In vitro expansion of Wilms' tumor protein 1 epitope-specific primary T cells from healthy human peripheral blood mononuclear cells.

Wilms' tumor protein 1 (WT1) is a tumor-associated antigen overexpressed in various cancers. As a self-antigen, negative selection reduces the number of WT1-specific T cell receptors (TCRs). Here, we provide a protocol to generate WT137-45-specific TCRs using healthy human peripheral blood mononuclear cells. We describe the expansion of WT1-specific T cell clones by two consecutive in vitro stimulations with autologous WT137-45-pulsed dendritic cells and peripheral blood lymphocytes. We then detail the detection with human leukocyte antigen/WT137-45 tetramers.

Treatment of in vitro generated Langerhans cells with JAK-STAT inhibitor reduces their inflammatory potential.

Alopecia areata (AA) is a condition in which hair is lost in small regions or over the entire body. It has a prevalence of 1 in 1000 and has a great impact on psychological wellbeing. AA is generally considered an autoimmune disease in which a collapse of the immune privilege system of the hair follicle has shown to play an important role, potentially driven by interferon gamma (IFN-γ). The most prominent cells located in or around the hair follicle in AA are Langerhans cells, CD4+ or CD8+ T cells, macrophages and mast cells. Langerhans cells, specialized dendritic cells, are resident in the epidermis and are known to associate with hair follicles. Therefore, we aimed to develop in vitro generated Langerhans cells contributing as an in vitro model of disease. In vitro models provide insight into the behaviour of cells and are a valuable tool before being in need of an animal model or patient samples. For this, Langerhans-like cells were generated from CD14+ monocytes in the presence of GM-CSF and TGF-ß. After 10 days of cell culture, Langerhans-like cells express CD207 and CD1a but lack CD209 expression as well as Birbeck granules. Next, Langerhans-like cells were exposed to inflammatory conditions and the effect of different AA treatments was investigated. All treatments-diphencyprone contact immunotherapy, UV-B light therapy and JAK-STAT inhibition-affect the expression of costimulatory and skin-homing markers on Langerhans-like cells. Importantly, also the T cell stimulatory capacity of Langerhans-like cells was significantly reduced following treatment under inflammatory conditions. Noteworthy, JAK-STAT inhibition outperformed conventional AA treatments. In conclusion, our findings demonstrate that in vitro generated Langerhans-like cells can be used as a model of disease. Moreover, JAK-STAT inhibition may become a valuable new approach for the treatment of AA.

Rapid Assessment of Functional Avidity of Tumor-Specific T Cell Receptors Using an Antigen-Presenting Tumor Cell Line Electroporated with Full-Length Tumor Antigen mRNA.

The functional avidity of T-cell receptor (TCR)-engineered T cells towards their cognate epitope plays a crucial role in successfully targeting and killing tumor cells expressing the tumor-associated antigen (TAA). When evaluating in vitro functional T-cell avidity, an important aspect that is often neglected is the antigen-presenting cell (APC) used in the assay. Cell-based models for antigen-presentation, such as tumor cell lines, represent a valid alternative to autologous APCs due to their availability, off-the-shelf capabilities, and the broad range of possibilities for modification via DNA or messenger RNA (mRNA) transfection. To find a valuable model APC for in vitro validation of TAA Wilms' tumor 1 (WT1)-specific TCRs, we tested four different WT1 peptide-pulsed HLA-A2+ tumor cell lines commonly used in T-cell stimulation assays. We found the multiple myeloma cell line U266 to be a suitable model APC to evaluate differences in mean functional avidity (EC50) values of transgenic TCRs following transfection in 2D3 Jurkat T cells. Next, to assess the dose-dependent antigen-specific responsiveness of WT1 TCR-engineered 2D3 T cells to endogenously processed epitopes, we electroporated U266 cells with different amounts of full-length antigen WT1 mRNA. Finally, we analyzed the functional avidity of WT1 TCR-transfected primary CD8 T cells towards WT1 mRNA-electroporated U266 cells. In this study, we demonstrate that both the APC and the antigen loading method (peptide pulsing versus full-length mRNA transfection) to analyze T-cell functional avidity have a significant impact on the EC50 values of a given TCR. For rapid assessment of the functional avidity of a cloned TCR towards its endogenously processed MHC I-restricted epitope, we showcase that the TAA mRNA-transfected U266 cell line is a suitable and versatile model APC.