Journal: bioRxiv

Article Title: Disruptors of sestrin-MAPK interactions rejuvenate T cells and expand TCR specificity

doi: 10.1101/2024.05.17.594698

Figure Lengend Snippet: (A) Senescence-associated β-galactosidase expression in DOS-treated (DOS-juvenated) or untreated T sen . Cells were purified and cultured for one week in the presence of anti-CD3 (0.5 μg/mL) and rh-IL-2 (5 ng/mL), then stained to detect β-galactosidase activity. Representative image on the inverted phase-contrast microscope (left) and relative quantification (right, n = 8 donors). (B) Population doublings of human T sen (transduced with irrelevant scramble) and sestrin null CD4 + T sen (transduced by triple lentiviral depletion of sestrins) and cultured as in (A) (left). Donor-matched CD27 + CD28 + CD4 + T cells (herafter, T erl ) were cultured in parallel but activated with anti-CD3 and anti-CD28 ( n = 5 donors). Cells were cultured over two weeks with restimulation every 7 days. DOS-driven population doublings (right) were calculated as delta between DOS treated and DOS untreated T cells, with or without depletion of sestrins. (C) DOS-juvenation of human CD4 + T cells. Terminally differentiated effector memory CD45RA - CD28 - CD27 - CD4 + T cells (hereafter, T EM ) were purified and cultured over 20 days, as in (A). At day 1 (18 hours), 7, and 21 cell phenotypes were assessed by flow cytometry. Quantifications of rejuvenated stem like (CD28 + CD45RA + CCR7 + CD95 + CD62L + TCF1 + ) among human CD4 + T cells are shown (left; n = 5 donors). Decay of T EM and CD28 - CD27 - CD45RA + CD4 + T cells (hereafter, T EMRA ) undergoing rejuvenation is shown (right). (D) Adoptive transfer of DOS-juvenated T cells, experimental design. Donor T cells were derived from twenty-month-old mice 15 days after Fluad vaccination with or without DOS treatment (0.1 mg/Kg throughout), labeled with Cell Trace Violet (CTV) dye or congenic CD45.1 tracking, then transferred into young naïve CD45.2 recipients (3 months). In parallel, young mice (3 months) were used as young donor control. Recipient animals were rested for 28 days, then analysed for donor T cell persistence and maintenance of stem phenotype after transfer. (E) Maintenance of donor DOS-juvenated CD45.1 CD4 + T cells, their aged-matched controls, and that of young donor T cells, 28 days after transfer (day 43) in recipient mouse lymph nodes. Representative flow cytometry plots and poled data ( n = 5 mice per group) are shown. (F) Assessment of mouse T cell memory programs in stem like transferred T cells (among CD45.1 CD44 - CD62L + CD95 + CD4 + T cells) and terminally differentiated cells (TE, among CD45.1 CD44 - CD62L - CD4 + T cells) following adoptive transfer as in (D) ( n = 5 mice). (G) IL7R gene expression and (H) CD95 and Sca-1 mean fluorescent intensity (MFI; throughout) in stem cells derived from CD4 + CD45.1 + transferred stem T cells among lymph nodes of recipient CD45.2 mice, 28 days after transfer ( n = 5 mice). (I) Representative plots of CD45.1 transferred cells in quiescent state before and after transfer assessed by cycle related intra-nuclear Ki67 staining. Representative of n = 5 mice per group. (J) G1 (Ki67 + ) to G0 (Ki67 - ) transition in stem like CD45.1 CD4 + T cells before and after adoptive transfer as indicated ( n = 5 mice per group). (K) Assessment of T cell longevity following DOS-juvenation. Cells from lymph nodes were stained using the Annexin-PI Apoptosis detection Kit 28 days after transfer. Representative FACS plot (left) and quantification of dead CD45.1 + CD4 + transferred T cells (right). (L) DOS-juvenated T cell maintenance, in vivo model. In (A and B, right) two tailed paired T test was used. In (B, left-C, E-H and J-K) one-way Anova with Bonferroni post-correction for multiple comparisons was used, *p<0,05, **P<0,01; ***P<0,001; ****P<0,0001. Error bars indicate SEM.

Article Snippet: Primary human CD4 + T cells were isolated using CD4 MicroBeads (130-045-101; Miltenyi); primary human senescent CD4 + CD27 - CD28 - T cells (thereafter T sen ) were isolated using CD4 + T Cell Isolation Kit (130-096-533; Miltenyi) followed by depletion of CD27 + and CD28 + T cells using CD27 MicroBeads (130-051-601; Miltenyi) and CD28 MicroBead Kit (130-093-247; Miltenyi).

Techniques: Expressing, Purification, Cell Culture, Staining, Activity Assay, Microscopy, Quantitative Proteomics, Transduction, Flow Cytometry, Adoptive Transfer Assay, Derivative Assay, Labeling, Control, Gene Expression, In Vivo, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Helper B cells promote cytotoxic T cell survival and proliferation independently of antigen presentation through CD27/CD70 interactions.

doi: 10.4049/jimmunol.180.3.1362

Figure Lengend Snippet: FIGURE 5. Function of TILs/B cell couplets. A, Representative expression of CD27, CD28, CD70, and CD80 in TILs, and in fresh CD19 cells. B, F.I. of TILs after a 4-day expansion alone or in the presence of fresh heterologous B cells. TILs, or CD19 cells, were blocked at the beginning of the culture with anti-CD70 (dashed bar), and anti-CD80 (diamond bar) mAbs, respectively, or with the proper Ig control (light-gray bar is the control for CD70 block, dark-gray bar for CD80 block) (n 4). C, F.I. of CTL after 9 days culture alone or after cross-linking of human recombinant CD27, anti-CD70 mAb, or a combination of the two. The anti-CD19 mAb used as an isotype matched negative control, whereas the pan T cell stimulator OKT3 mAb was used as positive control. PBMC were expanded in presence of rIL-2 with (CD8Flu, black bars) or without (CD8 alone, white bars) Ag-specific stimulation with the Flu peptide. Student’s t test p values refer to significant differences between OKT-3 stimulated cultures and CD8 alone cultures (* 0.05, ** 0.01); all other conditions did not yield significant differences compared with CD8 alone cultures.

Article Snippet: The specificity of the requirements for CD27 stimulation of T cell proliferation by B cells through CD27 was tested by cross-linking human recombinant CD27 (R&D Systems) 10 ng/ml, anti-human CD70 mAb (Ancell), or antiCD3/OKT3 mAb (Ortho Biotech) to culture plates.

Techniques: Expressing, Control, Blocking Assay, Recombinant, Negative Control, Positive Control