XCR1 expression distinguishes human conventional dendritic cell type 1 with full effector functions from their immediate precursors.

Dendritic cells (DCs) are major regulators of innate and adaptive immune responses. DCs can be classified into plasmacytoid DCs and conventional DCs (cDCs) type 1 and 2. Murine and human cDC1 share the mRNA expression of XCR1. Murine studies indicated a specific role of the XCR1-XCL1 axis in the induction of immune responses. Here, we describe that human cDC1 can be distinguished into XCR1- and XCR1+ cDC1 in lymphoid as well as nonlymphoid tissues. Steady-state XCR1+ cDC1 display a preactivated phenotype compared to XCR1- cDC1. Upon stimulation, XCR1+ cDC1, but not XCR1- cDC1, secreted high levels of inflammatory cytokines as well as chemokines. This was associated with enhanced activation of NK cells mediated by XCR1+ cDC1. Moreover, XCR1+ cDC1 excelled in inhibiting replication of Influenza A virus. Further, under DC differentiation conditions, XCR1- cDC1 developed into XCR1+ cDC1. After acquisition of XCR1 expression, XCR1- cDC1 secreted comparable level of inflammatory cytokines. Thus, XCR1 is a marker of terminally differentiated cDC1 that licenses the antiviral effector functions of human cDC1, while XCR1- cDC1 seem to represent a late immediate precursor of cDC1.

Guidelines for mouse and human DC generation.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34+ cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM-derived DC with GM-CSF. Protocols for human DC generation focus on CD34+ cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Non-Coding RNA in Tumor Cells and Tumor-Associated Myeloid Cells-Function and Therapeutic Potential.

The RNA world is wide, and besides mRNA, there is a variety of other RNA types, such as non-coding (nc)RNAs, which harbor various intracellular regulatory functions. This review focuses on small interfering (si)RNA and micro (mi)RNA, which form a complex network regulating mRNA translation and, consequently, gene expression. In fact, these RNAs are critically involved in the function and phenotype of all cells in the human body, including malignant cells. In cancer, the two main targets for therapy are dysregulated cancer cells and dysfunctional immune cells. To exploit the potential of mi- or siRNA therapeutics in cancer therapy, a profound understanding of the regulatory mechanisms of RNAs and following targeted intervention is needed to re-program cancer cells and immune cell functions in vivo. The first part focuses on the function of less well-known RNAs, including siRNA and miRNA, and presents RNA-based technologies. In the second part, the therapeutic potential of these technologies in treating cancer is discussed, with particular attention on manipulating tumor-associated immune cells, especially tumor-associated myeloid cells.

Tumor Antigens beyond the Human Exome.

With the advent of immunotherapeutics, a new era in the combat against cancer has begun. Particularly promising are neo-epitope-targeted therapies as the expression of neo-antigens is tumor-specific. In turn, this allows the selective targeting and killing of cancer cells whilst healthy cells remain largely unaffected. So far, many advances have been made in the development of treatment options which are tailored to the individual neo-epitope repertoire. The next big step is the achievement of efficacious "off-the-shelf" immunotherapies. For this, shared neo-epitopes propose an optimal target. Given the tremendous potential, a thorough understanding of the underlying mechanisms which lead to the formation of neo-antigens is of fundamental importance. Here, we review the various processes which result in the formation of neo-epitopes. Broadly, the origin of neo-epitopes can be categorized into three groups: canonical, noncanonical, and viral neo-epitopes. For the canonical neo-antigens that arise in direct consequence of somatic mutations, we summarize past and recent findings. Beyond that, our main focus is put on the discussion of noncanonical and viral neo-epitopes as we believe that targeting those provides an encouraging perspective to shape the future of cancer immunotherapeutics.

CARs and Drugs: Pharmacological Ways of Boosting CAR-T-Cell Therapy.

The development of chimeric antigen receptor T cells (CAR-T cells) has marked a new era in cancer immunotherapy. Based on a multitude of durable complete remissions in patients with hematological malignancies, FDA and EMA approval was issued to several CAR products targeting lymphoid leukemias and lymphomas. Nevertheless, about 50% of patients treated with these approved CAR products experience relapse or refractory disease necessitating salvage strategies. Moreover, in the vast majority of patients suffering from solid tumors, CAR-T-cell infusions could not induce durable complete remissions so far. Crucial obstacles to CAR-T-cell therapy resulting in a priori CAR-T-cell refractory disease or relapse after initially successful CAR-T-cell therapy encompass antigen shutdown and CAR-T-cell dysfunctionality. Antigen shutdown predominately rationalizes disease relapse in hematological malignancies, and CAR-T-cell dysfunctionality is characterized by insufficient CAR-T-cell proliferation and cytotoxicity frequently observed in patients with solid tumors. Thus, strategies to surmount those obstacles are being developed with high urgency. In this review, we want to highlight different approaches to combine CAR-T cells with drugs, such as small molecules and antibodies, to pharmacologically boost CAR-T-cell therapy. In particular, we discuss how certain drugs may help to counteract antigen shutdown and CAR-T-cell dysfunctionality in both hematological malignancies and solid tumors.

Beyond Cancer: Regulation and Function of PD-L1 in Health and Immune-Related Diseases.

Programmed Cell Death 1 Ligand 1 (PD-L1, CD274, B7-H1) is a transmembrane protein which is strongly involved in immune modulation, serving as checkpoint regulator. Interaction with its receptor, Programmed Cell Death Protein 1 (PD-1), induces an immune-suppressive signal, which modulates the activity of T cells and other effector cells. This mediates peripheral tolerance and contributes to tumor immune escape. PD-L1 became famous due to its deployment in cancer therapy, where blockage of PD-L1 with the help of therapeutic antagonistic antibodies achieved impressive clinical responses by reactivating effector cell functions against tumor cells. Therefore, in the past, the focus has been placed on PD-L1 expression and its function in various malignant cells, whereas its role in healthy tissue and diseases apart from cancer remained largely neglected. In this review, we summarize the function of PD-L1 in non-cancerous cells, outlining its discovery and origin, as well as its involvement in different cellular and immune-related processes. We provide an overview of transcriptional and translational regulation, and expression patterns of PD-L1 in different cells and organs, and illuminate the involvement of PD-L1 in different autoimmune diseases as well as in the context of transplantation and pregnancy.

T-Cell Responses in Merkel Cell Carcinoma: Implications for Improved Immune Checkpoint Blockade and Other Therapeutic Options.

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer with rising incidence and high mortality. Approximately 80% of the cases are caused by the human Merkel cell polyomavirus, while the remaining 20% are induced by UV light leading to mutations. The standard treatment of metastatic MCC is the use of anti-PD-1/-PD-L1-immune checkpoint inhibitors (ICI) such as Pembrolizumab or Avelumab, which in comparison with conventional chemotherapy show better overall response rates and longer duration of responses in patients. Nevertheless, 50% of the patients do not respond or develop ICI-induced, immune-related adverse events (irAEs), due to diverse mechanisms, such as down-regulation of MHC complexes or the induction of anti-inflammatory cytokines. Other immunotherapeutic options such as cytokines and pro-inflammatory agents or the use of therapeutic vaccination offer great ameliorations to ICI. Cytotoxic T-cells play a major role in the effectiveness of ICI, and tumour-infiltrating CD8+ T-cells and their phenotype contribute to the clinical outcome. This literature review presents a summary of current and future checkpoint inhibitor therapies in MCC and demonstrates alternative therapeutic options. Moreover, the importance of T-cell responses and their beneficial role in MCC treatment is discussed.

BRAF and MEK Inhibitors Affect Dendritic-Cell Maturation and T-Cell Stimulation.

BRAF and MEK inhibitor (BRAFi/MEKi) combinations are currently the standard treatment for patients with BRAFV600 mutant metastatic melanoma. Since the RAS/RAF/MEK/ERK-pathway is crucial for the function of different immune cells, we postulated an effect on their function and thus interference with anti-tumor immunity. Therefore, we examined the influence of BRAFi/MEKi, either as single agent or in combination, on the maturation of monocyte-derived dendritic cells (moDCs) and their interaction with T cells. DCs matured in the presence of vemurafenib or vemurafenib/cobimetinib altered their cytokine secretion and surface marker expression profile. Upon the antigen-specific stimulation of CD8+ and CD4+ T cells with these DCs or with T2.A1 cells in the presence of BRAFi/MEKi, we detected a lower expression of activation markers on and a lower cytokine secretion by these T cells. However, treatment with any of the inhibitors alone or in combination did not change the avidity of CD8+ T cells in peptide titration assays with T2.A1 cells. T-helper cell/DC interaction is a bi-directional process that normally results in DC activation. Vemurafenib and vemurafenib/cobimetinib completely abolished the helper T-cell-mediated upregulation of CD70, CD80, and CD86 but not CD25 on the DCs. The combination of dabrafenib/trametinib affected DC maturation and activation as well as T-cell activation less than combined vemurafenib/cobimetinib did. Hence, for a potential combination with immunotherapy, our data indicate the superiority of dabrafenib/trametinib treatment.

A Chimeric IL-15/IL-15Rα Molecule Expressed on NFκB-Activated Dendritic Cells Supports Their Capability to Activate Natural Killer Cells.

Natural killer (NK) cells, members of the innate immune system, play an important role in the rejection of HLA class I negative tumor cells. Hence, a therapeutic vaccine, which can activate NK cells in addition to cells of the adaptive immune system might induce a more comprehensive cellular response, which could lead to increased tumor elimination. Dendritic cells (DCs) are capable of activating and expanding NK cells, especially when the NFκB pathway is activated in the DCs thereby leading to the secretion of the cytokine IL-12. Another prominent NK cell activator is IL-15, which can be bound by the IL-15 receptor alpha-chain (IL-15Rα) to be transpresented to the NK cells. However, monocyte-derived DCs do neither secrete IL-15, nor express the IL-15Rα. Hence, we designed a chimeric protein consisting of IL-15 and the IL-15Rα. Upon mRNA electroporation, the fusion protein was detectable on the surface of the DCs, and increased the potential of NFκB-activated, IL-12-producing DC to activate NK cells in an autologous cell culture system with ex vivo-generated cells from healthy donors. These data show that a chimeric IL-15/IL-15Rα molecule can be expressed by monocyte-derived DCs, is trafficked to the cell surface, and is functional regarding the activation of NK cells. These data represent an initial proof-of-concept for an additional possibility of further improving cellular DC-based immunotherapies of cancer.

CARs: Beyond T Cells and T Cell-Derived Signaling Domains.

When optimizing chimeric antigen receptor (CAR) therapy in terms of efficacy, safety, and broadening its application to new malignancies, there are two main clusters of topics to be addressed: the CAR design and the choice of transfected cells. The former focuses on the CAR construct itself. The utilized transmembrane and intracellular domains determine the signaling pathways induced by antigen binding and thereby the cell-specific effector functions triggered. The main part of this review summarizes our understanding of common signaling domains employed in CARs, their interactions among another, and their effects on different cell types. It will, moreover, highlight several less common extracellular and intracellular domains that might permit unique new opportunities. Different antibody-based extracellular antigen-binding domains have been pursued and optimized to strike a balance between specificity, affinity, and toxicity, but these have been reviewed elsewhere. The second cluster of topics is about the cellular vessels expressing the CAR. It is essential to understand the specific attributes of each cell type influencing anti-tumor efficacy, persistence, and safety, and how CAR cells crosstalk with each other and bystander cells. The first part of this review focuses on the progress achieved in adopting different leukocytes for CAR therapy.

CSPG4 as Target for CAR-T-Cell Therapy of Various Tumor Entities-Merits and Challenges.

Targeting cancer cells using chimeric-antigen-receptor (CAR-)T cells has propelled adoptive T-cell therapy (ATT) to the next level. A plentitude of durable complete responses using CD19-specific CAR-T cells in patients suffering from various lymphoid malignancies resulted in the approval by the food and drug administration (FDA) of CD19-directed CAR-T cells for the treatment of acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). A substantial portion of this success in hematological malignancies can be traced back to the beneficial properties of the target antigen CD19, which combines a universal presence on target cells with no detectable expression on indispensable host cells. Hence, to replicate response rates achieved in ALL and DLBCL in the realm of solid tumors, where ideal target antigens are scant and CAR-T cells are still lagging behind expectations, the quest for appropriate target antigens represents a crucial task to expedite the next steps in the evolution of CAR-T-cell therapy. In this review, we want to highlight the potential of chondroitin sulfate proteoglycan 4 (CSPG4) as a CAR-target antigen for a variety of different cancer entities. In particular, we discuss merits and challenges associated with CSPG4-CAR-T cells for the ATT of melanoma, leukemia, glioblastoma, and triple-negative breast cancer.

CSPG4-Specific CAR T Cells for High-Risk Childhood B Cell Precursor Leukemia.

The advent of CD19-specific chimeric antigen receptor (CAR) T cells has proven to be a powerful asset in the arsenal of cancer immunotherapy of acute lymphoblastic leukemia and certain B cell lymphomas. However, a sizable portion of patients treated with CD19-CAR T cells relapse with CD19-negative cancer cells, necessitating the quest for back-up antigens. Chondroitin sulfate proteoglycan 4 (CSPG4) expression has been reported on leukemic blasts bearing the ill-fated MLL 11q23 rearrangement. We aimed at exploring the use of CSPG4-specific CAR T cells against mixed-lineage leukemia (MLL)-rearranged leukemic blasts, using the precursor B cell leukemia cell line KOPN8 (MLL-MLLT1 translocation) as a model. First, we confirmed CSPG4 expression on KOPN8 cells. Bulk T cells electroporated with mRNA encoding a CSPG4-specific CAR upregulated activation markers and secreted the Th1 cytokines TNF and IFNγ in an antigen-specific manner upon co-culture with KOPN8 cells. More importantly, CSPG4-specific CAR T cells evinced specific degranulation towards KOPN8 cells and specifically lysed KOPN8 target cells in chromium lysis experiments. CSPG4 is a well-established CAR target in cutaneous melanoma. Here, we provide proof-of-principle data for the use of CSPG4-specific CAR T cells against MLL-translocated leukemias.

The siRNA-mediated downregulation of PD-1 alone or simultaneously with CTLA-4 shows enhanced in vitro CAR-T-cell functionality for further clinical development towards the potential use in immunotherapy of melanoma.

Chimeric antigen receptor (CAR)-T cells have been used successfully for cancer immunotherapy. While substantial tumor regression was observed in leukaemia and lymphoma, CAR therapy of solid tumors needs further improvement. A major obstacle to the efficiency of engineered T cells is posed by triggering of inhibitory receptors, for example programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), leading to an impaired antitumor activity. To boost CAR-T-cell function, we co-electroporated T cells with both, mRNA encoding a CAR specific for chondroitin sulphate proteoglycan 4 (CSPG4) and small-interfering RNAs (siRNAs) to downregulate PD-1 (siPD-1) and CTLA-4 (siCTLA-4). Flow cytometry revealed that activation-induced upregulation of both PD-1 and CTLA-4 was suppressed when compared to CAR-T cells electroporated with negative control siRNA. The siRNA transfection showed no influence on CAR expression of engineered T cells. Functionality assays were performed using PD-L1- and CD80-transfected melanoma cells endogenously expressing CSPG4. CAR-T cells transfected with siPD-1 alone showed improvement in cytokine secretion. Additionally, CAR-T cells transfected with either siPD-1 alone or together with siCTLA-4 exhibited a significantly increased cytotoxicity. No or only little effects were observed when CAR-T cells were co-transfected with siCTLA-4 only. Taken together, it is feasible to optimize CAR-T cells by co-transfection of CAR-encoding mRNA and siRNAs to downregulate inhibitory receptors. Our in vitro data indicate an improvement of the functionality of these CAR-T cells, suggesting that this strategy could represent a novel method to enhance CAR-T-cell immunotherapy of cancer.

The Generation of CAR-Transfected Natural Killer T Cells for the Immunotherapy of Melanoma.

Natural killer T (NKT) cells represent a cell subpopulation that combines characteristics of natural killer (NK) cells and T cells. Through their endogenous T-cell receptors (TCRs), they reveal a pronounced intrinsic anti-tumor activity. Thus, a NKT cell transfected with a chimeric antigen receptor (CAR), which recognizes a tumor-specific surface antigen, could attack tumor cells antigen-specifically via the CAR and additionally through its endogenous TCR. NKT cells were isolated from peripheral blood mononuclear cells (PBMCs), expanded, and electroporated with mRNA encoding a chondroitin sulfate proteoglycan 4 (CSPG4)-specific CAR. The CAR expression on NKT cells and their in vitro functionality were analyzed. A transfection efficiency of more than 80% was achieved. Upon stimulation with melanoma cells, CAR-NKT cells produced cytokines antigen-specifically. Compared with conventional CAR-T cells, cytokine secretion of CAR-NKT cells was generally lower. Specific cytotoxicity, however, was similar with CAR-NKT cells showing a trend towards improved cytotoxicity. Additionally, CAR-NKT cells could kill target cells through their endogenous TCRs. In summary, it is feasible to generate CAR-NKT cells by using mRNA electroporation. Their CAR-mediated cytotoxicity is at least equal to that of conventional CAR-T cells, while their intrinsic cytotoxic activity is maintained. Thus, CAR-NKT cells may represent a valuable alternative to conventional CAR-T cells for cancer immunotherapy.

BRAF and MEK Inhibitors Influence the Function of Reprogrammed T Cells: Consequences for Adoptive T-Cell Therapy.

BRAF and MEK inhibitors (BRAFi/MEKi), the standard treatment for patients with BRAFV600 mutated melanoma, are currently explored in combination with various immunotherapies, notably checkpoint inhibitors and adoptive transfer of receptor-transfected T cells. Since two BRAFi/MEKi combinations with similar efficacy are approved, potential differences in their effects on immune cells would enable a rational choice for triple therapies. Therefore, we characterized the influence of the clinically approved BRAFi/MEKi combinations dabrafenib (Dabra) and trametinib (Tram) vs. vemurafenib (Vem) and cobimetinib (Cobi) on the activation and functionality of chimeric antigen receptor (CAR)-transfected T cells. We co-cultured CAR-transfected CD8⁺ T cells and target cells with clinically relevant concentrations of the inhibitors and determined the antigen-induced cytokine secretion. All BRAFi/MEKi reduced this release as single agents, with Dabra having the mildest inhibitory effect, and Dabra + Tram having a clearly milder inhibitory effect than Vem + Cobi. A similar picture was observed for the upregulation of the activation markers CD25 and CD69 on CAR-transfected T cells after antigen-specific stimulation. Most importantly, the cytolytic capacity of the CAR-T cells was significantly inhibited by Cobi and Vem + Cobi, whereas the other kinase inhibitors showed no effect. Therefore, the combination Dabra + Tram would be more suitable for combining with T-cell-based immunotherapy than Vem + Cobi.

Proteomic Response of Human Umbilical Vein Endothelial Cells to Histamine Stimulation.

The histamine receptors (HRs) represent a subclass of G protein-coupled receptors (GPCRs) and comprise four subtypes. Due to their numerous physiological and pathological effects, HRs are popular drug targets for the treatment of allergic reactions or the regulation of gastric acid secretion. Hence, an understanding of the functional selectivity of HR ligands has gained importance. These ligands can bind to specific GPCRs and selectively activate defined pathways. Supporting the activation of a therapeutically necessary pathway without the activation of other signaling cascades can result in drugs with more specific activity and fewer side effects. To evaluate the cellular consequences resulting from receptor binding, comprehensive analyses of cellular protein alterations upon incubation with ligands are required. For this purpose, endothelial cells are treated with histamine, as the endogenous ligand of HRs, to obtain a global overview of its cellular effects. Quantitative proteomics and pathway analyses of histamine-treated and untreated cells reveal enrichment of the nuclear factor-κB and tumor necrosis factor signaling pathways, cytokine-cytokine receptor interactions, complement and coagulation cascades, and acute inflammatory processes upon histamine treatment. This strategy offers the opportunity to monitor HR-mediated signaling in a multidimensional manner.

RNA-transfection of γ/δ T cells with a chimeric antigen receptor or an α/ß T-cell receptor: a safer alternative to genetically engineered α/ß T cells for the immunotherapy of melanoma.

BACKGROUND: Adoptive T-cell therapy relying on conventional T cells transduced with T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has caused substantial tumor regression in several clinical trials. However, genetically engineered T cells have been associated with serious side-effects due to off-target toxicities and massive cytokine release. To obviate these concerns, we established a protocol adaptable to GMP to expand and transiently transfect γ/δ T cells with mRNA. METHODS: PBMC from healthy donors were stimulated using zoledronic-acid or OKT3 to expand γ/δ T cells and bulk T cells, respectively. Additionally, CD8+ T cells and γ/δ T cells were MACS-isolated from PBMC and expanded with OKT3. Next, these four populations were electroporated with RNA encoding a gp100/HLA-A2-specific TCR or a CAR specific for MCSP. Thereafter, receptor expression, antigen-specific cytokine secretion, specific cytotoxicity, and killing of the endogenous γ/δ T cell-target Daudi were analyzed. RESULTS: Using zoledronic-acid in average 6 million of γ/δ T cells with a purity of 85% were generated from one million PBMC. MACS-isolation and OKT3-mediated expansion of γ/δ T cells yielded approximately ten times less cells. OKT3-expanded and CD8+ MACS-isolated conventional T cells behaved correspondingly similar. All employed T cells were efficiently transfected with the TCR or the CAR. Upon respective stimulation, γ/δ T cells produced IFNγ and TNF, but little IL-2 and the zoledronic-acid expanded T cells exceeded MACS-γ/δ T cells in antigen-specific cytokine secretion. While the cytokine production of γ/δ T cells was in general lower than that of conventional T cells, specific cytotoxicity against melanoma cell lines was similar. In contrast to OKT3-expanded and MACS-CD8+ T cells, mock-electroporated γ/δ T cells also lysed tumor cells reflecting the γ/δ T cell-intrinsic anti-tumor activity. After transfection, γ/δ T cells were still able to kill MHC-deficient Daudi cells. CONCLUSION: We present a protocol adaptable to GMP for the expansion of γ/δ T cells and their subsequent RNA-transfection with tumor-specific TCRs or CARs. Given the transient receptor expression, the reduced cytokine release, and the equivalent cytotoxicity, these γ/δ T cells may represent a safer complementation to genetically engineered conventional T cells in the immunotherapy of melanoma (Exper Dermatol 26: 157, 2017, J Investig Dermatol 136: A173, 2016).

Combining a chimeric antigen receptor and a conventional T-cell receptor to generate T cells expressing two additional receptors (TETARs) for a multi-hit immunotherapy of melanoma.

The adoptive transfer of engineered T cells represents an important approach in immunotherapy of melanoma. However, relapse of the tumor can occur due to immune-escape mechanisms developed by the tumor cells, for example antigen loss, downregulation of the major histocompatibility complex presentation machinery and defects in antigen processing. To counteract these mechanisms, we combined a T-cell receptor and a chimeric antigen receptor, specific for different common melanoma antigens, gp100 (PMEL) and MCSP (HMW-MAA), to generate functional CD8+ T cells expressing two additional receptors (TETARs) by electroporation of receptor-encoding mRNA. These TETARs produced cytokines and were lytic upon recognition of each of their cognate antigens, while no reciprocal inhibition of the receptors occurred. When stimulated with target cells, which express both antigens, an enhanced effect was suggested. The confirmation that chimeric antigen receptors and T-cell receptors can be functionally combined opens up new avenues in cancer immunotherapy, and the generation of TETARs helps by-passing major mechanisms by which tumor cells escape immune recognition.

Concurrent interaction of DCs with CD4(+) and CD8(+) T cells improves secondary CTL expansion: It takes three to tango.

T-cell help is essential for CTL-memory formation. Nevertheless, it is unclear whether the continuous presence of CD4(+) T-helper (Th) cells is required during dendritic cell (DC)/CD8(+) T-cell encounters, or whether a DC will remember the helper signal after the Th cell has departed. This question is relevant for the design of therapeutic cancer vaccines. Therefore, we investigated how human DCs need to interact with CD4(+) T cells to mediate efficient repetitive CTL expansion in vitro. We established an autologous antigen-specific in vitro system with monocyte-derived DCs, as these are primarily used for cancer vaccination. Contrary to common belief, a sequential interaction of licensed DCs with CD8(+) T cells barely improved CTL expansion. In sharp contrast, simultaneous encounter of Th cells and CTLs with the same DC during the first in vitro encounter is a prerequisite for optimal subsequent CTL expansion in our in vitro system. These data suggest that, in contrast to DC maturation, the activation of DCs by Th cells, which is necessary for optimal CTL stimulation, is transient. This knowledge has significant implications for the design of new and more effective DC-based vaccination strategies. Furthermore, our in vitro system could be a valuable tool for preclinical immunotherapeutical studies.

Triggering of NF-κB in cytokine-matured human DCs generates superior DCs for T-cell priming in cancer immunotherapy.

Understanding the signaling that governs the immunogenicity of human dendritic cells (DCs) is a prerequisite for improving DC-based therapeutic vaccination strategies, in which the ability of DCs to induce robust and lasting Ag-specific CTL responses is of critical importance. Cytokine-matured DCs are regularly used, but to induce memory-type CTLs, they require additional activation stimuli, such as CD4+ T-cell help or TLR activation. One common denominator of these stimuli is the activation of NF-κB. Here, we show that human monocyte-derived, cytokine cocktail-matured DCs transfected with constitutively active mutants of IκB kinases (caIKKs) by mRNA electroporation, further upregulated maturation markers, and secreted enhanced amounts of cytokines, including IL-12p70, which was produced for more than 48 h after transfection. Most importantly, cytotoxic T cells induced by caIKK-transfected DCs combined high CD27 expression, indicating a more memory-like phenotype, and a markedly enhanced secondary expandability with a high lytic capacity. In contrast, CTLs primed and expanded with unmodified cytokine cocktail-matured DCs did not maintain their proliferative capacity upon repetitive stimulations. We hypothesize that "designer" DCs expressing constitutively active IκB kinases will prove highly immunogenic also in vivo and possibly emerge as a new strategy to improve the clinical efficacy of therapeutic vaccinations against cancer and other chronic diseases.