rosettasep kits  (STEMCELL Technologies Inc)

Journal: Frontiers in Aging

Article Title: The influence of three-dimensional structure on naïve T cell homeostasis and aging

doi: 10.3389/fragi.2022.1045648

Figure Lengend Snippet: Naïve T cells are maintained in a resting state in SLT-like organoids. (A) Enrichment of FRCs (CD45 neg CD31 neg podoplanin + ) from tonsil tissue of young adults. (B) Viability of total T cells and (C) frequency of naïve (CD45RA + CCR7+) CD4 T cells in 2D culture conditions with and without FRC co-culture (mean± SEM). ( n = 3) (D) Frequencies of live T cells based on viability dye over time across all culture conditions. (E) Frequencies of naïve CD4 and CD8 T cells ( n = 12) over time across 2DF and 3DF conditions. Mean ± sem. p -values compare 2DF vs. 3DF within one time point. (F) Frequencies of naïve (CD45RA + CCR7+) CD4 and CD8 T cells at day 14 in 2DF versus 3DF conditions. ( n = 12) (G) Frequencies of central memory (CD45RA-CCR7+) CD4 and CD8 T cells ( n = 12) over time across 2DF and 3DF conditions. Mean ± sem. (H) Expression of activation markers CD69 and CD71 on naïve CD4 T cells at day 14, representative of three individual donors. (I,J) CFSE proliferation analysis of (I) naïve CD4 T cells and (J) naïve CD8 T cells at day 14 ( n = 6 young donors, paired). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. p -values determined by paired t -test.

Article Snippet: CD3 + T cells were isolated directly from whole blood using Total T cells RosettaSep kit (Stem Cell Technologies Cat #15061).

Techniques: Co-Culture Assay, Expressing, Activation Assay

Journal: Frontiers in Aging

Article Title: The influence of three-dimensional structure on naïve T cell homeostasis and aging

doi: 10.3389/fragi.2022.1045648

Figure Lengend Snippet: SLT-organoids prevent T cells from acquiring features of immune aging and naïve cell-specific decline in CD45RA expression. (A) CD25 + naïve CD4 T cell frequencies in naïve CD4 T cells in 2DF and 3DF ( n = 5 young adult T cell donors) after 14 days of culturing. (B) CD39 + CD45RA neg CD4 T cells frequencies in total CD4 T cells in 2DF and 3DF ( n = 8 young adult T cell donors) after 14 days of culturing. (C) Effector memory (CD45RA neg CCR7 neg ) CD4 T cell and (D) TEMRA (CD45RA + CCR7 neg ) CD8 T cell frequencies in 2DF and 3DF ( n = 6 young adult T cell donors) after 14 days of culturing. (E,F) Distribution of (E) CD4 and (F) CD8 T cells by CD45RA and CCR7 expression after 14 days of culturing. (G,H) Representative histograms of CD45RA expression on naïve (CD45RA + CCR7+) (G) CD4 and (H) CD8 T cells in 2DF and 3DF after 14 days of culturing. (I,J) CD45RA and CCR7 MFI on naïve (I) CD4 and (J) CD8 T cells in 2DF and 3DF ( n = 6 young adult T cell donors done in duplicate) after 14 days of culturing. (K, L) CD45RA and CCR7 MFI over time on naïve (K) CD4 and (L) CD8 T cells in 2DF and 3DF (n = 6 young adult T cell donors done in duplicate). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.000. p -values determined paired t -test.

Article Snippet: CD3 + T cells were isolated directly from whole blood using Total T cells RosettaSep kit (Stem Cell Technologies Cat #15061).

Techniques: Expressing

Journal: Frontiers in Aging

Article Title: The influence of three-dimensional structure on naïve T cell homeostasis and aging

doi: 10.3389/fragi.2022.1045648

Figure Lengend Snippet: CD45RA expression is globally reduced on antigen-inexperienced naïve T cells in older adults. (A) Overview of young (YA; <35 years old) and older (OA; >60 years old) adult cohort design for peripheral blood mononuclear cell flow cytometry. Flow analysis was done same day, same instrument for each independent cohort. (B) Cohort 1 (YA = 10, OA = 8) and (C) cohort 2 (YA = 13, OA = 14) analysis of CD45RA expression on the surface of naïve CD4 and CD8 T cells. (D) Frequencies of and (E) CD45RA MFI on Mart-1-specific naïve CD8 T cells from young ( n = 10) and older ( n = 9) HLA-A2 + adults. CMV pp65 495-503 (NLVPMVATV) tetramer was used as a control for non-specific binding. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant. p -values determined by Mann-Whitney test.

Article Snippet: CD3 + T cells were isolated directly from whole blood using Total T cells RosettaSep kit (Stem Cell Technologies Cat #15061).

Techniques: Expressing, Flow Cytometry, Control, Binding Assay, MANN-WHITNEY

Journal: Frontiers in Aging

Article Title: The influence of three-dimensional structure on naïve T cell homeostasis and aging

doi: 10.3389/fragi.2022.1045648

Figure Lengend Snippet: CD45RA high subset of naïve T cells is replaced by a CD45RA low , CD25 low/+ subset with age. (A) General experimental outline and Phenograph clustering of naïve CD4 T cells from middle-aged (MA; n = 5; 40–49 years) and older (OA; n = 5; >60 years) adults. (B) UMAP of differential clusters between MA and OA naïve CD4 T cells. (C) Histograms of select markers delineating age-related clusters. (D) Relative markers expression in naïve CD4 and CD8 clusters that decrease with age compared with those that increase with age. (E) Hand-gated frequencies of population lost with age (termed “youthful;” CD45RA high CD27 high CD38 + CD25 neg naïve T cells). (F,G) Frequencies of (F) youthful and (G) CD45RA low CD25 + naïve T cells in a follow-up cohort of young ( n = 20; <35 years) and older ( n = 20; >60 years) individuals. p -values determined by Mann-Whitney test.

Article Snippet: CD3 + T cells were isolated directly from whole blood using Total T cells RosettaSep kit (Stem Cell Technologies Cat #15061).

Techniques: Expressing, MANN-WHITNEY

Journal: Frontiers in Aging

Article Title: The influence of three-dimensional structure on naïve T cell homeostasis and aging

doi: 10.3389/fragi.2022.1045648

Figure Lengend Snippet: CD45RA high naïve T cells are enriched in human blood, lymph nodes and SLT-like organoids. (A) UMAP of naïve CD4 T cells across different tissue types from young adults ( n = 3). (B) Markers expression within naïve CD4 T cells from tissues. (C–E) Frequencies of (C) CD45RA high CD27 high CD38 + CD25 neg and (D) CD25 + CD45RA low CD27 low naïve CD4 and (E) CD45RA high CD27 high CD38 + CD25 neg naïve CD8 T cells across tissue type. Mass cytometry dataset is from publicly available source ImmPort SDY1389. (F) Frequencies of CD45RA high CD27 high CD38 + CD25 neg naive CD4 T cells over time across different culture conditions ( n = 3 young donors). (G) Frequency of CD45RA high CD27 high naïve CD4 and CD8 T cells in larger cohort of young donors ( n = 15) at day 14 in either 2DF or 3DF conditions. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. p -values were determined by (C–E) one-way ANOVA and (F,G) paired t -test.

Article Snippet: CD3 + T cells were isolated directly from whole blood using Total T cells RosettaSep kit (Stem Cell Technologies Cat #15061).

Techniques: Expressing, Mass Cytometry

Journal: Frontiers in Aging

Article Title: The influence of three-dimensional structure on naïve T cell homeostasis and aging

doi: 10.3389/fragi.2022.1045648

Figure Lengend Snippet: Effects of FRC aging, senescence, and lymphopenia on naïve T cell aging. (A) Overview of fibroblastic reticular cell isolation from young ( n = 3; 27, 31, and 41 years old) and older ( n = 3; 85, 94, and 96 years old) adults. (B) T cell viability and (C) CD45RA high CD27 high naïve CD4 T cells after 14 days organoid cultures ( n = 3 young T cell donors) with FRCs isolated from young or older adults. (D) Overview and characterization of senescent FRCs generated from FRCs from young tissue donor. Relative gene expression of IL-6, p21 and p16 determined by qPCR, normalized to RPLP0 control gene. (E) Percent Live T cells and (F) frequencies of CD45RA high CD27 high naïve CD4 and CD8 T cells in 2DF (comparison control) or 3DF with proliferating (PRO) or senescent (SEN) FRCs. T cells are from four different young donors, performed in duplicate. (G) Effect of T cell input number on CD45RA high CD27 high naïve CD4 T cell frequencies in young donors ( n = 4) after 14 days organoid cultures. (H) Frequencies of CD45RA high CD27 high CD38 + CD25 neg naïve CD4 and CD8 T cells in cohort of patients 1–2 years after chemotherapy induced lymphopenia ( n = 10) compared with control cohort ( n = 10). ns, not significant. p -values determined by (B,C) paired t -test, (E–G) RM one-way ANOVA with Holm-Sidak multiple comparisons tests and (H) Mann-Whitney test.

Article Snippet: CD3 + T cells were isolated directly from whole blood using Total T cells RosettaSep kit (Stem Cell Technologies Cat #15061).

Techniques: Cell Isolation, Isolation, Generated, Gene Expression, Control, Comparison, MANN-WHITNEY

Journal: Frontiers in Aging

Article Title: The influence of three-dimensional structure on naïve T cell homeostasis and aging

doi: 10.3389/fragi.2022.1045648

Figure Lengend Snippet: Structure and cell density effect naïve T cell phenotype and the conversion from CD45RA high to CD45RA low . (A) Clustering of T cells in organoids at different cell densities after 7 days. Representative of three independent experiments. (B,C) Effect of cell density/contact capability on (B) CD45RA high CD27 high naïve CD4 T cells and (C) T cell viability in young donor T cells ( n = 3). T cells were cultured with FRCs (from young adult) in flat, U-bottom, V-bottom or organoids for 14 days. (D) T cells were isolate from young (YA; 35 years or less) and older (OA; 60 years or older) adult peripheral blood and cultured in organoids with FRCs (from young adult). (E,F) Analysis of naïve CD4 T cell similarly at (E) day 1 and (F) day 14 post-culturing. CD45RA expression map is on the right with arrow indicating low to high expression. (G,H) Frequencies of CD45RA high CD27 high (G) CD4 and (H) CD8 naïve T cells from young ( n = 6 CD4, n = 5 CD8) and older ( n = 6 CD4, n = 5 CD8) donors. (I) Clustering of T cells from young or old donors in organoids. Representative of more than three independent experiments. * p < 0.05 p -values were determined by (B,C) Friedman test with Dunn’s multiple comparison test, and (G,H) Mixed-effects analysis with Holm-Sidak multiple comparison.

Article Snippet: CD3 + T cells were isolated directly from whole blood using Total T cells RosettaSep kit (Stem Cell Technologies Cat #15061).

Techniques: Cell Culture, Expressing, Comparison

Journal: Frontiers in Aging

Article Title: The influence of three-dimensional structure on naïve T cell homeostasis and aging

doi: 10.3389/fragi.2022.1045648

Figure Lengend Snippet: Reductions in CD45RA is caused by alternative splicing of CD45 not total protein loss. (A) Overview of CD45 isoforms. Bold indicated which exons specific antibodies bind to. (B) RNA expression of CD45 (PTPRC) in bulk naïve (CD45RA + CCR7+) CD4 and CD8 T cells from young ( n = 10) and older ( n = 9) adults. (C) Representative flow histogram of CD45 protein expression on naïve CD4 T cells from a young (blue) and older (orange) adult. Gray is negative control. (D) Naïve CD4 and (E) naïve CD8 T cell expression of total protein levels for CD45, CD45RA, −RB, −RC, and −RO in young ( n = 7) and older ( n = 8) adults detected by antibody. (F) Naïve CD4 and (G) naïve CD8 T cell expression of CD45 isoforms via RT-PCR analysis. Cell input was normalized for total CD45 expression. CD45RO isoform expression was too low in naïve T cells to be accurately quantified. (H) Expression of potential CD45 splice factors in naïve CD4 and CD8 T cells from young ( n = 10) and older ( n = 9) adults. p -values determined by (B,D,E,H) Mann-Whitney test and (F,G) unpaired t -test.

Article Snippet: CD3 + T cells were isolated directly from whole blood using Total T cells RosettaSep kit (Stem Cell Technologies Cat #15061).

Techniques: Alternative Splicing, RNA Expression, Expressing, Negative Control, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY