Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Improving the ex vivo expansion of human tumor-reactive CD8 + T cells by targeting toll-like receptors

doi: 10.3389/fbioe.2022.1027619

Figure Lengend Snippet: Co-stimulation with TLR1/2, TLR2/6, and TLR5 agonists enhances the proliferative response of human blood-derived CD8 + T cells to TCR triggering. (A) Expression profiles of surface TLRs in resting CD8 + T cells within human blood mononuclear cells (PBMCs). The T cell subpopulations of human PBMCs were identified by staining the cells with antibodies against CD3, CD4, and CD8; the TLR expression was measured by flow cytometry and expressed as the percentage of positively stained cells. Shown is aggregated data of 12 samples. (B and C) Co-stimulatory effects of different TLR agonists on the proliferation of human PBMC-derived CD8 + T cells. CD8+T cells were purified from human PBMCs, stained with eFluor 670, and then stimulated with ant-CD3 beads, anti-CD3/CD28 beads, or anti-CD3 beads coupled with the indicated TLRs agonists. Cells were harvested 7 days later and dilution of eFluor 670 was determined by flow cytometry to measure cellular proliferation, with representative flow cytometry plot and data summary (n = 3) shown in (B) and (C) , respectively. (D,E) Determination of optimal dosages of TLR agonists required for co-stimulation of ex vivo proliferation of human CD8 + T cells. CD8+T cells purified from human PBMCs were stimulated with anti-CD3 beads in presence of various concentration of TLR1/2 agonist, TLR2/6 agonist, or TLR5 agonist after labelling with eFluor 670. Flow cytometry analyses were performed on day 4 and 6 after stimulation to determine the cellular proliferation through assessment of eFluor 670 dilution. Shown is aggregated data of 3 samples.

Article Snippet: Human TLR1-9 Agonist Kit, TLR1/TLR2 agonist Pam3CSK4, TLR5 agonist flagellin (FLA), and TLR2/TLR6 agonist FSL-1 were purchased from InvivoGen.

Techniques: Derivative Assay, Expressing, Staining, Flow Cytometry, Purification, Ex Vivo, Concentration Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Improving the ex vivo expansion of human tumor-reactive CD8 + T cells by targeting toll-like receptors

doi: 10.3389/fbioe.2022.1027619

Figure Lengend Snippet: Co-stimulation with a combination of TLR1/2, TLR2/6, and TLR5 agonists enhances the transcriptional induction of proliferative-related genes in human blood-derived CD8 + T cells responding to TCR triggering. CD8+T cells purified from human PBMCs were stimulated with anti-CD3 beads in the presence or absence of a combination of TLR1/2 agonist, TLR2/6 agonist, and TLR5 agonist. Cells were harvested 24 h later for extracting total RNA, which was subjected to RNA sequencing. The differentially expressed genes showing >2-fold change between minus and plus TLR agonists groups were clustered based on Gene Ontology (GO) enrichment analysis (A) . Shown in (B) is the heatmap of representative proliferation-related genes that were identified to be co-stimulated by TLR agonists. H1 to H3 represent samples from three healthy individuals.

Article Snippet: Human TLR1-9 Agonist Kit, TLR1/TLR2 agonist Pam3CSK4, TLR5 agonist flagellin (FLA), and TLR2/TLR6 agonist FSL-1 were purchased from InvivoGen.

Techniques: Derivative Assay, Purification, RNA Sequencing Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Improving the ex vivo expansion of human tumor-reactive CD8 + T cells by targeting toll-like receptors

doi: 10.3389/fbioe.2022.1027619

Figure Lengend Snippet: TLR agonists is an advantageous supplement benefiting the ex vivo expansion of human blood-derived PD1+ CD8 + T cells. (A) Exploration of optimal combination of TLR agonists in facilitating the ex vivo expansion of human blood-derived PD1+ CD8 + T cells. PD1+ CD8 + T cells isolated from blood samples of health individuals were continuously cultured for 21 days in medium containing anti-CD3/anti-CD28 beads and IL-7/IL-15, either alone or in the presence of different combination of TLR1/2, TLR2/6, and TLR5 agonists. Cell counting was performed at various time points throughout the culture period to generate the cell expansion curve. (B) Expansion curves of PD1+ CD8 + T cells derived from blood samples of cancer patients with different tumor types. Among the total of fifteen samples, seven were with lung cancer, four with pancreatic cancer, and the rest four with other types of cancers. Five samples from healthy individuals were also included as controls.

Article Snippet: Human TLR1-9 Agonist Kit, TLR1/TLR2 agonist Pam3CSK4, TLR5 agonist flagellin (FLA), and TLR2/TLR6 agonist FSL-1 were purchased from InvivoGen.

Techniques: Ex Vivo, Derivative Assay, Isolation, Cell Culture, Cell Counting