1. SAP controls the cytolytic activity of CD8+ T cells against EBV-infected cells
by Loïc Dupré, Grazia Andolfi, Stuart G. Tangye, Rita Clementi, Franco Locatelli, Maurizio Aricò, Alessandro Aiuti, and Maria-Grazia Roncarolo
Blood
Volume 105(11):
June 1, 2005
©2005 by American Society of Hematology

2. Regulation of SAP expression in CD8+ T cells.
Regulation of SAP expression in CD8+ T cells. Western blot analysis of SAP expression in CD8+ T cells from healthy donors (ND1, ND2, and ND3), and patients with HLH (SAP1) and XLP (SAP2 and SAP3) at a resting state and at 24 or 72 hours after activation with 10 μg/mL immobilized anti-CD3 mAbs.
Loïc Dupré et al. Blood 2005;105:
©2005 by American Society of Hematology

3. Lytic activity of SAP-deficient CD8+ T cells.
Lytic activity of SAP-deficient CD8+ T cells. (A) Lytic activity of EBV-specific CD8+ T-cell lines from a SAP-deficient HLH patient (SAP1) and from a healthy control donor. The targets are autologous EBV-transformed B-cell lines used at the indicated effector-target ratio. (B) Mean (± SD) of the lytic activity of allospecific CD8+ T-cell lines from 3 SAP-deficient patients (SAP1, SAP2, and SAP3) and from 3 healthy donors (ND1, ND2, and ND3) against the EBV-transformed B-cell line JY. (A-B) ▪ indicates ND; □, SAP deficient. (C) Mean (± SD) of the lytic activity of allospecific CD8+ T-cell lines from 3 healthy donors (ND1, ND2, and ND3) against JY cells in the presence of either blocking anti-2B4 mAbs (▨) or isotype control Abs (+IgG1; ▪). (D) Mean (± SD) of the lysis of allospecific CD8+ T-cell lines from 3 SAP-deficient patients (SAP1, SAP2, and SAP3) and from 3 healthy donors (ND1, ND2, and ND3) against the CD48- cell line K562. ▪ indicates ND; □, SAP deficient. One representative experiment of 5 performed is shown. Each mean (± SD) was obtained from 6 data points (3 cell lines studied in duplicate), and statistical analysis was performed using an unpaired t test. Significant differences of lysis between SAP and control T-cell lines are indicated by asterisks (*P < .05; **P < .01).
Loïc Dupré et al. Blood 2005;105:
©2005 by American Society of Hematology

4. Distribution of perforin and GM1 in SAP-deficient CD8+ T cells.
Distribution of perforin and GM1 in SAP-deficient CD8+ T cells. (A) GM1 and perforin distribution in normal and SAP-deficient CD8+ T cells (T) forming conjugates with EBV-positive B-cell line JY (B). The effects of blocking anti-2B4 mAbs on normal CD8+ T cells are also shown. One representative conjugate is shown in parallel as bright field (i-iii), GM1 staining (iv-vi), and perforin staining (vii-ix). (B) Quantitative analysis of GM1 and perforin coclustering at the area of contact with B-cell targets in T cells from 3 SAP-deficient patients and T cells from 3 healthy donors, either untreated or treated with blocking anti-2B4 mAbs. Cells were considered positive for coclustered GM1 and perforin if the staining was centered at the site of contact with the B cell and occupied less than one third of the cell surface. Data are represented as mean percentages (± SDs) of 3 experiments counting T cells forming clusters with a single JY B-cell target (for each cell line a total of cells was counted). Statistical analysis was performed using an unpaired t test, and P values corresponding to the comparison of 1 group with the other are indicated.
Loïc Dupré et al. Blood 2005;105:
©2005 by American Society of Hematology

5. Distribution of 2B4 in normal and SAP-deficient CD8+ T cells.
Distribution of 2B4 in normal and SAP-deficient CD8+ T cells. (A) 2B4 distribution in normal CD8+ CTLs (T) either alone or at the contact with EBV+/CD48+ JY B cells or EBV-/CD48-K562 cells. Representative cells are shown as bright field (i-iv) and 2B4 staining (v-viii). The white arrows indicate 2B4 clustering at the cell contact area. (B) 2B4 distribution in SAP-deficient CD8+ T cells either alone or at the contact with EBV+/CD48+ JY B cells. Representative cells are shown as bright field (i-ii) and 2B4 staining (iii-iv). (C) Quantitative analysis of the percentage of CTLs (normal or SAP deficient) with clustered 2B4 at the cell contact area. ▪ indicates normal CTL + JY; ▦, normal CTL + K562; and □, SAP-deficient CTL + JY cells. Mean percentages (± SDs) of 3 experiments counting T cells forming clusters with single target cells (100 conjugates were counted). Statistical analysis was performed using an unpaired t test, and P values corresponding to the comparison of 1 group with the other are indicated.
Loïc Dupré et al. Blood 2005;105:
©2005 by American Society of Hematology

6. Phenotype of SAP-deficient CTLs
Phenotype of SAP-deficient CTLs. Phenotype of CD8+ T cells from SAP-deficient patients (SAP; □) and healthy donors (ND; ▪) in a resting state or after activation with 1 μg/mL anti-CD3 mAbs or stimulation with 100 ng/mL IL-15 (mean ± SD of 3 SAP-deficient pa...
Phenotype of SAP-deficient CTLs. Phenotype of CD8+ T cells from SAP-deficient patients (SAP; □) and healthy donors (ND; ▪) in a resting state or after activation with 1 μg/mL anti-CD3 mAbs or stimulation with 100 ng/mL IL-15 (mean ± SD of 3 SAP-deficient patients and 3 healthy donors is shown). (A) Mean fluorescence intensity of the intracytoplasmic staining of the lytic molecules perforin and granzyme-B. (B) Percentage of CD8+ T cells staining positive for the CD2-superfamily receptors SLAM, 2B4, and CD84. (C) Percentage of CD8+ T cells staining positive for the T-cell activation markers CD25, CD69, and CD56. One representative experiment of 3 performed is shown.
Loïc Dupré et al. Blood 2005;105:
©2005 by American Society of Hematology

7. Proliferation and cytokine production of SAP-deficient CD8+ T cells.
Proliferation and cytokine production of SAP-deficient CD8+ T cells. (A) Proliferation of CD8+ T-cell lines from healthy donors (mean ± SD of ND1, ND2, and ND3; ▪) and SAP-deficient patients (mean ± SD of SAP1, SAP2, and SAP3; □) after stimulation with the indicated doses of immobilized anti-CD3 mAbs. Proliferation is expressed as cpm values corresponding to 3H-thymidine uptake after a 72-hour stimulation. (B) Intracytoplasmic staining for cytokine production in T cells of healthy donors and SAP-deficient patients 6 hours after stimulation with immobilized anti-CD3 mAbs plus anti-CD28 mAbs or TPA/ionomycin. Numbers in dot plot quadrants refer to percentages of T cells positive for the indicated cytokines. One representative experiment of 3 performed is shown.
Loïc Dupré et al. Blood 2005;105:
©2005 by American Society of Hematology