The skin microbiome stratifies patients with cutaneous T cell lymphoma and determines event-free survival.

Mycosis fungoides (MF) is the most common entity of Cutaneous T cell lymphomas (CTCL) and is characterized by the presence of clonal malignant T cells in the skin. The role of the skin microbiome for MF development and progression are currently poorly understood. Using shotgun metagenomic profiling, real-time qPCR, and T cell receptor sequencing, we compared lesional and nonlesional skin of 20 MF patients with early and advanced MF. Additionally, we isolated Staphylococcus aureus and other bacteria from MF skin for functional profiling and to study the S. aureus virulence factor spa. We identified a subgroup of MF patients with substantial dysbiosis on MF lesions and concomitant outgrowth of S. aureus on plaque-staged lesions, while the other MF patients had a balanced microbiome on lesional skin. Dysbiosis and S. aureus outgrowth were accompanied by ectopic levels of cutaneous antimicrobial peptides (AMPs), including adaptation of the plaque-derived S. aureus strain. Furthermore, the plaque-derived S. aureus strain showed a reduced susceptibility towards antibiotics and an upregulation of the virulence factor spa, which may activate the NF-κB pathway. Remarkably, patients with dysbiosis on MF lesions had a restricted T cell receptor repertoire and significantly lower event-free survival. Our study highlights the potential for microbiome-modulating treatments targeting S. aureus to prevent MF progression.

Manipulation of Serum Protein Adsorption by Nanoengineered Biomaterials Influences Subsequent Immune Responses.

The adsorption of serum proteins on biomaterial surfaces is a critical determinant for the outcome of medical procedures and therapies, which involve inserting materials and devices into the body. In this study, we aimed to understand how surface topography at the nanoscale influences the composition of the protein corona that forms on the (bio)material surface when placed in contact with serum proteins. To achieve that, we developed nanoengineered model surfaces with finely tuned topography of 16, 40, and 70 nm, overcoated with methyl oxazoline to ensure uniform outermost chemistry across all surfaces. Our findings revealed that within the studied height range, surface nanotopography had no major influence on the overall quantity of adsorbed proteins. However, significant alterations were observed in the composition of the adsorbed protein corona. For instance, clusterin adsorption decreased on all the nanotopography-modified surfaces. Conversely, there was a notable increase in the adsorption of ApoB and IgG gamma on the 70 nm nanotopography. In comparison, the adsorption of albumin was greater on surfaces that had a topography scale of 40 nm. Analysis of the gene enrichment data revealed a reduction in protein adsorption across all immune response-related biological pathways on nanotopography-modified surfaces. This reduction became more pronounced for larger surface nanoprotrusions. Macrophages were used as representative immune cells to assess the influence of the protein corona composition on inflammatory outcomes. Gene expression analysis demonstrated reduced inflammatory responses on the nanotopographically modified surface, a trend further corroborated by cytokine analysis. These findings underscore the potential of precisely engineered nanotopography-coated surfaces for augmenting biomaterial functionality.

Interfacial Denaturation at the Droplet Simplifies the Formation of Drug-Loaded Protein Nanocapsules to Enhance Immune Response of Cells.

Nanocapsules enable multicomponent encapsulation of therapeutic cargoes with high encapsulation content and efficiency, which is vital for cancer immunotherapy. In the past, chemical crosslinking is used to synthesize nanocapsules, which can impede the regulatory approval process. Therefore, a new class of protein nanocapsules is developed by eliminating the need for chemical crosslinking by utilizing protein denaturation through a process that is referred to as "baking at the droplet interface". Such protein nanocapsules with antigens incorporated in the shell and a combination of encapsulated drugs showed an enhancement in the immune response of cells.

Effect of Protein Corona on the Specificity and Efficacy of Nanobioconjugates to Treat Intracellular Infections.

Encapsulating drugs into functionalized nanoparticles (NPs) is an alternative to reach the specific therapeutic target with lower doses. However, when the NPs are in contact with physiological media, proteins adsorb on their surfaces, forming a protein corona (PC) biomolecular layer, acquiring a distinct biological identity that alters their interactions with cells. Itraconazole (ITZ), an antifungal agent, is encapsulated into PEGylated and/or functionalized NPs with high specificity for macrophages. It is evaluated how the PC impacts their cell uptake and antifungal effect. The minimum inhibitory concentration and colony-forming unit assays demonstrate that encapsulated ITZ into poly(ethylene glycol) (PEG) NPs improves the antifungal effect compared with NPs lacking PEGylation. The improvement can be related to the synergistic effect of the encapsulated ITZ and NPs composition and the reduction of PC formation in PEG NPs. Functionalized NPs with anti-F4/80 and anti-MARCO antibodies, or mannose without PEG and treated with PC, show an improved uptake but, in the presence of PEG, significantly reduce the endocytosis, dominating the stealth effect from PEG. Therefore, the PC plays a crucial role in the nanosystem uptake and antifungal effects, which suggests the need for in vivo model studies to evaluate the effect of PC in the specificity and biodistribution.

Uptake of extracellular vesicles into immune cells is enhanced by the protein corona.

The influence of a protein corona on the uptake of nanoparticles in cells has been demonstrated in various publications over the last years. Extracellular vesicles (EVs), can be seen as natural nanoparticles. However, EVs are produced under different cell culture conditions and little is known about the protein corona forming on EVs and its influence on their uptake by target cells. Here, we use a proteomic approach in order to analyze the protein composition of the EVs themselves and the protein composition of a human blood plasma protein corona around EVs. Moreover, we analyze the influence of the protein corona on EV uptake into human monocytes and compare it with the influence on the uptake of engineered liposomes. We show that the presence of a protein corona increases the uptake of EVs in human monocytes. While for liposomes this seems to be triggered by the presence of immunoglobulins in the protein corona, for EVs blocking the Fc receptors on monocytes did not show an influence of uptake. Therefore, other mechanisms of docking to the cell membrane and uptake are most like involved, demonstrating a clear difference between EVs and liposomes as technically produced nanocarriers.

Nuclear Glycoprotein A Repetitions Predominant (GARP) Is a Common Trait of Glioblastoma Stem-like Cells and Correlates with Poor Survival in Glioblastoma Patients.

Glioblastoma (GB) is notoriously resistant to therapy. GB genesis and progression are driven by glioblastoma stem-like cells (GSCs). One goal for improving treatment efficacy and patient outcomes is targeting GSCs. Currently, there are no universal markers for GSCs. Glycoprotein A repetitions predominant (GARP), an anti-inflammatory protein expressed by activated regulatory T cells, was identified as a possible marker for GSCs. This study evaluated GARP for the detection of human GSCs utilizing a multidimensional experimental design that replicated several features of GB: (1) intratumoral heterogeneity, (2) cellular hierarchy (GSCs with varied degrees of self-renewal and differentiation), and (3) longitudinal GSC evolution during GB recurrence (GSCs from patient-matched newly diagnosed and recurrent GB). Our results indicate that GARP is expressed by GSCs across various cellular states and disease stages. GSCs with an increased GARP expression had reduced self-renewal but no alterations in proliferative capacity or differentiation commitment. Rather, GARP correlated inversely with the expression of GFAP and PDGFR-α, markers of astrocyte or oligodendrocyte differentiation. GARP had an abnormal nuclear localization (GARPNU+) in GSCs and was negatively associated with patient survival. The uniformity of GARP/GARPNU+ expression across different types of GSCs suggests a potential use of GARP as a marker to identify GSCs.

Proteomics reveals time-dependent protein corona changes in the intracellular pathway.

The intracellular protein corona has not been fully investigated in the field of nanotechnology-biology (nano-bio) interactions. To effectively understand intracellular protein corona formation and dynamics, we established a workflow to isolate the intracellular protein corona at different uptake times of two nanoparticles - magnetic hydroxyethyl starch nanoparticles (HES-NPs) and magnetic human serum albumin nanocapsules (HSA-NCs). We performed label-free quantitative LC-MS proteomics to analyze the composition of the intracellular protein corona and correlated our findings with results from conventional methods for intracellular trafficking of nanocarriers, such as flow cytometry, transmission electron microscopy (TEM), and confocal microscopy (cLSM). We determined the evolution of the intracellular protein corona. At different time stages the protein corona of the HES-NPs with a slower uptake changed, but there were fewer changes in that of the HSA-NCs with a more rapid uptake. We identified proteins that are involved in macropinocytosis (RAC1, ASAP2) as well as caveolin. This was confirmed by blocking experiments and by TEM studies. The investigated nanocarrier predominantly trafficked from early endosomes as determined by RAB5 identification in proteomics and in cLSM to late endosomes/lysosomes (RAB7, LAMP1, cathepsin K and HSP 90-beta) We further demonstrated differences between nanoparticles with slower and faster uptake kinetics and determined the associated proteome at different time points. Analysis of the intracellular protein corona provides us with effective data to examine the intracellular trafficking of nanocarriers used in efficient drug delivery and intracellular applications. STATEMENT OF SIGNIFICANCE: Many research papers focus on the protein corona on nanoparticles formed in biological fluids, but there are hardly any articles dealing with proteins that come in contact with nanoparticles inside cells. The "intracellular protein corona" studied here is a far more complex and highly demanding field. Most nanocarriers are designed to be taken up into cells. Given this, we chose two different nanocarriers to reveal changes in the proteins in dendritic cells during contact at specific times. Further studies will allow us to examine molecular target proteins using these methods. Our research is a significant addition towards the goal of understanding and thus improving the efficacy of drug nanocarriers.

Delivery of Immunostimulatory Cargos in Nanocarriers Enhances Anti-Tumoral Nanovaccine Efficacy.

Finding a long-term cure for tumor patients still represents a major challenge. Immunotherapies offer promising therapy options, since they are designed to specifically prime the immune system against the tumor and modulate the immunosuppressive tumor microenvironment. Using nucleic-acid-based vaccines or cellular vaccines often does not achieve sufficient activation of the immune system in clinical trials. Additionally, the rapid degradation of drugs and their non-specific uptake into tissues and cells as well as their severe side effects pose a challenge. The encapsulation of immunomodulatory molecules into nanocarriers provides the opportunity of protected cargo transport and targeted uptake by antigen-presenting cells. In addition, different immunomodulatory cargos can be co-delivered, which enables versatile stimulation of the immune system, enhances anti-tumor immune responses and improves the toxicity profile of conventional chemotherapeutic agents.

Anti-PEG antibodies enriched in the protein corona of PEGylated nanocarriers impact the cell uptake.

Poly(ethylene glycol) (PEG) is the gold standard used to reduce unspecific protein adsorption and prolong nanocarrier circulation time. However, this stealth effect could be counteracted by the increasing prevalence of anti-PEG antibodies in the bloodstream. Up to now, the presence of anti-PEG antibodies in the protein corona and their effect on cell uptake has not been investigated yet. Our results showed a high concentration and prevalence of anti-PEG antibodies in the German population. PEGylated nanocarriers exhibited a higher level of anti-PEG antibodies in the protein corona compared to non-PEGylated, which lead to higher uptake in macrophages. Consequently, the anti-PEG antibodies in the protein corona could mitigate the stealth effect of PEG, leading to accelerated blood clearance and unwanted side effects.

Systematic modulation of the lipid composition enables the tuning of liposome cellular uptake.

As liposomes have been widely explored as drug delivery carriers over the past decades, they are one of the most promising platforms due to their biocompatibility and versatility for surface functionalization. However, to improve the specific design of liposomes for future biomedical applications such as nanovaccines, it is necessary to understand how these systems interact with cell membranes, as most of their potential applications require them to be internalized by cells. Even though several investigations on the cellular uptake of liposomes were conducted, the effect of the liposome membrane properties on internalization in different cell lines remains unclear. Here, we demonstrate how the cellular uptake behavior of liposomes can be driven towards preferential interaction with dendritic cells (DC2.4) as compared to macrophages (RAW264.7) by tuning the lipid composition with varied molar ratios of the lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Cellular internalization efficiency was analyzed by flow cytometry, as well as liposome-cell membrane co-localization by confocal laser scanning microscopy. The corresponding proteomic analysis of the protein corona was performed in order to unravel the possible effect on the internalization. The obtained results of this work reveal that it is possible to modulate the cellular uptake towards enhanced internalization by dendritic cells just by modifying the applied lipids and, thus, mainly the physico-chemical properties of the liposomes. STATEMENT OF SIGNIFICANCE: In the field of nanomedicine, it is of key importance to develop new specific and efficient drug carriers. In this sense, liposomes are one of the most widely known carrier types and used in clinics with good results. However, the exact interaction mechanisms of liposomes with cells remain unclear, which is of great importance for the design of new drug delivery platforms. Therefore, in this work we demonstrate that cellular uptake depends on the lipid composition. We are able to enhance the uptake in a specific cell type just by tuning the content of a lipid in the liposome membrane. This finding could be a step towards the selective design of liposomes to be internalized by specific cells with promising applications in biomedicine.

Endosomal sorting results in a selective separation of the protein corona from nanoparticles.

The formation of the protein corona is a well-known effect when nanoparticles (NP) are exposed to biological environments. The protein corona is the most important factor, which determines the rate and route of endocytosis, and decisively impacts cellular processes and even the release of the active pharmaceutical ingredient from the nanoparticles. While many studies concentrate on the effect of the protein corona formation extracellularly or the uptake consequences, little is known about the fate of the protein corona inside of cells. Here, we reconstruct for the first time the separation of the protein corona from the NPs by the cell and their further fate. Ultimately, the NPs and protein corona are separated from each other and end up in morphologically different cellular compartments. The cell directs the NPs towards recycling endosomes, whereas the protein corona gathers in multivesicular bodies. From this, we conclude that the NPs are prepared for subsequent exocytosis, while the protein corona remains in the cell and is finally metabolized there.

A new methodology combining QCM-D and proteomic profiling enables characterization of protein adsorption on 2D surfaces.

One of the critical features of biomedical material design is controlling the plasma protein adsorption to modulate the material behavior in biological media. Protein adsorption is highly influenced by the material surfaces and the proteins present in the biological medium. Thus, it is necessary to study protein-surface interactions that eventually take place on nanomaterials introduced into the body by the use of human plasma. However, very little information is available about human plasma interaction with planar surfaces under physiological conditions. Due to the limitation of the current characterization techniques to investigate the complicated interaction between the complex milieu of plasma proteins and planar materials, most efforts have focused on single proteins. To face this challenge, we have developed a new methodology based on the combination of quartz crystal microbalance with dissipation monitoring (QCM-D) and liquid chromatography coupled with mass spectrometry (LC-MS) to obtain information about protein-surface interactions on planar surfaces. First, QCM-D allowed us to determine the adsorbed protein mass and layer thickness. After detaching the proteins by a surfactant treatment, LC-MS analysis revealed the proteomic profile. Here, we have investigated three base materials, polystyrene (PS), gold (Au), and silica (SiO2) with or without precoating and compared the protein profiles.

Antibacterial Nanostructured Surfaces Modulate Protein Adsorption, Inflammatory Responses, and Fibrous Capsule Formation.

The present study interrogates the interaction of highly efficient antibacterial surfaces containing sharp nanostructures with blood proteins and the subsequent immunological consequences, processes that are of key importance for the fate of every implantable biomaterial. Studies with human serum and plasma pointed to significant differences in the composition of the protein corona that formed on control and nanostructured surfaces. Quantitative analysis using liquid chromatography-mass spectrometry demonstrated that the nanostructured surface attracted more vitronectin and less complement proteins compared to the untreated control. In turn, the protein corona composition modulated the adhesion and cytokine expression by immune cells. Monocytes produced lower amounts of pro-inflammatory cytokines and expressed more anti-inflammatory factors on the nanostructured surface. Studies using an in vivo subcutaneous mouse model showed reduced fibrous capsule thickness which could be a consequence of the attenuated inflammatory response. The results from this work suggest that antibacterial surface modification with sharp spike-like nanostructures may not only lead to the reduction of inflammation but also more favorable foreign body response and enhanced healing, processes that are beneficial for most medical devices implanted in patients.

The Role of the Immune Phenotype in Tumor Progression and Prognosis of Patients with Mycosis Fungoides: A Quantitative Immunohistology Whole Slide Approach.

BACKGROUND AND OBJECTIVES: Mycosis fungoides (MF) is the most common type of cutaneous T-cell lymphomas, characterized by mature, skin-tropic CD4+ T-helper cells. In order to study the immune tumor microenvironment in MF patients, we performed immunohistochemical stains on MF biopsies, digitized whole-slide tissue sections, and performed quantitative analysis of the different immune cell subsets to correlate tissue parameters with the clinical data of patients, such as progression-free survival or overall survival. PATIENTS AND METHODS: Overall, 35 patients who were treated between 2009 and 2019 and for whom one or more paraffin tissue blocks were available have been included in the present study (58 tissue specimens in total). Conventional immunohistochemistry stains for CD3, CD4, CD8, CD20 and CD30 were used for the analysis of the immune phenotype, and quantitative analysis was performed using QuPath as a quantitative digital pathology tool for bioimage analysis of whole slides. RESULTS: Analysis of tissue parameters for prognostic significance revealed that patients with a stronger infiltration by CD8+ lymphocytes within the tumor cell compartment had a higher risk of disease progression (p = 0.031) and showed a shorter progress-free survival (p = 0.038). Furthermore, a significant association of the percentage of CD30+ cells (median: 7.8%) with the risk of disease progression (p = 0.023) and progression-free survival (p = 0.023) was found. In relation to the clinical features of our patient cohort, a higher risk of disease progression (p = 0.015) and a shorter progression-free survival (p = 0.032) for older patients (>61 years) were observed. CONCLUSIONS: Our results demonstrated the prognostic relevance of large-cell transformation in mycosis fungoides and its strong association with the presence of CD30+ lymphocytes. Unlike previous reports, our study suggests an adverse prognostic role for CD8+ T cells in patients with mycosis fungoides. Moreover, our data indicate that the immune phenotype within the tumor microenvironment shows strong temporal heterogeneity and is altered in the course of tumor progression.

Mechanically Robust and Flame-Retardant Superhydrophobic Textiles with Anti-Biofouling Performance.

The attachment of bio-fluids to surfaces promotes the transmission of diseases. Superhydrophobic textiles may offer significant advantages for reducing the adhesion of bio-fluids. However, they have not yet found widespread use because dried remnants adhere strongly and have poor mechanical or chemical robustness. In addition, with the massive use of polymer textiles, features such as fire and heat resistance can reduce the injuries and losses suffered by people in a fire accident. We developed a superhydrophobic textile covered with a hybrid coating of titanium dioxide and polydimethylsiloxane (TiO2/PDMS). Such a textile exhibits low adhesion to not only bio-fluids but also dry blood. Compared to a hydrophilic textile, the peeling force of the coated textile on dried blood is 20 times lower. The textile's superhydrophobicity survives severe treatment by sandpaper (400 mesh) at high pressure (8 kPa) even if some of its microstructures break. Furthermore, the textile shows excellent heat resistance (350 °C) and flame-retardant properties as compared to those of the untreated textile. These benefits can greatly inhibit the flame spread and reduce severe burns caused by polymer textiles adhering to the skin when melted at high temperatures.

Proteomics reveals differential adsorption of angiogenic platelet lysate proteins on calcium phosphate bone substitute materials.

Protein adsorption on biomaterials for bone substitution, such as calcium phosphates (CaP), evokes biological responses and shapes the interactions of biomaterials with the surrounding biological environment. Proteins adsorb when CaP materials are combined with growth factor-rich hemoderivatives prior to implantation to achieve enhanced angiogenesis and stimulate new bone formation. However, the identification of the adsorbed proteins and their angiogenic effect on bone homeostasis remain incompletely investigated. In this study, we analyzed the adsorbed complex protein composition on CaP surfaces when using the hemoderivatives plasma, platelet lysate in plasma (PL), and washed platelet lysate proteins (wPL). We detected highly abundant, non-regenerative proteins and anti-angiogenic proteins adsorbed on CaP surfaces after incubation with PL and wPL by liquid chromatography and mass spectrometry (LC-MS) proteomics. Additionally, we measured a decreased amount of adsorbed pro-angiogenic growth factors. Tube formation assays with human umbilical endothelial cells demonstrated that the CaP surfaces only stimulate an angiogenic response when kept in the hemoderivative medium but not after washing with PBS. Our results highlight the necessity to correlate biomaterial surfaces with complex adsorbed protein compositions to tailor the biomaterial surface toward an enrichment of pro-angiogenic factors.

PEG Spacer Length Substantially Affects Antibody-Based Nanocarrier Targeting of Dendritic Cell Subsets.

Successful cell targeting depends on the controlled positioning of cell-type-specific antibodies on the nanocarrier's (NC) surface. Uncontrolled antibody immobilization results in unintended cell uptake due to Fc-mediated cell interaction. Consequently, precise immobilization of the Fc region towards the nanocarrier surface is needed with the Fab regions staying freely accessible for antigen binding. Moreover, the antibody needs to be a certain distance from the nanocarrier surface, influencing the targeting performance after formation of the biomolecular corona. This can be achieved by using PEG linker molecules. Here we demonstrate cell type-specific targeting for dendritic cells (DC) as cellular key regulators of immune responses. However, to date, dendritic cell targeting experiments using different linker lengths still need to be conducted. Consequently, we focused on the surface modification of nanocarriers with different molecular weight PEG linkers (0.65, 2, and 5 kDa), and their ability to reduce undesired cell uptake, while achieving efficient DC targeting via covalently immobilized antibodies (stealth targeting). Our findings demonstrate that the PEG linker length significantly affects active dendritic cell targeting from cell lines (DC2.4) to primary cells (BMDCs, splenocytic conventional DCs type 1 (cDC1)). While antibody-functionalized nanocarriers with a shorter PEG length (0.65 kDa) showed the best targeting in DC2.4, a longer PEG length (5 kDa) was required to specifically accumulate in BMDCs and splenocytic cDC1. Our study highlights that these crucial aspects must be considered when targeting dendritic cell subsets, which are of great importance in the fields of cancer immunotherapy and vaccine development.

Higher Loading of Gold Nanoparticles in PAD Mesenchymal-like Stromal Cells Leads to a Decreased Exocytosis.

Cell therapy is an important new method in medicine and is being used for the treatment of an increasing number of diseases. The challenge here is the precise tracking of cells in the body and their visualization. One method to visualize cells more easily with current methods is their labeling with nanoparticles before injection. However, for a safe and sufficient cell labeling, the nanoparticles need to remain in the cell and not be exocytosed. Here, we test a glucose-PEG-coated gold nanoparticle for the use of such a cell labeling. To this end, we investigated the nanoparticle exocytosis behavior from PLX-PAD cells, a cell type currently in clinical trials as a potential therapeutic agent. We showed that the amount of exocytosed gold from the cells was influenced by the uptake time and loading amount. This observation will facilitate the safe labeling of cells with nanoparticles in the future and contribute to stem cell therapy research.

Peptidomimetic inhibitors of TMPRSS2 block SARS-CoV-2 infection in cell culture.

The transmembrane serine protease 2 (TMPRSS2) primes the SARS-CoV-2 Spike (S) protein for host cell entry and represents a promising target for COVID-19 therapy. Here we describe the in silico development and in vitro characterization of peptidomimetic TMPRSS2 inhibitors. Molecular docking studies identified peptidomimetic binders of the TMPRSS2 catalytic site, which were synthesized and coupled to an electrophilic serine trap. The compounds inhibit TMPRSS2 while demonstrating good off-target selectivity against selected coagulation proteases. Lead candidates are stable in blood serum and plasma for at least ten days. Finally, we show that selected peptidomimetics inhibit SARS-CoV-2 Spike-driven pseudovirus entry and authentic SARS-CoV-2 infection with comparable efficacy as camostat mesylate. The peptidomimetic TMPRSS2 inhibitors also prevent entry of recent SARS-CoV-2 variants of concern Delta and Omicron BA.1. In sum, our study reports antivirally active and stable TMPRSS2 inhibitors with prospects for further preclinical and clinical development as antiviral agents against SARS-CoV-2 and other TMPRSS2-dependent viruses.

Nanodrugs Targeting T Cells in Tumor Therapy.

In contrast to conventional anti-tumor agents, nano-carriers allow co-delivery of distinct drugs in a cell type-specific manner. So far, many nanodrug-based immunotherapeutic approaches aim to target and kill tumor cells directly or to address antigen presenting cells (APC) like dendritic cells (DC) in order to elicit tumor antigen-specific T cell responses. Regulatory T cells (Treg) constitute a major obstacle in tumor therapy by inducing a pro-tolerogenic state in APC and inhibiting T cell activation and T effector cell activity. This review aims to summarize nanodrug-based strategies that aim to address and reprogram Treg to overcome their immunomodulatory activity and to revert the exhaustive state of T effector cells. Further, we will also discuss nano-carrier-based approaches to introduce tumor antigen-specific chimeric antigen receptors (CAR) into T cells for CAR-T cell therapy which constitutes a complementary approach to DC-focused vaccination.