Antigen-specific Memory CD8 Research Articles

Metabolic Reprogramming by the Excessive AMPK Activation Exacerbates Antigen-Specific Memory CD8+ T Cell Differentiation after Acute Lymphocytic Choriomeningitis Virus Infection.

During virus infection, T cells must be adapted to activation and lineage differentiation states via metabolic reprogramming. Whereas effector CD8+ T cells preferentially use glycolysis for their rapid proliferation, memory CD8+ T cells utilize oxidative phosphorylation for their homeostatic maintenance. Particularly, enhanced AMP-activated protein kinase (AMPK) activity promotes the memory T cell response through different pathways. However, the level of AMPK activation required for optimal memory T cell differentiation remains unclear. A new metformin derivative, IM156, formerly known as HL156A, has been reported to ameliorate various types of fibrosis and inhibit in vitro and in vivo tumors by inducing AMPK activation more potently than metformin. Here, we evaluated the in vivo effects of IM156 on antigen-specific CD8+ T cells during their effector and memory differentiation after acute lymphocytic choriomeningitis virus infection. Unexpectedly, our results showed that in vivo treatment of IM156 exacerbated the memory differentiation of virus-specific CD8+ T cells, resulting in an increase in short-lived effector cells but decrease in memory precursor effector cells. Thus, IM156 treatment impaired the function of virus-specific memory CD8+ T cells, indicating that excessive AMPK activation weakens memory T cell differentiation, thereby suppressing recall immune responses. This study suggests that metabolic reprogramming of antigen-specific CD8+ T cells by regulating the AMPK pathway should be carefully performed and managed to improve the efficacy of T cell vaccine.

Selective Targeting of Multiple Myeloma By Bcma-Specific Central Memory CD8+ cytotoxic T Lymphocytes: A Potential Immunotherapeutic Application in Multiple Myeloma and Other Plasma Cell Disorders

Background:Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation and accumulation of malignant plasma cells in the bone marrow and development of osteolytic bone lesions. Despite recent advances in treatment using novel therapeutics, MM remains incurable with high mortality rates. We have demonstrated in preclinical studies that CD8+ cytotoxic T lymphocytes (CTL) generated with immunogenic HLA-A2 or HLA-A24 peptides targeting XBP1(X-box binding protein 1), CD138 (Syndecan-1) and CS1 (SLAMF7) antigens induces robust cytotoxic activities against MM. The Phase 1/2a trials were completed or are in progress in the patients with smoldering multiple myeloma or triple negative breast cancer, respectively, using the HLA-A2 XBP1/CD138/CS1 multipeptide vaccine. The clinical data demonstrated that the multipeptide vaccine is safe and induces the XBP1/CD138/CS1-specific immune responses, evidenced by expansion of peptides-specific Tetramer+/CD45RO+memory CTL and Th-1 specific immune responses. Moreover, clinical trials combining with Lenalidomide or checkpoint inhibitor enhanced the CD8+ CTL activities induced by multipeptide vaccine, indicating the benefit of combination therapy. To expand the breadth and extent of the antigens-specific immunotherapy beyond XBP1/CD138/CS1, we have recently identified additional tumor-associated antigens (TAA) on tumor cells obtained from newly diagnosed MM patients (N=616). Here, we introduce a novel heteroclitic peptides specific to BCMA, the receptor forbinding of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL). Due to its restricted expression pattern on MM cells and plasma cells along with its critical role in promoting MM cell growth, survival and drug resistance, we are currently in development of novel immunotherapeutic to target BCMA on MM cells with different therapeutic approaches.Objective:The aims of current study were to target BCMA as a TAA by generating the antigen-specific memory CD8+ CTL to induce effective and long-lasting immune response against MM.Findings:We report on immunogenic HLA-A2-restricted peptides derived from BCMA, which are capable of evoking antigen-specific immune responses against MM. The heteroclitic BCMA peptides displayed improved binding affinity/stability to HLA-A2 molecules from their native BCMA peptides. To define immunogenicity of the selected peptides, we generated BCMA-specific CTL (BCMA-CTL) by repeated stimulationof CD3+ T cells with respective heteroclitic peptide. The BCMA-specific engineered peptides evoked the expansion of antigen-specific CD8+ CTL and generated BCMA-CTL displaying high T cell activation (CD38, CD69) and co-stimulatory (CD40L, OX40, GITR) molecules expression. Additionally, a gradual expansion was observed in BCMA-specific memory CD8+ T cells, with a corresponding decrease in naïve CD8+ T cells. The BCMA-CTL demonstrated robust poly-functional immune responses with Th1-specific anti-MM activities [high IFN-g/IL-2/TNF-aproduction, CD8+ T cells proliferation, cytotoxicity] in antigen-specific and HLA-A2-restricted manner. The functional activities were directly correlated with the expansion of central memory CD8+ CTL in the BCMA-CTL generated from different HLA-A2+ individuals (Donor 1 BCMA-CTL: 81.0%, Donor 2 BCMA-CTL: 82.6%, Donor 3 BCMA-CTL: 67.0%). Finally, the combination with checkpoint inhibitor (anti-LAG3) or immune agonist (anti-OX40) enhanced the anti-tumor activities of BCMA-CTL, along with the induction of cytotoxic activities by central memory CD8+ T cell subset. Therefore, these studies suggest that heteroclitic BCMA peptides offer a therapeutic potential to effectively generate BCMA-specific CD8+ CTL targeting MM.Significance:Here, we introduce novel immunogenic engineered heteroclitic BCMA peptides capable of inducing antigen-specific memory CD8+ CTL with robust poly-functional immune responses against MM. These results provide the framework for therapeutic application of heteroclitic BCMA peptides in MM patients. They further support combination treatment options incorporating BCMA peptides-specific vaccine or BCMA peptides-specific adoptive T cells immunotherapy with anti-LAG3 and/or anti-OX40for patients with myeloma or other diseases expressing BCMA. DisclosuresMunshi:OncoPep: Other: Board of director. Anderson:Celgene: Consultancy; Takeda Millennium: Consultancy; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership.

Bcma Heteroclitic Peptide Encapsulated Nanoparticle Enhances Antigen Stimulatory Capacity and Tumor-Specific CD8+ cytotoxic T Lymphocytes Against Multiple Myeloma

Background:B-cell Maturation Antigen (BCMA), a member of the tumor necrosis factor (TNF) receptor superfamily and the receptor for binding of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), is a promising therapeutic target for MM. Due to its restricted expression pattern on MM cells and plasma cells along with its role in promoting MM cells growth, survival, and drug resistance, BCMA is being targeted by several immunotherapeutic strategies including antibodies, immunotoxins, bispecific T-cell engagers, and CAR-T cells. Recently, we have identified nanomedicine-based therapeutics targeting BCMA as a promising area of translational research to effectively evoke and augment anti-tumor responses in MM patients. Several nanomedicines are available and more advanced nanoparticle constructs are under development for antigen encapsulation. To this end, we have designed a heteroclitic BCMA peptide encapsulated nanoparticle-based cancer vaccine to overcome the limitations of free peptide vaccines including poor peptide stability, susceptibility to enzyme degradation, and low antigen uptake and delivery. Furthermore, the nanotechnology-based cancer vaccine was developed to induce more robust BCMA-specific CD8+ cytotoxic T lymphocytes (CTL) activities in MM patients, with more sustained antigen release and increased bioavailability and presentation of the immunogenic peptide. Here, we examine the potential of a novel nanomedicine-based therapeutic delivery system specific to BCMA antigen to treat the patients with MM.Objective:The purpose of this study was to design the optimal nanoparticle encapsulated BCMA antigen constructs to efficiently evoke and develop the BCMA-specific CD8+ CTL with functional anti-myeloma activities.Findings:Nanoparticles [liposome or poly(D,L-lactide-co-glycolide) (PLGA)] with different antigen-release kinetics demonstrated their capacity to effectively deliver heteroclitic BCMA peptideto antigen-presenting cells and evoke BCMA antigen-specific CTL with anti-MM activities. The heteroclitic BCMA peptide encapsulated nanoparticles demonstrated a higher uptake by human dendritic cells than free peptide, with the highest uptake mediated with liposome-based nanoparticles. In contrast, BCMA-specific CTL induced with PLGA-based nanoparticle demonstrated the highest functional activities and specific immune responses against MM cells. The PLGA/BCMA peptide nanoparticle induced BCMA-specific CTL displayed the highest increases in CD107a degranulation, the antigen-specific CD8+ T cells proliferation and Th-1 type cytokines (IFN-g, IL-2, TNF-a) production to MM patients' tumor cells and MM cell lines compared to BCMA-CTL generated with free BCMA peptide or liposome/BCMA peptide nanoparticle. These observations were aligned with the highest level of CD28 costimulatory molecules upregulation, Tetramer+ CTL generation and peptide-specific responses within the BCMA-CTL generated by PLGA/BCMA nanoparticles. Furthermore, the PLGA/BCMA nanoparticles triggered a more robust induction of antigen-specific memory CD8+ T cells, which demonstrated significantly higher anti-tumor activities, evidenced by CD107a degranulation and IFN-g production, compared to non-memory CD8+ T cells within the BCMA-CTL. Especially, the increased central memory CTL development and their anti-tumor activities evoked by PLGA/BCMA peptide were associated with the optimal peptide release kinetics and enhanced immunogenicity of the antigen via this nanotechnology. Thus, these results demonstrate that the heteroclitic BCMA peptide encapsulated nanoparticle strategy supports the peptide delivery into dendritic cells and then subsequently to T cells, resulting in effective induction of BCMA-specific central memory CTL with poly-functional activities against MM.Significance:These results demonstrate the utility of nanotechnology using encapsulated heteroclitic BCMA peptide to enhance the immunogenicity of BCMA peptide-specific therapeutics against MM. Importantly, our observations provide the framework for therapeutic application of PLGA-based heteroclitic BCMA peptide delivery to enhance the BCMA-specific memory T cell immune responses, overcome the limitations of current peptide-based cancer vaccine, and improve the patient outcome in MM. DisclosuresMunshi:OncoPep: Other: Board of director. Anderson:Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Millennium Takeda: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Gilead: Membership on an entity's Board of Directors or advisory committees.

A Highly Active Form of XCL1/Lymphotactin Functions as an Effective Adjuvant to Recruit Cross-Presenting Dendritic Cells for Induction of Effector and Memory CD8+ T Cells.

The chemokine receptor XCR1 is known to be selectively expressed by cross-presenting dendritic cells (DCs), while its ligand XCL1/lymphotactin is mainly produced by activated CD8+ T cells and natural killer cells. Recent studies have shown that XCL1-antigen fusion proteins efficiently induce CD8+ T cell responses by preferentially delivering antigens to XCR1+ DCs. However, XCL1 per se was found to be a poor adjuvant for induction of CD8+ T cell responses. XCL1 is unique because of its lack of one of the two disulfide bonds commonly conserved in all other chemokines and thus has an unstable structure with a relatively weak chemokine activity. In the present study, we generated a variant form of murine XCL1 termed mXCL1-V21C/A59C that contained a second disulfide bond to stabilize its chemokine structure. We confirmed that mXCL1-V21C/A59C had much more potent chemotactic and calcium mobilization activities than the wild type XCL1 (mXCL1-WT). Intradermal injection of mXCL1-V21C/A59C, but not that of mXCL1-WT, significantly increased the accumulation of XCR1+CD103+ DCs in the injection site, and most of the accumulated XCR1+CD103+ DCs were found to take up co-injected ovalbumin (OVA). Furthermore, recruited XCR1+CD103+ DCs efficiently migrated to the draining lymph nodes and stayed for a prolonged period of time. Consequently, mXCL1-V21C/A59C strongly induced OVA-specific CD8+ T cells. The combination of OVA and mXCL1-V21C/A59C well protected mice from E.G7-OVA tumor growth in both prophylactic and therapeutic protocols. Finally, memory CTL responses were efficiently induced in mice immunized with OVA and mXCL1-V21C/A59C. Although intradermal injection of OVA and polyinosinic-polycytidylic acid (poly(I:C)) as an adjuvant also induced CD8+ T cell responses to OVA, poly (I:C) poorly recruited XCR1+CD103+ DCs in the injection site and failed to induce significant memory CTL responses to OVA. Collectively, our findings demonstrate that a highly active form of XCL1 is a promising vaccine adjuvant for cross-presenting DCs to induce antigen-specific effector and memory CD8+ T cells.

Defining Memory CD8 T Cell.

CD8 T cells comprising the memory pool display considerable heterogeneity, with individual cells differing in phenotype and function. This review will focus on our current understanding of heterogeneity within the antigen-specific memory CD8 T cell compartment and classifications of memory CD8 T cell subsets with defined and discrete functionalities. Recent data suggest that phenotype and/or function of numerically stable circulatory memory CD8 T cells are defined by the age of memory CD8 T cell (or time after initial antigen-encounter). In addition, history of antigen stimulations has a profound effect on memory CD8 T cell populations, suggesting that repeated infections (or vaccination) have the capacity to further shape the memory CD8 T cell pool. Finally, genetic background of hosts and history of exposure to diverse microorganisms likely contribute to the observed heterogeneity in the memory CD8 T cell compartment. Extending our tool box and exploring alternative mouse models (i.e., “dirty” and/or outbred mice) to encompass and better model diversity observed in humans will remain an important goal for the near future that will likely shed new light into the mechanisms that govern biology of memory CD8 T cells.

Bacterial Superantigens Expand and Activate, Rather than Delete or Incapacitate, Preexisting Antigen-Specific Memory CD8+ T Cells.

Superantigens (SAgs) released by common Gram-positive bacterial pathogens have been reported to delete, anergize, or activate mouse T cells. However, little is known about their effects on preexisting memory CD8+ T cell (TCD8) pools. Furthermore, whether SAgs manipulate human memory TCD8 responses to cognate antigens is unknown. We used a human peripheral blood mononuclear cell culture system and a nontransgenic mouse model in which the impact of stimulation by two fundamentally distinct SAgs, staphylococcal enterotoxin B and Mycoplasma arthritidis mitogen, on influenza virus- and/or cytomegalovirus-specific memory TCD8 could be monitored. Bacterial SAgs surprisingly expanded antiviral memory TCD8 generated naturally through infection or artificially through vaccination. Mechanistically, this was a T cell-intrinsic and T cell receptor β-chain variable-dependent phenomenon. Importantly, SAg-expanded TCD8 displayed an effector memory phenotype and were capable of producing interferon-γ and destroying target cells ex vivo or in vivo. These findings have clear implications for antimicrobial defense and rational vaccine design.

Abstract 4558: In vitro and in vivo characterization of the PD-1 targeting antibody BI 754091

Abstract The programmed cell death-1 (PD-1) receptor provides inhibitory checkpoint signals to activated T cells upon binding to its ligands, PD-L1 and PD-L2, which are expressed on antigen-presenting cells and cancer cells leading to suppression of T-cell effector function and tumor immune evasion. Blockade of the PD-1 axis using either anti-PD-1 or anti-PD-L1 approved monoclonal antibodies (mAbs) results in improved T-cell effector function and anti-tumor immune responses. Durable tumor responses occur in 15-30% of cancer patients. BI 754091, a humanized IgG4 mAb with high affinity against hPD-1 blocks the interaction between PD-1 and PD-L1 or PD-L2. BI 754091 was characterized in a panel of binding, blocking and functional cell-based assays. In addition, efficacy and safety was assessed in mice and in cynomolgus monkeys, respectively. The ability of BI 754091 to stimulate cytokine production in exhausted human T cells in vitro was tested in an autologous assay system with antigen-specific memory CD4+ T cells being re-stimulated by antigen-pulsed dendritic cells in the presence of BI 754091 or isotype control. Under these assay conditions the majority of T cells co-expressed the exhaustion markers PD-1 and LAG-3 on their surface. Furthermore, PD-L1 and PD-L2 were expressed on the dendritic cells. At the end of the experiment supernatants were harvested and analyzed for IFNγ secretion as a measure for T-cell activation. BI 754091 showed a potent dose dependent T-cell activation. The average fold increase of IFNγ was 7.9 as compared to isotype control, with an average EC50 of 0.9 nM. The in vivo activity of BI 754091 was determined in MC-38 tumor-bearing mice, using a mouse strain where parts of the extracellular domain of murine PD-1 was replaced by the corresponding human PD-1 domain (C57BL/6NTac-PDCD1tm(PDCD1)Arte mice). A dose of 10 mg/kg BI 754091, given either as single treatment or in a twice weekly schedule, induced significant tumor growth inhibition (median TGI of 83% and 90%, respectively) and complete responses (CRs) in some tumors (3 CRs out of 10 and 2 CRs out of 10, respectively). BI 754091 binds to PD-1 from cynomolgus monkeys with comparable affinities as to human PD-1, thus allowing pharmacokinetic and toxicological assessment in this species. Repeated high doses of BI 754091 were well tolerated without adverse immune-related effects. BI 754091 is currently undergoing clinical investigations (NCT02952248). Citation Format: Markus Zettl, Melanie Wurm, Otmar Schaaf, Iñigo Tirapu, Sven Mostböck, Markus Reschke, Stephan-Michael Schmidbauer, Lee Frego, Ivo C. Lorenz, Michael Thibodeau, Diann Blanset, Elisa Oquendo Cifuentes, Jürgen Moll, Norbert Kraut, Eric Borges, Anne Vogt, Jonathon Sedgwick, Irene C. Waizenegger. In vitro and in vivo characterization of the PD-1 targeting antibody BI 754091 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4558.

Effector responses of cytotoxic CD8+ T cells generated by utilizing an FcR-targeting vaccine strategy in an intracellular bacterial infection model.

Abstract F. tularensis is a highly virulent (Category A) gram-negative intracellular mucosal pathogen. It is believed that while humoral immunity delays the progression of infection, cellular immunity is critical for protection. Consequently, by identifying a vaccine strategy that would generate a pathogen-specific, cell-mediated, cytotoxic immune response will be key for protection against intracellular bacterial pathogens such as F. tularensis, which has no FDA approved vaccine available. For this purpose, we have successfully developed an unadjuvanted vaccine delivery system, which targets Ag to FcγR on antigen presenting cells (APC). However, there is a significant lack of information regarding the role of FcR in the generation of mucosal immunity and more specifically the protective role played by cytotoxic CD8+ T cells. For this reason, we have utilized our F. tularensis FcR-targeting vaccine platform to analyze the generation of F. tularensis-specific, memory CD8+ T lymphocytes and their effector, cytotoxic functions during lethal respiratory challenge with the bacterium. In our studies, we have concluded that our FcR-targeting strategy generates antigen-specific effector memory CD8+ T cells that have significantly improved cytotoxic capabilities against macrophages infected by the bacterium and are capable to transfer protection in naïve recipients.

Intranasal delivery of adjuvant-free peptide nanofibers elicits resident CD8+ T cell responses

Influenza vaccines that can be administered intranasally or by other needle-free delivery routes have potential advantages over injected formulations in terms of patient compliance, cost, and ease of global distribution. Supramolecular peptide nanofibers have been investigated previously as platforms for vaccines and immunotherapies and have been shown to raise immune responses in the absence of exogenous adjuvants and without measurable inflammation. However, at present it has not been tested whether the immunogenicity of these materials extends to the intranasal route. Here we investigated the extent to which self-assembled peptide nanofibers bearing an influenza peptide epitope elicit antigen-specific CD8+ T cell responses when delivered intranasally, and we compared these responses with those elicited by subcutaneous immunization. Peptides containing an epitope from influenza acid polymerase (PA) and the Q11 self-assembly domain formed nanofibers that were avidly taken up by dendritic cells in lung-draining mediastinal lymph nodes after intranasal immunization. Intranasally delivered nanofibers generated greater antigen-specific CD8+ T cell responses in the lung-draining lymph nodes than subcutaneous immunizations while retaining the non-inflammatory character of the materials observed in other delivery sites. The CD8+ T cells elicited systemically were functional as assessed by their ability to produce IFN-γ ex vivo, lyse epitope-pulsed target cells in vivo, and diminish viral loads in infected mice. Compared to subcutaneously delivered nanofibers, intranasally delivered peptide nanofibers significantly increased the number of persisting antigen-specific tissue resident memory CD8+ T cells in the lung, allowing for a more rapid response to infection at 6 weeks post-vaccination. These results indicate that intranasally delivered self-assembled peptide nanofibers are immunogenic when delivering CD8+ epitopes without adjuvant or CD4+ epitopes, are non-inflammatory, and promote more lung-resident memory CD8+ T cells compared to subcutaneous immunization.

Analysis of adenovirus-induced immunity to infection with Listeria monocytogenes: Fading protection coincides with declining CD8 T cell numbers and phenotypic changes

Defining correlates of T cell mediated protection is important in order to accelerate the development of efficient T cell based vaccines conferring long-term immunity.Extensive studies have provided important insight regarding the characteristics and functional properties of the effector and memory CD8 T cells induced by viral vector based vaccines. However, long-term protection has been difficult to achieve with T cell inducing vaccines, and the determinants underlying this loss in protection over time are still not fully defined.In this study we analyzed different parameters of the CD8 T cell response as a function of time after vaccination with a human serotype 5 adenovector expressing the glycoprotein (GP) of LCMV tethered to the MHC class II-associated invariant chain. Using this vector we have previously found that CD8 T cells mediate protection from challenge with GP-expressing Listeria monocytogenes at 60 days post vaccination, but only little protection after further 60 days, and we now confirm this observation. A comparison of vaccine-primed CD8 T cells early and late after vaccination revealed a minor decline in the overall numbers of antigen specific memory CD8 T cells during this interval. More importantly, we also observed phenotypic changes over time with a distinct decline in the frequency and number of KLRG1+ CD8 T cells, and, notably, adoptive transfer studies confirmed that memory CD8 T cells expressing KLRG1 are central to protection from systemic L. monocytogenes infection. Together these findings imply that multiple factors including changes in memory T cell numbers and phenotypic composition over time influence the longevity of CD8 T-cell mediated protection.

Nonfollicular reactivation of bone marrow resident memory CD4 T cells in immune clusters of the bone marrow

The bone marrow maintains memory CD4 T cells, which provide memory to systemic antigens. Here we demonstrate that memory CD4 T cells are reactivated by antigen in the bone marrow. In a secondary immune response, antigen-specific T cells of the bone marrow mobilize and aggregate in immune clusters together with MHC class II-expressing cells, mostly B lymphocytes. They proliferate vigorously and express effector cytokines, but they do not develop into follicular T-helper cells. Neither do the B lymphocytes develop into germinal center B cells in the bone marrow. Within 10 days, the immune clusters disappear again. Within 30 days, the expanded antigen-specific memory CD4 T cells return to memory niches and are maintained again individually as resting cells. Thus, in secondary immune responses in the bone marrow T-cell memory is amplified, while in germinal center reactions of secondary lymphoid organs humoral memory is adapted by affinity maturation.

Identification and characterization of HLA-A24-specific XBP1, CD138 (Syndecan-1) and CS1 (SLAMF7) peptides inducing antigens-specific memory cytotoxic T lymphocytes targeting multiple myeloma.

X-box binding protein 1 (XBP1), CD138 (Syndecan-1) and CS1 (SLAMF7) are highly expressed antigens in cancers including multiple myeloma (MM). Here, we identify and characterize immunogenic HLA-A24 peptides derived from these antigens for potential vaccination therapy of HLA-A24+ patients with MM. The identified immunogenic HLA-A24-specific XBP1 unspliced (UN)185-193 (I S P W I L A V L), XBP1 spliced (SP)223-231 (V Y P E G P S S L), CD138265-273 (I F A V C L V G F) and CS1240-248 (L F V L G L F L W) peptides induced antigen-specific CTL with anti-MM activity in an HLA-A24 restricted manner. Furthermore, a cocktail containing the four HLA-A24 peptides evoked MM-specific CTL with distinct phenotypic profiles (CD28, CD40L, 41BB, CD38, CD69) and anti-tumor activities, evidenced by perforin upregulation, CD107a degranulation (cytotoxicity) and Th1-type cytokines (IFN-γ/IL-2/TNF-α) production in response to HLA-A24+ MM cells. The multipeptide-specific CTL included antigen-specific memory CD8+ T cells expressing both T-cell activation (CD38, CD69) and immune checkpoints antigens (CTLA, PD-1, LAG-3, TIM-3). These results provide the framework for a multipeptide vaccination therapy to induce tumor-specific CTL in HLA-A24-positive patients with myeloma and other cancers expressing these antigens.

Adjuvanting a viral vectored vaccine against pre-erythrocytic malaria

The majority of routinely given vaccines require two or three immunisations for full protective efficacy. Single dose vaccination has long been considered a key solution to improving the global immunisation coverage. Recent infectious disease outbreaks have further highlighted the need for vaccines that can achieve full efficacy after a single administration. Viral vectors are a potent immunisation platform, benefiting from intrinsic immuno-stimulatory features while retaining excellent safety profile through the use of non-replicating viruses. We investigated the scope for enhancing the protective efficacy of a single dose adenovirus-vectored malaria vaccine in a mouse model of malaria by co-administering it with vaccine adjuvants. Out of 11 adjuvants, only two, Abisco®-100 and CoVaccineHTTM, enhanced vaccine efficacy and sterile protection following malaria challenge. The CoVaccineHTTM adjuvanted vaccine induced significantly higher proportion of antigen specific central memory CD8+ cells, and both adjuvants resulted in increased proportion of CD8+ T cells expressing the CD107a degranulation marker in the absence of IFNγ, TNFα and IL2 production. Our results show that the efficacy of vaccines designed to induce protective T cell responses can be positively modulated with chemical adjuvants and open the possibility of achieving full protection with a single dose immunisation.

Neutrophils acquire the capacity for antigen presentation to memory CD4+ T cells in vitro and ex vivo

AbstractNeutrophils are critical cells of the innate immune system and rapidly respond to tissue injury and infection. Increasing evidence also indicates that neutrophils have versatile functions in contributing to adaptive immunity by internalizing and transporting antigen and influencing antigen-specific responses. Here, we demonstrate that freshly isolated human neutrophils can function as antigen-presenting cells (APCs) to memory CD4+ T cells. Neutrophils pulsed with the cognate antigens cytomegalovirus pp65 or influenza hemagglutinin were able to present the antigens to autologous antigen-specific CD4+ T cells in a major histocompatibility complex class II (MHC-II; HLA-DR)-dependent manner. Although myeloid dendritic cells and monocytes showed superior presenting ability, neutrophils consistently displayed antigen presentation capability. Upregulation of HLA-DR on neutrophils required the presence of the antigen-specific or activated T cells whereas exposure to innate stimuli such as Toll-like receptor ligands was not sufficient. Neutrophils sorted from vaccine-draining lymph nodes from rhesus macaques also showed expression of HLA-DR and were capable of presenting vaccine antigen to autologous antigen-specific memory CD4+ T cells ex vivo. Altogether, the data demonstrate that neutrophils can adapt a function as APCs and, in combination with their abundance in the immune system, may have a significant role in regulating antigen-specific T-cell responses.

NK cells require antigen-specific memory CD4+ T cells to mediate superior effector functions during HSV-2 recall responses in vitro.

Natural killer (NK) cells have an important role in mounting protective innate responses against genital herpes simplex virus type 2 (HSV-2) infections. However their role as effectors in adaptive immune responses against HSV-2 is unclear. Here, we demonstrate that NK cells from C57BL/6 mice in an ex vivo splenocyte culture produce significantly more interferon γ (IFN-γ) upon re-exposure to HSV-2 antigens in a mouse model of genital HSV-2 immunization. We find that naïve NK cells do not require any prior stimulation or priming to be activated to produce IFN-γ. Our results demonstrate that HSV-2-experienced CD4+ T cells have a crucial role in coordinating NK cell activation and that their presence during HSV-2 antigen presentation is required to activate NK cells in this model of secondary immune response. We also examined the requirement of cell-to-cell contacts for both CD4+ T cells and NK cells. NK cells are dependent on direct interactions with other HSV-2-experienced splenocytes, and CD4+ T cells need to be in close proximity to NK cells to activate them. This study revealed that NK cells do not exhibit any memory toward HSV-2 antigens and, in fact, require specific interactions with HSV-2-experienced CD4+ T cells to produce IFN-γ.

CD8+ T Cells Utilize Highly Dynamic Enhancer Repertoires and Regulatory Circuitry in Response to Infections

Differentiation of effector and memory CD8+ Tcells isaccompanied by extensive changes in the transcriptome and histone modifications at gene promoters; however, the enhancer repertoire and associated gene regulatory networks are poorly defined. Using histone mark chromatin immunoprecipitation coupled with deep sequencing, we mapped the enhancer and super-enhancer landscapes in antigen-specific naive, differentiated effector, and central memory CD8+ Tcells during LCMV infection. Epigenomics-based annotation revealed a highly dynamic repertoire of enhancers, which were inherited, de novo activated, decommissioned and re-activated during CD8+ Tcell responses. We employed a computational algorithm to pair enhancers with target gene promoters. On average, each enhancer targeted three promoters and each promoter was regulated by two enhancers. By identifying enriched transcription factor motifs in enhancers, we defined transcriptional regulatory circuitry at each CD8+ Tcell response stage. These multi-dimensional datasets provide a blueprint for delineating molecular mechanisms underlying functional differentiation of CD8+ Tcells.

IL-15 enhances the antitumor effect of human antigen-specific CD8+ T cells by cellular senescence delay

ABSTRACTOptimal expansion protocols for adoptive human T-cell therapy often include interleukin (IL)-15; however, the mechanism by which IL-15 improves the in vivo antitumor effect of T cells remains to be elucidated. Using human T cells generated from HLA-A2+ donors against novel T-cell epitopes derived from the human U266 myeloma cell line Ig light chain V-region (idiotype) as a model, we found that T cells cultured with IL-15 provided superior resistance to tumor growth in vivo, compared with IL-2, after adoptive transfer into immunodeficient hosts. This effect of IL-15 was associated with delayed/reversed senescence in tumor antigen-specific memory CD8+ T cells mediated through downregulation of P21WAF1, P16INK4a, and P53 expression. Compared to IL-2, IL-15 stimulation dramatically activated JAK3-STAT5 signaling and inhibited the expression of DNA damage genes. Thus, our study elucidates a new mechanism for IL-15 in the regulation of STAT signaling pathways and CD8+ T-cell senescence.

Effect of memory CD4+ T cells' signal transducer and activator of transcription (STATs) functional shift on cytokine-releasing properties in asthma.

Recent data have demonstrated that long-lived memory T cells are present in the human lung and can play significant roles in the pathogenesis of specific allergic and autoimmune diseases. However, most evidence has been obtained from mouse studies, and the potential roles of memory T cells in human allergic diseases, such as asthma, remain largely unknown. Thirty-three asthmatics, 26 chronic obstructive pulmonary disease (COPD) patients, and 22 healthy volunteers were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood, and cell surface staining (CD4, CD45RO, CRTH2, CD62L, and CCR7) was performed for the detection of memory CD4+ T cells in blood. After stimulation with interleukin-27 (IL-27) or IL-4 for 15min, the STAT1/STAT6 phosphorylation of memory CD4+ T cells was measured separately by flow cytometric techniques. The cytokine-releasing profiles after 6days of culture under neutralization, TH2, TH2+lipopolysaccharide (LPS), and TH2+house dust mite (HDM) conditions were detected by intracellular protein (IL-5, IL-17, and interferon (IFN)-γ) staining. Correlation analyses between the profile of memory CD4+ T cells and clinical characteristics of asthma were performed. The number of circulating memory CD4+ T (CD4+ Tm) cells in asthmatics was increased compared with that in the healthy subjects (48±5.7% vs. 32±4.1%, p<0.05). Compared with COPD and healthy subjects, the phosphorylation of signal transducer and activator of transcription 1 (STAT1-py) was impaired in asthmatics, whereas the phosphorylation of signal transducer and activator of transcription 6 (STAT6-py) was slightly enhanced. This imbalance of STAT1-py/STAT6-py was attributed to TH2 memory cells but not non-TH2 memory cells in blood. The cytokine-releasing profiles of asthmatics was unique, specifically IL-5high, IL-17high, and IFN-rlow, compared with those of COPD patients and healthy subjects. The IL-17 production levels in CD4+ Tm cells are associated with disease severity and positively correlated with medication consumption in asthma. The long-lived, antigen-specific memory CD4+ T cells, rather than PBMCs or peripheral lymphocytes, might be the ideal T cell subset candidates for analyzing the endotype of asthma. Memory CD4+ T cells exhibiting a shift in STAT phosphorylation and specific cytokine-releasing profiles have the potential to facilitate the understanding of disease heterogeneity and severity, allowing the more personalized treatment of patients.

CD8+ T cells produce a dialyzable antigen-specific activator of dendritic cells

Cellular lysates from PPD+ donors have been reported to transfer tuberculin reactivity to naïve recipients, but not diphtheria reactivity, and vice versa. A historically controversial topic, the terms "transfer factor" and "DLE" were used to characterize the reactivity-transferring properties of lysates. Intrigued by these reported phenomena, we found that the cellular extract derived from antigen-specific memory CD8+ T cells induces IL-6 from antigen-matched APCs. This ultimately elicits IL-17 from bystander memory CD8+ T cells. We have identified that dialyzable peptide sequences, S100a9, and the TCR β chain from CD8+ T cells contribute to the molecular nature of this activity. We further show that extracts from antigen-targeted T cells enhance immunity to Staphylococcus aureus and Candida albicans These effects are sensitive to immunization protocols and extraction methodology in ways that may explain past discrepancies in the reproducibility of passive cellular immunity.

The adaptor molecule BCAP is essential for the formation of CD8+ T cell memory

Abstract Generation of effector and memory CD8+ T cells is pivotal for the clearance of intracellular infections and forms the basis for vaccination strategies against a wide range of pathogens and anti-cancer immunotherapy. We have generated novel data which shows that the signaling scaffold protein B cell adaptor for PI3-kinase (BCAP) is upregulated in activated CD8+ T cells, links TCR/CD3 engagement to the PI3K signaling cascade and is essential for CD8+ T cell memory formation. To analyze the cell intrinsic properties of BCAP in CD8+ T cells, we used OT-1+ mice which have a high affinity CD8+ T Cell Receptor (TCR) for the ova peptide. We transferred 5,000 WT OT-1+CD45.1+cells and 5,000 BCAPKO OT-1+CD45.2+ cells into CD45.1+CD45.2+ recipients, then infected with Listeria Monocytogenes expressing the ova peptide. We found that BCAP is essential for the proper clonal expansion of CD8+ T cells and BCAP deficiency in CD8+ T cells resulted in a loss of KLRG1+CD127− Terminal Effector Cells (TECs) in favor of KLRG1− CD127+ Memory Precursor Cells (MPCs) in vivo. Strikingly, we observed an enhanced CD8+ T cell contraction phenotype in BCAP deficient CD8+ T cells which led to a significant loss of antigen-specific memory CD8+ T cells. BCAPKO CD8+ T cells were undetectable in 50% of all mice analyzed at day 100 post infection. Therefore BCAP is critical for the formation of CD8+ T cell memory. We found the greatest loss of cells was confined to the recently described KLRG1+CD27loCXCR3loEomeslo effector memory cells, which have potent cytolytic function and anti-cancer capabilities. Finally we found that the ability of BCAPKO memory CD8+ T cells to respond to secondary infection was dysregulated. To conclude, BCAP is essential for the formation of CD8+ T cell memory.