Enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy.

Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) targeting neoantigens can mediate tumor regression in selected patients with metastatic epithelial cancer. However, effectively identifying and harnessing neoantigen-reactive T cells for patient treatment remains a challenge and it is unknown whether current methods to detect neoantigen-reactive T cells are missing potentially clinically relevant neoantigen reactivities. We thus investigated whether the detection of neoantigen-reactive TILs could be enhanced by enriching T cells that express PD-1 and/or T cell activation markers followed by microwell culturing to avoid overgrowth of nonreactive T cells. In 6 patients with metastatic epithelial cancer, this method led to the detection of CD4+ and CD8+ T cells targeting 18 and 1 neoantigens, respectively, compared with 6 and 2 neoantigens recognized by CD4+ and CD8+ T cells, respectively, when using our standard TIL fragment screening approach. In 2 patients, no recognition of mutated peptides was observed using our conventional screen, while our high-throughput approach led to the identification of 5 neoantigen-reactive T cell receptors (TCRs) against 5 different mutations from one patient and a highly potent MHC class II-restricted KRASG12V-reactive TCR from a second patient. In addition, in a metastatic tumor sample from a patient with serous ovarian cancer, we isolated 3 MHC class II-restricted TCRs targeting the TP53G245S hot-spot mutation. In conclusion, this approach provides a highly sensitive platform to isolate clinically relevant neoantigen-reactive T cells or their TCRs for cancer treatment

Production of LacZ inducible T cell hybridoma specific for human and mouse gp100₂₅₋₃₃ peptides.

Identification and quantification of immunogenic peptides and tumor-derived epitopes presented on MHC-I molecules are essential for basic studies and vaccines generation. Although lymphocytes derived from transgenic mice can serve as sensitive detectors of processes of antigen presentation and recognition, they are not always available. The use of cell lines might be extremely useful. In this study, we generated a lacZ inducible CD8⁺ hybridoma (BUSA14) capable of recognizing both human and mouse gp100₂₅₋₃₃ melanoma antigens presented on dendritic and tumor cell lines. This hybridoma expresses a variety of membranal T cell markers and secretes IL-2 and TNFα. Thus, BUSA14 offers a quantifiable, cheap and straightforward tool for studying peptide presentation by MHC-I molecules on the cell surface

BUSA14 are activated by hgp100_25–33 and mgp100_25–33 presented on melanoma cell lines.

<p><b>A.</b> B16-MO5, F10.9, D122 and EL4 tumor cell lines were analyzed by flow cytometry with monoclonal antibodies to H-2K^b and H-2D^b to analyze MHC-I membranal expression. MFI values are presented in the figure. <b>B.</b> Twenty thousand B16-MO5, F10.9 and D122 cells were loaded with 30 µg/ml hgp100_25–33 or SIINFEKL peptides. Cells were washed and co-incubated with 6×10⁴ BUSA14 and BWZ.36/CD8α for 12 hours. Cells were then lysed and β-Gal enzymatic activity was monitored with CPRG. Cultures with D122 served as reference for CPRG background levels. Representative results (1 of 3 experiments) are presented as ΔOD (sample OD-background OD) measured after 12 hours. <b>C.</b> Sixty thousand BUSA14 and BWZ.36/CD8α cells/well were incubated overnight, in triplicates, with 2×10⁴ B16-MO5, F10.9 or D122 tumor cell lines. Representative results (1 of 2 experiments) are presented as ΔOD (sample OD-background OD) measured after 24 hours. Statistical analysis was done using student T test (*p<0.05, **p<0.01, ***p<0.001).</p

Detection of cytokines produced by BUSA14 cells.

<p>BUSA14 or BWZ.36/CD8α were co-incubated with DC2.4 cells loaded with hgp100_25–33 or SIINFEKL. Cells alone or co-cultured with unloaded DC2.4 or with PMA and ionomycin served as negative and positive controls, respectively. All cells were intracellulary stained with antibodies to CD8, IL-2/TNFα (<b>A</b>), IL-4/IFNγ (<b>B</b>) and analyzed by flow cytometry. Cells were gated for CD8 to exclude DC2.4 cells.</p

T cell markers expressed by BUSA14 cells.

<p><b>A.</b> BUSA14, B3Z and BWZ.36/CD8α cells were analyzed by flow cytometry and monoclonal antibodies against CD8 and TCR Vβ13 to confirm the Pmel-1 TCR expression. <b>B.</b> BUSA14, BWZ.36/CD8α and B3Z cells were co-cultured with hgp100_25–33 or SIINFEKL for 12 hours, or remained untreated. Cells were stained with antibodies to CD8, CD62L and CD44 and analyzed by flow cytometry. Cells were gated for CD8 to exclude DC2.4 cells. <b>C.</b> Two million BUSA14, BWZ.36/CD8α and B3Z cells were co-cultured with 6×10⁵ DC2.4 loaded with hgp100_25–33 or SIINFEKL for 12 hours, or remained untreated. Then stained with antibodies against CD8, CD69, CD279 and analyzed by flow cytometry. Cells were gated for CD8 to exclude DC2.4 cells. Mean fluorescent intensity (MFI) values are presented in the figure. This figure is a representative of three experimental repeats.</p

Comparison of Transcatheter Aortic Valve Implantation Devices in Aortic Stenosis: A Network Meta-Analysis of 42,105 Patients

Background: In recent years, trans-catheter aortic valve implantation (TAVI) has emerged as an excellent alternative to surgical aortic valve replacement (SAVR). Currently, there are several approved devices on the market, yet comparisons among them are scarce. We aimed to compare the various devices via a network meta-analysis. Methods: We performed a network meta-analysis including randomized controlled trials (RCTs) and propensity-matched studies that provide comparisons of either a single TAVI with SAVR or two different TAVI devices and report clinical outcomes. Results: We included 12 RCT and 13 propensity-matched studies comprising 42,105 patients, among whom 27,134 underwent TAVI using various valve systems (Sapien &amp; Sapien XT, Sapien 3, Corvalve, Evolut &amp; Evolut Pro, Acurate Neo, Portico). The mean follow-up time was 23.4 months. Sapien 3 was superior over SAVR in the reduction of all-cause mortality (OR = 0.53; 95%CrI 0.31&ndash;0.91), while no significant difference existed between other devices and SAVR. Aortic regurgitation was more frequent among TAVI devices compared to SAVR. There was no significant difference between the various THVs and SAVR in cardiovascular mortality, myocardial infarction, NYHA class III-IV, and endocarditis. Conclusions: Newer generation TAVI devices, especially Sapien 3 and Evolut R/Pro are associated with improved outcomes compared to SAVR and other devices of the older generation