A single amino acid change, A91V, leads to conformational changes that can impair processing to the active form of perforin by Christina Trambas, Federico Gallo, Daniela Pende, Stefania Marcenaro, Lorenzo Moretta, Carmela De Fusco, Alessandra Santoro, Luigi Notarangelo, Maurizio Arico, and Gillian M. Griffiths Blood Volume 106(3):932-937 August 1, 2005 ©2005 by American Society of Hematology

Defective lytic activity in CTLs and NK cells. Defective lytic activity in CTLs and NK cells. (A) A CD8+ CTL clone derived from a healthy donor (▴) is compared with 2 different clones, derived from patient 612 expressing perforin A91V and W374X (□ and ⋄) and 1 clone derived from patient 112 lacking perforin expression (▵), for their cytolytic activity against P815 in the presence of anti-CD3 monoclonal antibody (mAb). (B-C) Polyclonal activated NK-cell populations derived from 2 healthy donors (▪ and ▴), from patient 6 (⋄), and patient 1 (▵) were tested in a cytolytic assay against the 51Cr-labeled FO-1 (B) and 721.221 (C) target cell lines. The percentages of target cell lysis at different effector-target (E/T) ratios are shown. Christina Trambas et al. Blood 2005;106:932-937 ©2005 by American Society of Hematology

Cytofluorimetric analysis of perforin using δG9 mAb. Cytofluorimetric analysis of perforin using δG9 mAb. FACS profiles of staining of control and patient CTLs with δG9 antiperforin. Nonspecific staining with an isotype-matched control antibody is shown by the open histograms. FL2-H indicates fluorescence channel 2. Christina Trambas et al. Blood 2005;106:932-937 ©2005 by American Society of Hematology

Patient CTLs do not stain with δG9 antiperforin antibody. Patient CTLs do not stain with δG9 antiperforin antibody. Confocal microscopy of CD8+ CTL clones from control and patients 6 and 112 stained with δG9 antiperforin and cathepsin D (includes brightfield and all images merged [right]). Scale bar: 10 micrometers. See “Immunofluorescence microscopy” for details. Images were visualized with a Nikon TE300 inverted microscope equipped with a Plan Apo 100 ×/1.4 oil-immersion objective lens (Nikon UK, Kingston Upon Thames, United Kingdom), and with a Radiance 2000 confocal laser scanning microscope (Carl Zeiss, Welwyn Garden City, United Kingdom). Christina Trambas et al. Blood 2005;106:932-937 ©2005 by American Society of Hematology

Processing of perforin to the mature form is impaired in patient CTLs Processing of perforin to the mature form is impaired in patient CTLs. Western blots of CD8+ CTL lysates from control (lanes 1,3) and patient (lanes 2,4), showing immature, intermediate, and mature forms of perforin in control CTLs, and only the immature an... Processing of perforin to the mature form is impaired in patient CTLs. Western blots of CD8+ CTL lysates from control (lanes 1,3) and patient (lanes 2,4), showing immature, intermediate, and mature forms of perforin in control CTLs, and only the immature and intermediate forms in patient 612. Longer (top) and shorter (bottom) exposures of lysates from NK cells from control (lanes 5-6) or patient 6 (lanes 7-8) were incubated in the presence (lanes 5,7) or absence (lanes 6,8) of Endo H and probed with 2d4-antiperforin. All samples were run on 7.5% acrylamide gels under nonreducing conditions. Christina Trambas et al. Blood 2005;106:932-937 ©2005 by American Society of Hematology