RESUMO
Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than two decades to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs) and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers (i.e. caIKK), and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs and mRNA-electroporated T cells for therapeutic applications in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types; (2) scalability from 106 to approximately 108 cells per shot; (3) high transfection efficiency (80-99%); (4) homogenous protein expression; (5) GMP compliance if the EP is performed in a class A clean room; and (6) no transgene integration into the genome. The provided protocol involves: OptiMEM® as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time has to be altered. Thus, we share an overview of proven electroporation settings (including recovery media), which we have established for various cell types. Next to the basic protocol, we also provide an extensive list of hints and tricks, which, in our opinion, are of great value for everyone who intends to use this transfection technique.
Assuntos
Células Dendríticas , Eletroporação , RNA Mensageiro , Transfecção , Eletroporação/métodos , Humanos , RNA Mensageiro/genética , Transfecção/métodos , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , Linfócitos T/metabolismo , Linfócitos T/imunologia , Antígenos/genética , Linfócitos B/metabolismo , Linfócitos B/imunologiaRESUMO
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.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Apoptose , Azetidinas/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Humanos , Imidazóis/farmacologia , Oximas/farmacologia , Piperidinas/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologiaRESUMO
INTRODUCTION: With the medical focus on disease, the problem of overdiagnosis inevitably increases with ageing. Considering the functional health of patients might help to discriminate between necessary and unnecessary medicine. The International Classification of Functioning, Disability and Health (ICF) is an internationally recognised tool for describing functional health. However, it is too detailed to be used in primary care practices. Consequently, the aim of this study is to identify relevant codes for an ICF core set for community-dwelling older adults (75 years and above) in primary care. METHODS AND ANALYSIS: The study will follow the methodology proposed by the ICF Research Branch to identify relevant concepts from different perspectives: (1) Research perspective: A systematic review of studies focusing on functional health in old age will be conducted in different databases. Relevant concepts will be extracted from the publications. (2) Patients' perspective: Relevant areas of functioning and disability will be identified conducting qualitative interviews and focus groups with community-dwelling older persons. The interviews will be transcribed verbatim and analysed using the documentary method of interpretation. (3) Experts' perspective: An online survey with open-ended questions will be conducted. Answers will be analysed using the qualitative content analysis of Mayring. (4) Clinical perspective: A cross-sectional empirical study will be performed to assess the health status of community-dwelling older adults using the extended ICF checklist and other measurement tools.Relevant concepts identified in each study will be linked to ICF categories resulting in four preliminary core sets. ETHICS AND DISSEMINATION: Ethical approval for the study was obtained (90_17B). All participants will provide written informed consent. Data will be pseudonymised for analysis. Results will be disseminated by conference presentations and journal publications. TRIAL REGISTRATION NUMBER: Projektdatenbank Versorgungsforschung Deutschland: VfD_17_003833,Clinicaltrials.gov: NCT03384732 and PROSPERO: CRD42017067784.
Assuntos
Medicina Geral/métodos , Vida Independente , Classificação Internacional de Funcionalidade, Incapacidade e Saúde , Idoso , Estudos Transversais , Grupos Focais , Avaliação Geriátrica/métodos , Humanos , Entrevistas como Assunto , Projetos de Pesquisa , Inquéritos e Questionários , Revisões Sistemáticas como AssuntoRESUMO
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⺠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.
Assuntos
Reprogramação Celular/efeitos dos fármacos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos , Proteoglicanas de Sulfatos de Condroitina/genética , Citocinas/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , MAP Quinase Quinase Quinases/metabolismo , Melanoma/terapia , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
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.
Assuntos
Biologia Computacional/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Histamina/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteômica/métodos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/metabolismo , Agonistas dos Receptores Histamínicos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Ligantes , Transdução de Sinais/efeitos dos fármacosRESUMO
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).
Assuntos
RNA , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genética , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Adulto , Técnicas de Cultura de Células , Citocinas/metabolismo , Citotoxicidade Imunológica , Eletroporação , Engenharia Genética , Antígeno HLA-A2/imunologia , Voluntários Saudáveis , Humanos , Separação Imunomagnética , Imunofenotipagem , Imunoterapia Adotiva , Melanoma/genética , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/terapia , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T , Transfecção , Adulto Jovem , Antígeno gp100 de Melanoma/imunologiaRESUMO
BACKGROUND: Merkel cell carcinoma (MCC) is a rare but very aggressive skin tumor that develops after integration of a truncated form of the large T-antigen (truncLT) of the Merkel cell polyomavirus (MCV) into the host's genome. Therapeutic vaccination with dendritic cells (DCs) loaded with tumor antigens is an active form of immunotherapy, which intends to direct the immune system towards tumors which express the respective vaccination antigens. METHODS: Cytokine-matured monocyte-derived DCs of healthy donors and MCC patients were electroporated with mRNA encoding the truncLT. To permit major histocompatibility complex (MHC) class II next to class I presentation, we used an RNA construct in which the antigen was fused to a DCLamp sequence in addition to the unmodified antigen. To further improve their immunogenicity, the DCs were additionally activated by co-transfection with the constitutively active nuclear factor (NF)-κB activator caIKK. These DCs were used to stimulate autologous CD8+ T-cells or a mixture of CD4+ and CD8+ T-cells. Then the percentage of T-cells, specific for the truncLT, was quantified by interferon (IFN)γ ELISpot assays. RESULTS: Both the truncLT and its DCLamp-fusion were detected within the DCs by flow cytometry, albeit the latter required blocking of the proteasome. The transfection with caIKK upregulated maturation markers and induced cytokine production. After 2-3 rounds of stimulation, the T-cells from 11 out of 13 healthy donors recognized the antigen. DCs without caIKK appeared in comparison less potent in inducing such responses. When using cells derived from MCC patients, we could induce responses for 3 out of 5 patients; however, here the caIKK-transfected DCs did not display their superiority. CONCLUSION: These results show that optimized DCs are able to induce MCV-antigen-specific T-cell responses. Therapeutic vaccination with such transfected DCs could direct the immune system against MCC.
RESUMO
Monocytes are a part of the innate immune system. Their differentiation into macrophages changes their cellular proteome and secretome. Particularly secretome components such as cytokines are crucial for immune response and inflammation in many diseases. Differentiation of human lymphoma cell line U937 can be triggered by phorbol 12-myristate 13-acetate (PMA). Screening of the cytokine release in U937 upon PMA stimulation by cytometric bead array almost exclusively showed interleukin-8 (IL-8). Next, a label-free nanoLC-ESI-MS/MS-sSRM method for quantification of IL-8 in the cell secretome was established and applied to monitor the time kinetics of PMA treatment in different concentrations. Targeted secretome analysis was achieved by scheduled SRM-MS using one proteotypic peptide as precursor ion and four mass transitions. Label-free quantification was performed by external calibration using IL-8 standard. Validation results indicated that the method was suited for the quantification of IL-8 in the secretome. The maximal IL-8 release of 62.4 ng/mL was observed after incubating cells treated by 50 ng/mL PMA for 48 h. The method can now be used for quantification of IL-8 release from different cells under various conditions. Furthermore, it can be easily expanded to other secreted proteins detected by untargeted screening methods.
Assuntos
Cromatografia Líquida/métodos , Interleucina-8/análise , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Acetato de Tetradecanoilforbol/farmacologia , Carcinógenos/farmacologia , Humanos , Proteoma/efeitos dos fármacos , Espectrometria de Massas por Ionização por Electrospray/métodos , Células U937RESUMO
Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than a decade to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs), and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers, and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs for therapeutic vaccination in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types, (2) scalability from 106 to approximately 108 cells per shot, (3) high transfection efficiency (80-99 %), (4) homogenous protein expression, (5) GMP compliance if the EP is performed in a class A clean room, and (6) no transgene integration into the genome. The provided protocol involves: Opti-MEM® as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time is altered. Next to the basic protocol, we also provide an extensive list of hints and tricks, which in our opinion are of great value for everyone who intends to use this transfection technique.
Assuntos
Antígenos/imunologia , RNA Mensageiro/imunologia , Linfócitos B/imunologia , Linhagem Celular , Células Dendríticas/imunologia , Eletroporação/métodos , Humanos , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Transfecção/métodosRESUMO
For therapeutic cancer vaccination, the adoptive transfer of mRNA-electroporated dendritic cells (DCs) is frequently performed, usually with monocyte-derived, cytokine-matured DCs (moDCs). However, DCs are rich in danger-sensing receptors which could recognize the exogenously delivered mRNA and induce DC activation, hence influencing the DCs' immunogenicity. Therefore, we examined whether electroporation of mRNA with a proper cap and a poly-A tail of at least 64 adenosines had any influence on cocktail-matured moDCs. We used 16 different RNAs, encoding tumor antigens (MelanA, NRAS, BRAF, GNAQ, GNA11, and WT1), and variants thereof. None of those RNAs induced changes in the expression of CD25, CD40, CD83, CD86, and CD70 or the secretion of the cytokines IL-8, IL-6, and TNFα of more than 1.5-fold compared to the control condition, while an mRNA encoding an NF-κB-activation protein as positive control induced massive secretion of the cytokines. To determine whether mRNA electroporation had any effect on the whole transcriptome of the DCs, we performed microarray analyses of DCs of 6 different donors. None of 60,000 probes was significantly different between mock-electroporated DCs and MelanA-transfected DCs. Hence, we conclude that no transcriptional programs were induced within cocktail-matured DCs by electroporation of single tumor-antigen-encoding mRNAs.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/fisiologia , Imunoterapia Adotiva/métodos , Antígeno MART-1/metabolismo , Monócitos/fisiologia , RNA Mensageiro/genética , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/transplante , Eletroporação , Perfilação da Expressão Gênica , Humanos , Antígeno MART-1/genética , Análise em MicrossériesRESUMO
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.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Comunicação Celular/fisiologia , Proliferação de Células/fisiologia , Células Dendríticas/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T CD8-Positivos/citologia , Vacinas Anticâncer/imunologia , Células Cultivadas , Células Dendríticas/citologia , Feminino , Humanos , Imunoterapia/métodos , Masculino , Linfócitos T Auxiliares-Indutores/citologiaRESUMO
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.
Assuntos
Células Dendríticas/imunologia , Quinase I-kappa B/genética , NF-kappa B/imunologia , Neoplasias/terapia , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Adulto , Idoso , Vacinas Anticâncer/imunologia , Diferenciação Celular/imunologia , Proliferação de Células , Células Dendríticas/enzimologia , Feminino , Humanos , Memória Imunológica , Imunoterapia/métodos , Interleucina-12/biossíntese , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , NF-kappa B/genética , Neoplasias/imunologia , Transdução de Sinais/imunologia , Transfecção , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Adulto JovemRESUMO
Sequencing, cloning and functional testing of T-cell-receptor (TCR) alpha- and beta-chains from T-cell clones is often required in immunotherapy and in immunological research. However, the determination of the TCR chains by a simple PCR is not possible, since, in contrast to the 3' constant domain and untranslated region (UTR), no conserved sequences are present in the 5' region. Furthermore, subsequent functional testing of cloned TCRs requires laborious cell culture experiments, often involving primary human material and time-consuming viral transduction strategies. Here we present a universal PCR-based protocol, adapted from the capswitch technology, that allows for amplification of the TCR alpha- and beta-chain mRNAs without knowledge of the TCR variable domain subtype by attaching a designed sequence to the mRNA's 5' end. Two different MelanA/HLA-A2-specific and one HIVgag/HLA-A2-specific TCR were cloned that way, and were functionally tested by a newly developed easy, fast, and low-cost method: we electroporated Jurkat T cells simultaneously with TCR-encoding RNA and an NFAT-reporter construct, and measured the activation status of the cells upon specific stimulation. The results of this assay correlated with the cytokine release, functional avidity, proliferative activity, and the ability to recognize MelanA/HLA-A2-presenting tumor cells of bulk T cells electroporated with RNA encoding the same TCR. Together these two protocols represent a rapid and low-cost tool for the identification and functional testing of TCRs of T-cell clones, which can then be applied in immunotherapy or immunological research.