Journal: Bone marrow transplantation

Article Title: Donor CD4 T cells convert mixed to full donor T-cell chimerism and replenish the CD52-positive T-cell pool after alemtuzumab-based T-cell-depleted allo-transplantation.

doi: 10.1038/bmt.2009.212

Figure Lengend Snippet: Figure 2 CD52 expression of lymphocytes before and after CD8depl donor lymphocyte infusion (DLI). Lymphocytes of a patient receiving three doses of CD8depl DLI during the first year after transplantation were analyzed for the expression levels of CD52. The plots were gated on lymphocytes with regard to forward and sideward scatters, and further gated as outlined in the figure. On day þ 110 before the first dose of DLI, the majority of CD8 and CD4 T cells was CD52-negative. DLI therapy was followed by increased proportions of CD52-positive T cells in both subsets at day þ 350. The positive CD52 status of B cells (CD3/CD19 þ) and NK cells (CD3/CD56 þ) remained unchanged over time.

Article Snippet: As recently described, this procedure leads to a 2.5–6-log reduction of CD8-positive T cells in the DLI preparation.13 Briefly, the leukapheresis product was diluted 1:3 in CliniMACS PBS/EDTA buffer and was centrifuged at 300 g. The cell pellet was resuspended in 95ml buffer and 7.5ml CliniMACS CD8 reagent (Miltenyi Biotec) was added.

Techniques: Expressing, Transplantation Assay

Journal: Nature

Article Title: Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I

doi: 10.1038/s41586-020-2229-5

Figure Lengend Snippet: a,b, Autophagy flux ( a ) and cell surface MHC-I levels in PDAC cells measured by flow cytometry. Mouse PDAC cells expressing the GFP-LC3-RFP reporter and doxycycline (Dox)-inducible mTurquoise2-Atg4B C74A were grown as organoids for 8 days and treated with Dox (1 μg/ml) for the indicated hours. a, Autophagy flux represented by GFP/RFP ratio. Note that increased GFP/RFP ratio indicates reduced autophagy flux. b, Cell surface MHC-I (H-2K ) levels. Representative flow cytometry plots were shown. Data are mean ± s.d. n = 3 biological replicates. Data are representative of at least four independent experiments. c, Fold changes of respective molecules on the cell surface quantified by flow cytometry. HY15549 cells expressing Dox-inducible mTurquoise2-tagged Atg4B C74A were grown as organoids for 8 days and treated ± Dox (1 μg/ml) for 72 hrs. Positive surface expression of each molecule was confirmed using respective isotype controls. Molecules found in immunological synapses are underlined. TFRC, transferrin receptor. Data are mean ± s.d. n = 4 biological replicates. Representative data from two independent experiments are shown. d-f, (Related to , ) Mouse PDAC cells expressing OVA and carrying doxycycline (Dox)-inducible mTurquoise2-Atg4B C74A were grown as organoids and treated ± Dox (1 μg/mL) for 96 hrs. d, Autophagy inhibition was confirmed by immunoblot. mTurquoise2-Atg4B C74A or endogenous Atg4B were detected by anti-ATG4B antibody. e,f, Flow cytometry plots for H-2K ( e ) and H-2K -SIINFEKL ( f ). Representative plots from , are shown. Grey, isotype control. g , Representative flow cytometry plots of the OT-I cells co-cultured with mouse PDAC cells from . h, (Related to ) Quantitative reverse transcription PCR (qRT-PCR) analysis of OT-I cells that were co-cultured with PDAC cells for 48 hrs. Data are mean ± s.d. n = 3 biological replicates. For g, h, Dox(+) or Dox(−) indicates that PDAC cells were grown ± Dox (1 μg/mL) before co-culture. Dox was not added in co-culture. A representative of at least three independent experiments is shown in d-g . P values determined by unpaired two-tailed t -tests ( a,b,h ). **** P < 0.0001. For gel source data of d , see .

Article Snippet: Organoids were dissociated into single cells, which were then incubated with either anti-H-2K b -SIINFEKL antibody (clone 25-D1.16, BioXCell, BE0207) or isotype control (clone MOPC-21, BioXCell, BE0083) at 100 μg/mL for 30 min at 4 °C.

Techniques: Flow Cytometry, Expressing, Inhibition, Western Blot, Control, Cell Culture, Reverse Transcription, Quantitative RT-PCR, Co-Culture Assay, Two Tailed Test

Journal: Nature

Article Title: Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I

doi: 10.1038/s41586-020-2229-5

Figure Lengend Snippet: a,b, Surface H-2K b ( a ) and H-2K b -SIINFEKL ( b ) measured by flow cytometry. Mouse PDAC cells expressing OVA and Dox-inducible mTurquoise2-Atg4B C74A were grown as organoids and treated ± Dox (1 μg/mL) for 96 hrs ( n = 4 per group). c,d, Co-culture of OT-I cells with HY19636 cells shown in a,b. After 48 hrs, OT-I proliferation was measured by CFSE dilution ( c ) ( n = 4 per group) and PDAC cell viability was measured by Cell-Titer Glo ( d ) ( n = 6 per group). e-r, HY15549 cells carrying Dox-inducible mStrawberry (mSt) or mSt-Atg4B C74A (4B) were orthotopically ( e-h, m-r ) or intrasplenically ( i-l ) injected into syngeneic mice (C57BL/6). MHC-I and PD-L1 expression on PDAC cells ( f,g,j,k,p ) and tumour-infiltrating CD8 + T cells ( h,l,n,q ) quantified by flow cytometry. e-h, Orthotopic tumours harvested on day 20 (mSt, n = 8; 4B, n = 7). i-l, Livers harvested on day 15 ( n = 4 per group). Weight of tumours ( e ) and livers ( i ). Flow cytometry analysis ( f-h , j-l ). m, Weight of tumours following treatment with isotype control IgG or neutralizing monoclonal antibody against CD8 ( n = 7 per group). n,o, Tumours in wild type mice (WT) or Batf3 −/− mice (KO) ( n = 8, 4, 8, and 5; left to right). Quantification of CD8 + T cells ( n ). Tumour weight ( o ). p-r, Tumours expressing control shRNA (Scr) or shRNA against B2m ( n = 8 per group) harvested on day 20. Flow cytometry analysis ( p,q ). Tumour weight ( r ). Data are mean ± s.d. ( a-d ) or s.e.m. ( e-r ). n indicates biological replicates ( a-d ) or individual mice ( e-r ). For a-m, p-r , experiments were performed at least twice and representative data of one experiment are shown. P values determined by unpaired two-tailed t -tests.

Article Snippet: Organoids were dissociated into single cells, which were then incubated with either anti-H-2K b -SIINFEKL antibody (clone 25-D1.16, BioXCell, BE0207) or isotype control (clone MOPC-21, BioXCell, BE0083) at 100 μg/mL for 30 min at 4 °C.

Techniques: Flow Cytometry, Expressing, Co-Culture Assay, Injection, Control, shRNA, Two Tailed Test