Figure A. RPE conjugated Mouse anti Human CD19 and Pacific Blue conjugated Mouse IgG2a isotype control . Figure B. RPE conjugated Mouse anti Human CD19 and Pacific Blue conjugated Mouse anti Human HLA ABC . All experiments performed on red cell lysed human blood gated on lymphocytes in the presence of Human Seroblock . Data acquired on the ZE5 cell analyzer.

Figure A. Alexa Fluor 488 conjugated Mouse anti Human CD19 and RPE-Alexa Fluor 647 conjugated Mouse IgG2a isotype control . Figure B. Alexa Fluor 488 conjugated Mouse anti Human CD19 and RPE-Alexa Fluor 647 conjugated Mouse anti Human HLA ABC . All experiments performed on red cell lysed human blood gated on lymphocytes in the presence of 10% human serum. Data acquired on the ZE5 Cell Analyzer.

Mouse anti Human HLA ABC antibody, clone W6/32 used to immunoprecipitate all MHC-1 complexes from phagosomal complexes.

Image caption:

HLA-E:peptide complexes are present in phagosomal fractions. (A,B) DC were pulsed with H37Rv-eGFP or CFP102–11 peptide and the homogenate was separated using 27% percoll as described. Each fraction was freeze-thawed and tested for its ability to stimulate D160 1-23 (A) or D160 1-1B (A&B) CD8+ T cell clones in the absence of additional APC. IFN-? production was measured using ELISPOT. The mean±SEM of duplicate wells is presented. Data are representative of four experiments at various timepoints in A, and four experiments after a one hour peptide pulse in B. (C) Plasma membrane/ER fractions from Mtb–infected DC (HLA-B15+/HLA-B44?) were incubated with HLA-I matched or mismatched CD8+ T cell clones and IFN-? production was measured using ELISPOT. The mean±SEM of duplicate wells is presented and data are representative of three experiments done similarly. (D,E) Individual fractions were analyzed by flow cytometry to assess HLA-II-PE (plasma membrane) and H37Rv-eGFP fluorescence. Selected fractions are shown (D) including the peak plasma membrane fraction (#8) and phagosomal fractions (#24–27). Prior to flow cytometry, fractions were mixed with a reference latex bead population at a known concentration. Equal numbers of latex bead events were collected for all fractions and used to quantify the number of plasma membrane and phagosome particles (E) as described in Materials and Methods. (F) HLA-I was immunoprecipitated from plasma membrane or phagosome fractions using W6/32. After dilution of the plasma membrane sample to give similar levels of HC10 staining, the presence of HLA-I alleles was assessed using antibodies to pan-HLA-I (HC10), HLA-A2 (HCA2), and HLA-E (MEM-E/02). The numbers below blots indicate the relative intensity of the bands as described in Materials and Methods.
From: Grotzke JE, Harriff MJ, Siler AC, Nolt D, Delepine J, et al. (2009)

The Mycobacterium tuberculosis Phagosome Is a HLA-I Processing Competent Organelle.
PLoS Pathog 5(4): e1000374.

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