectors with the aim of inducing robust CD8+ T cell immunity. While much of the pioneering work was done with virulent strains, such as Western Reserve (WR), attenuated strains such as modified vaccinia virus Ankara (MVA) are more realistic vectors for clinical use. To unify this literature, side-by-side comparisons of virus strains are required. Here, we compare the form of antigen that supports optimal CD8+ T cell responses for VACV strains WR and MVA using equivalent constructs. We found that for multiple antigens, minimal antigenic constructs (epitope minigenes) that prime CD8+ T cells via the direct presentation pathway elicited optimal responses from both vectors, which was surprising because this finding contradicts the prevailing view in the literature for MVA. We then went on to explore the discrepancy between current and published data for MVA, finding evidence that the expression locus and in some cases the presence of the viral thymidine kinase may influence the ability of this strain to prime optimal responses from antigens that require direct presentation. This extends our knowledge of the design parameters for VACV vectored vaccines, especially those based on MVA.IMPORTANCE Recombinant vaccines based on vaccinia virus and particularly attenuated strains such as MVA are in human clinical trials, but due to the complexity of these large vectors much remains to be understood about the design parameters that alter their immunogenicity. Previous work had found that MVA vectors should be designed to express stable protein in order to induce robust immunity by CD8+ (cytotoxic) T cells. Here, we found that the primacy of stable antigen is not generalizable to all designs of MVA and may depend where a foreign antigen is inserted into the MVA genome. This unexpected finding suggests that there is an interaction between genome location and the best form of antigen for optimal T cell priming in MVA and thus possibly other vaccine vectors. It also highlights that our understanding of antigen presentation by even the best studied of vaccine vectors remains incomplete.

Route of Antigen Presentation Can Determine the Selection of Foxp3-Dependent or Foxp3-Independent Dominant Immune Tolerance.

It has been shown that dominant tolerance, namely in transplantation, requires Foxp3+ regulatory T cells. Although most tolerance-inducing regimens rely on regulatory T cells, we found that induction of tolerance to proteins in aluminum hydroxide can be achieved in Foxp3-deficient mice using nondepleting anti-CD4 Abs. This type of tolerance is Ag specific, and tolerant mice retain immune competence to respond to unrelated Ags. We demonstrated with chicken OVA-specific TCR-transgenic mice that the same tolerizing protocol (CD4 blockade) and the same target Ag (OVA) achieves Foxp3-dependent transplantation tolerance to OVA-expressing skin grafts, but Foxp3-independent tolerance when the Ag is provided as OVA-aluminum hydroxide. In the latter case, we found that tolerance induction triggered recessive mechanisms leading to elimination of effector cells and, simultaneously, a dominant mechanism associated with the emergence of an anergic and regulatory CTLA-4+IL-2lowFoxp3- T cell population, where the tolerance state is IL-10 dependent. Such Foxp3-independent mechanisms can improve the efficacy of tolerance-inducing protocols.

T helper cells subtypes and their cytokine gene expression affected by carvacrol in sensitized mice administered during sensitization period.

Th1, Th2, Th17, and Treg cells and their cytokine gene expressions in splenocytes of control mice, ovalbumin sensitized (S), and S treated with dexamethasone and carvacrol during a sensitization period were examined. Th2 and Th17 population as well as the gene expression of IL-4, IL-17, and TGF-ß were increased, but Th1, Th1/Th2 ratio, the gene expression of IFN-γ and FOXP3 as well as the IFN-γ/IL-4 ratio were decreased in S compared with control group ( P < 0.001 for all cases). Carvacrol treatment caused significant reduction of Th2 and Th17 population as well as gene expression of IL-4, IL-17, and TGF-ß but increase in Treg cells, Th1/Th2 ratio, gene expressions of FOXP3, IFN-γ, and IFN-γ/IL-4 ratio ( P < 0.05 to P < 0.001). The population of Th1, Th2, Th17 cells as well as the gene expression of IL-4, IL-17, and TGF-ß were significantly decreased, but only Treg was increased in the dexamethasone treatment group ( P < 0.05 to P < 0.001). Carvacrol treatment during the sensitization period showed a more specific effect on Th1/Th2 imbalance in sensitized mice than dexamethasone, which may indicate the therapeutic potentials of carvacrol in disorders associated with Th1/Th2 imbalance such as asthma.

Cystitis-induced bladder pain is Toll-like receptor 4 dependent in a transgenic autoimmune cystitis murine model: a MAPP Research Network animal study.

Altered Toll-like receptor (TLR)4 activation has been identified in several chronic pain conditions but has not been well studied in interstitial cystitis/bladder pain syndrome (IC/BPS). Our previously published human studies indicated that patients with IC/BPS present altered systemic TLR4-mediated inflammatory responses, which were significantly correlated with reported pain severity. In the present study, we sought to determine whether altered TLR4 activation plays a role in pelvic/bladder pain seen in patients with IC/BPS using our validated IC/BPS-like transgenic autoimmune cystitis model (URO-OVA). URO-OVA mice developed responses consistent with pelvic and bladder pain after cystitis induction, which was associated with increased splenocyte production of TLR4-mediated proinflammatory cytokines IL-1ß, IL-6, and TNF-α. Increased spinal expression of mRNAs for proinflammatory cytokines IL-6 and TNF-α, glial activation markers CD11b and glial fibrillary acidic protein, and endogenous TLR4 ligand high mobility group box 1 was also observed after cystitis induction. Compared with URO-OVA mice, TLR4-deficient URO-OVA mice developed significantly reduced nociceptive responses, although similar bladder inflammation and voiding dysfunction, after cystitis induction. Intravenous administration of TAK-242 (a TLR4-selective antagonist) significantly attenuated nociceptive responses in cystitis-induced URO-OVA mice, which was associated with reduced splenocyte production of TLR4-mediated IL-1ß, IL-6, and TNF-α as well as reduced spinal expression of mRNAs for IL-6, TNF-α, CD11b, glial fibrillary acidic protein, and high mobility group box 1. Our results indicate that altered TLR4 activation plays a critical role in bladder nociception independent of inflammation and voiding dysfunction in the URO-OVA model, providing a potential mechanistic insight and therapeutic target for IC/BPS pain.

mRNA Polyplexes with Post-Conjugated GALA Peptides Efficiently Target, Transfect, and Activate Antigen Presenting Cells.

Vaccines based on mRNA have emerged as potent systems to elicit CD8+ T cell responses against various cancers and viral infectious diseases. The efficient intracellular delivery of mRNA molecules encoding antigens into the cytosol of antigen-presenting cells (APCs) is still challenging, requiring cell attachment, active uptake, and subsequent endosomal escape. Here, we report a facile approach for the formulation of peptide-functionalized mRNA polyplexes using copper-free click chemistry to promote presentation of mRNA antigen by dendritic cells (DCs). After screening different membrane active peptides, GALA modified mRNA polyplexes (PPx-GALA) with a size around 350 nm and with a slightly negative surface charge (-7 mV), exhibited the highest EGFP-mRNA transfection in RAW 246.7 macrophages (∼36%) and D1 dendritic cells (∼50%) as compared to polyplexes decorated with melittin or LEDE peptides. Interestingly, we found that PPx-GALA enters DCs through sialic acid mediated endo/phagocytosis, which was not influenced by DC maturation. The PPx-GALA formulation exhibited 18-fold higher cellular uptake compared to a lipofectamine mRNA formulation without inducing cytotoxicity. Live cell imaging showed that PPx-GALA that were taken up by endocytosis induced calcein release from endosomes into the cytosol. DCs treated with PPx-GALA containing mRNA encoding for OVA displayed enhanced T cell responses and DC maturation. Collectively, these data provide a strong rationale for further study of this PPx-GALA formulation in vivo as a promising mRNA vaccine platform.

Repetitive antigen stimulation induces stepwise transcriptome diversification but preserves a core signature of memory CD8(+) T cell differentiation.

Repetitive antigen stimulation by prime-boost vaccination or pathogen reencounter increases memory CD8(+) T cell numbers, but the impact on memory CD8(+) T cell differentiation is unknown. Here we showed that repetitive antigen stimulations induced accumulation of memory CD8(+) T cells with uniform effector memory characteristics. However, genome-wide microarray analyses revealed that each additional antigen challenge resulted in the differential regulation of several hundred new genes in the ensuing memory CD8(+) T cell populations and, therefore, in stepwise diversification of CD8(+) T cell transcriptomes. Thus, primary and repeatedly stimulated (secondary, tertiary, and quaternary) memory CD8(+) T cells differed substantially in their molecular signature while sharing expression of a small group of genes and biological pathways, which may constitute a core signature of memory differentiation. These results reveal the complex regulation of memory CD8(+) T cell differentiation and identify potential new molecular targets to dissect the function of memory cells generated by repeated antigen stimulation.

Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier.

BACKGROUND: Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. RESULTS: We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. CONCLUSION: We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development.