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Adjuvants contribute to enhancing and shaping the vaccine immune response through different modes of action. Here early biomarkers of adjuvanticity after primary immunization were investigated using four different adjuvants combined with the chimeric tuberculosis vaccine antigen H56. C57BL/6 mice were immunized by the subcutaneous route with different vaccine formulations, and the modulation of primary CD4 T cell and B cell responses was assessed within draining lymph nodes, blood, and spleen, 7 and 12 days after priming. Vaccine formulations containing the liposome system CAF01 or a squalene-based oil-in-water emulsion (o/w squalene), but not aluminum hydroxide (alum) or CpG ODN 1826, elicited a significant primary antigen-specific CD4 T cell response compared to antigen alone, 7 days after immunization. The effector function of activated CD4 T cells was skewed toward a Th1/Th17 response by CAF01, while a Th1/Th2 response was elicited by o/w squalene. Differentiation of B cells in short-lived plasma cells, and subsequent early H56-specific IgG secretion, was observed in mice immunized with o/w squalene or CpG adjuvants. Tested adjuvants promoted the germinal center reaction with different magnitude. These results show that the immunological activity of different adjuvants can be characterized by profiling early immunization biomarkers after primary immunization. These data and this approach could give an important contribution to the rational development of heterologous prime-boost vaccine immunization protocols.

•An in vitro assay to detect de novo cross-primed CD8+ T-cell responses against HCV antigens.•10-day cocultures, allow rapid identification of functional HCV epitopes.•Identification of functional immunogenic and less immunogenic epitopes for cancer vaccines. For the rational design of epitope-specific vaccines, identifying epitopes that can be processed and presented is essential. As algorithm-based epitope prediction is frequently discordant with actually recognized CD8+ T-cell epitopes, we developed an in vitro CD8 T-cell priming protocol to enable the identification of truly and functionally expressed HLA class I epitopes. The assay was established and validated to identify epitopes presented by hepatitis C virus (HCV)-infected cells. In vitro priming of naïve CD8 T cells was achieved by culturing unfractionated PBMCs in the presence of a specific cocktail of growth factors and cytokines, and next exposing the cells to hepatic cells expressing the NS3 protein of HCV. After a 10-day co-culture, HCV-specific T-cell responses were identified based on IFN-γ ELISpot analysis. For this, the T cells were restimulated with long synthetic peptides (SLPs) spanning the whole NS3 protein sequence allowing the identification of HCV-specificity. We demonstrated that this protocol resulted in the in vitro priming of naïve precursors to antigen-experienced T-cells specific for 11 out of 98 SLPs tested. These 11 SLPs contain 12 different HLA-A*02:01-restricted epitopes, as predicted by a combination of three epitope prediction algorithms. Furthermore, we identified responses against 3 peptides that were not predicted to contain any immunogenic HLA class I epitopes, yet showed HCV-specific responses in vitro. Separation of CD8+ and CD8− T cells from PBMCs primed in vitro showed responses only upon restimulation with short peptides. We established an in vitro method that enables the identification of HLA class I epitopes resulting from cross-presented antigens and that can cross-prime T cells and allows the effective selection of functional immunogenic epitopes, but also less immunogenic ones, for the design of tailored therapeutic vaccines against persistent viral infections and tumor antigens.

Immunotherapy, represented by immune checkpoint inhibitors (ICIs), has greatly improved the clinical efficacy of malignant tumor therapy. ICI-mediated antitumor responses depend on the infiltration of T cells capable of recognizing and killing tumor cells. ICIs are not effective in \"cold tumors\", which are characterized by the lack of T-cell infiltration. To realize the full potential of immunotherapy and solve this obstacle, it is essential to understand the drivers of T-cell infiltration into tumors. We present a critical review of our understanding of the mechanisms underlying \"cold tumors\", including impaired T-cell priming and deficient T-cell homing to tumor beds. \"Hot tumors\" with significant T-cell infiltration are associated with better ICI efficacy. In this review, we summarize multiple strategies that promote the transformation of \"cold tumors\" into \"hot tumors\" and discuss the mechanisms by which these strategies lead to increased T-cell infiltration. Finally, we discuss the application of nanomaterials to tumor immunotherapy and provide an outlook on the future of this emerging field. The combination of nanomedicines and immunotherapy enhances cross-presentation of tumor antigens and promotes T-cell priming and infiltration. A deeper understanding of these mechanisms opens new possibilities for the development of multiple T cell-based combination therapies to improve ICI effectiveness.

Combinations of different delivery routes for priming and boosting represent vaccination strategies that can modulate magnitude, quality, and localization of the immune response. A murine model was used to study T cell clonal expansion following intranasal (IN) or subcutaneous (SC) priming, and secondary immune responses after boosting by either homologous or heterologous routes. T cell primary activation was studied by using the adoptive transfer model of ovalbumin-specific transgenic CD4(+) T cells. Both IN and SC immunization efficiently elicited, in the respective draining lymph nodes, primary clonal expansion of antigen-specific CD4(+) T cells that disseminated toward distal lymph nodes (mesenteric and iliac) and the spleen. After boosting, a significant serum IgG response was induced in all groups independent of the combination of immunization routes used, while significant levels of local IgA were detected only in mice boosted by the IN route. Mucosal priming drove a stronger Th1 polarization than the systemic route, as shown by serum IgG subclass analysis. IFN-gamma production was observed in splenocytes of all groups, while prime-boost vaccine combinations that included the mucosal route, yielded higher levels of IL-17. Memory lymphocytes were identified in both spleen and draining lymph nodes in all immunized mice, with the highest number of IL-2 producing cells detected in mice primed and boosted by the nasal route. This work shows the critical role of immunization routes in modulating quality and localization of immune responses in prime-boost vaccine strategies.

The design of heterologous prime-boost vaccine combinations that optimally shape the immune response is of critical importance for the development of next generation vaccines. Here, we tested different prime-boost combinations using the tuberculosis vaccine antigen H56 with CAF01 or CpG ODN 1826 adjuvants, administered by the parenteral and nasal routes. Using peptide-MHC class II tetramers, antigen-specific CD4(+) T cells were tracked following primary and booster immunizations. Both parenteral priming with H56 plus CAF01 and nasal priming with H56 plus CpG elicited significant expansion of CD4(+) tetramer-positive T cells in the spleen; however, only parenterally primed cells responded to booster immunization. Subcutaneous (SC) priming with H56 and CAF01 followed by nasal boosting with H56 and CpG showed the greater expansion of CD4(+) tetramer-positive T cells in the spleen and lungs compared to all the other homologous and heterologous prime-boost combinations. Nasal boosting exerted a recruitment of primed CD4(+) T cells into lungs that was stronger in subcutaneously than nasally primed mice, in accordance with different chemokine receptor expression induced by primary immunization. These data demonstrate that SC priming is fundamental for eliciting CD4(+) T cells that can be efficiently boosted by the nasal route and results in the recruitment of antigen-experienced cells into the lungs. Combination of different vaccine formulations and routes of delivery for priming and boosting is a strategic approach for improving and directing vaccine-induced immune responses.

Tumors that lack T cell infiltration are less likely to respond to immune checkpoint inhibition and could benefit from cancer vaccination for the initiation of anti-tumor T cell responses. An attractive vaccine strategy is in vivo targeting of dendritic cells (DCs), key initiators of antigen-specific T cell responses. In this study we generated tumor-derived apoptotic extracellular vesicles (ApoEVs), which are potentially an abundant source of tumor-specific neo-antigens and other tumor-associated antigens (TAAs), and which can be manipulated to express DC-targeting ligands for efficient antigen delivery. Our data demonstrates that by specifically modifying the glycocalyx of tumor cells, high-mannose glycans can be expressed on their cell surface and on extracellular vesicles derived after the induction of apoptosis. High-mannose glycans are the natural ligands of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), a dendritic cell associated C-type lectin receptor (CLR), which has the ability to efficiently internalize its cargo and direct it to both major histocompatibility complex (MHC)-I and MHC-II pathways for the induction of CD8 and CD4 T cell responses, respectively. Compared to unmodified ApoEVs, ApoEVs carrying DC-SIGN ligands are internalized to a higher extent, resulting in enhanced priming of tumor-specific CD8 T cells. This approach thus presents a promising vaccination strategy in support of T cell-based immunotherapy of cancer.

T cell priming is a critical event in the initiation of the immune response to vaccination since it deeply influences both the magnitude and the quality of the immune response induced. CD4(+) T cell priming, required for the induction of high-affinity antibodies and immune memory, represents a key target for improving and modulating vaccine immunogenicity. A major challenge in the study of in vivo T cell priming is due to the low frequency of antigen-specific T cells. This review discusses the current knowledge on antigen-specific CD4(+) T cell priming in the context of vaccination, as well as the most advanced tools for the characterization of the in vivo T cell priming and the opportunities offered by the application of systems biology.

CD4+ T-cell priming is an essential step in vaccination due to the key role of T helper cells in driving both effector and memory immune responses. Here we have characterized in C57BL/6 mice the T helper subtype differentiation among tetramer-specific CD4+ T cells primed by subcutaneous immunization with the tuberculosis vaccine antigen H56 plus the adjuvant CAF01. Peptide-specific population identified by the MHC class II tetramers differentiated into several T helper subtypes upon antigen encounter, and the frequency of subpopulations differed according to their localization. Th1 (CXCR3+T-bet+), Tfh (CXCR5+PD-1+Bcl-6+) and RORγt+ cells were induced in the lymph nodes draining the immunization site (dLN), while Th1 cells were the predominant subtype in the spleen. In addition, CD4+ T cells co-expressing multiple T-cell lineage-specifying transcription factors were also detected. In the lungs, most of the tetramer-binding T cells were RORγt+, while Tfh and Th1 cells were absent. After boosting, a higher frequency of tetramer-binding cells co-expressing the markers CD44 and CD127 was detected compared to primed cells, and cells showed a prevalent Th1 phenotype in both dLN and spleens, while Tfh cells were significantly reduced. In conclusion, these data demonstrate that parenteral immunization with H56 and CAF01 elicits a distribution of antigen-specific CD4+ T cells in both lymphoid tissues and lungs, and gives rise to multiple T helper subtypes, that differ depending on localization and following reactivation.

Dendritic cells (DC) are required for priming antigen‐specific T cells and acquired immunity to many important human pathogens, including Mycobacteriuim tuberculosis (TB) and influenza. However, inappropriate priming of auto‐reactive T cells is linked with autoimmune disease. Understanding the molecular mechanisms that regulate the priming and activation of naïve T cells is critical for development of new improved vaccines and understanding the pathogenesis of autoimmune diseases. The serine/threonine kinase IKKα (CHUK) has previously been shown to have anti‐inflammatory activity and inhibit innate immunity. Here, we show that IKKα is required in DC for priming antigen‐specific T cells and acquired immunity to the human pathogen Listeria monocytogenes . We describe a new role for IKKα in regulation of IRF3 activity and the functional maturation of DC. This presents a unique role for IKKα in dampening inflammation while simultaneously promoting adaptive immunity that could have important implications for the development of new vaccine adjuvants and treatment of autoimmune diseases. IKKα kinase has anti‐inflammatory activity. Here, IKKα is shown to be required in dendritic cells for T‐cell priming and acquired immunity to Listeria monocytogenes . IKKα thus inhibits the innate and activates the adaptive immune systems.

Triple negative breast cancer (TNBC) is a significant clinical problem to which immunotherapeutic strategies have been applied with limited success. Using the syngeneic E0771 TNBC mouse model, this work explores the potential for antitumor CD8+ T cell immunity to be primed extratumorally in lymphoid tissues and therapeutically leveraged. CD8+ T cell viability and responses within the tumor microenvironment (TME) were found to be severely impaired, effects coincident with local immunosuppression that is recapitulated in lymphoid tissues in late stage disease. Prior to onset of a locally suppressed immune microenvironment, however, CD8+ T cell priming within lymph nodes (LN) that depended on tumor lymphatic drainage remained intact. These results demonstrate tumor-draining LNs (TdLN) to be lymphoid tissue niches that support the survival and antigenic priming of CD8+ T lymphocytes against lymph-draining antigen. The therapeutic effects of and CD8+ T cells response to immune checkpoint blockade were furthermore improved when directed to LNs within the tumor-draining lymphatic basin. Therefore, TdLNs represent a unique potential tumor immunity reservoir in TNBC for which strategies may be developed to improve the effects of ICB immunotherapy.