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Blood, Vol. 105, Issue 4, 1640-1647, February 15, 2005
Cutaneous T-cell lymphoma: malignant proliferation of T-regulatory cells
Blood Berger et al.
105: 1640
Supplemental materials for: Berger et al, Vol 105, Issue 4, 1640-1647
Files in this Data Supplement:
- Figure S1. Cultured CTCL cells express CTLA-4 when grown in the presence of high numbers of apoptotic cells (PDF, 63 KB) -
(A) Patient 1, isotype controls. (B) On primary isolation, CTCL cells express membrane CD3/cytoplasmic CTLA-4. (C) CTCL cells and DCs purified and recultured overnight; CD3+ CTCL cells lose expression of cytoplasmic CTLA-4. (D) DCs were pulsed with apoptotic tumor and recultured with purified CTCL cells; CD3/CTLA-4 expression was re-induced. (E) High levels of apoptotic cells were present in the initial cultures (D0), as shown by annexin-FITC staining. (F) Repurified and recultured CTCL cells and DCs contain low numbers of apoptotic cells as shown by annexin-FITC binding. (G) Patient 2, isotype controls. (H) On primary isolation, CTCL cells express membrane CD3/cytoplasmic CTLA-4. (I) Repurification and overnight reculture of the CTCL cells and DCs led to a reduction in CTLA-4 expression in the cytoplasm of CD3+ CTCL cells. (J) CTCL cells were recultured overnight with DCs that had been fed apoptotic tumor; a re-emergence of the CD3/CTLA-4+ phenotype was induced. (K) High levels of apoptotic cells were found in the primary cultures as shown by annexin-FITC staining. (L) After overnight reculture of purified CTCL cells and DCs, the number of apoptotic cells in the culture was reduced as determined by annexin-FITC binding.
- Figure S2A. CD3 dose response curve (PDF, 99 KB) -
(i) CTCL cells were purified and incubated for 1 hour with 33 µg/mL CD3, and were cultured overnight. Apoptosis was determined by annexin-FITC binding. (ii) CD3 dose of 3 µg/mL. (iii) CD3 dose of 0.3 µg/mL. (iv) A 33-µg/mL dose of CD4 isotype-matched control antibody was incubated for 1 hour with purified CTCL cells. (v) CD4 dose of 3 µg/mL. (vi) CD4 dose of 0.3 µg/mL. (vii) Under identical conditions, 33 µg/mL CD3 antibody was added to purified CTCL cells (20 minutes), and apoptosis measured by annexin-FITC. (viii) CTCL cells treated with 33 µg/mL CD4 control antibody. (ix) Dose response curve of viable cells/mL (as determined by trypan blue staining and hemacytometer count) recovered after incubation for 1 hour of purified CTCL cells with 33 µg/mL, 3 µg/mL, and 0.3 µg/mL CD3 or CD4 antibodies.
- Figure S2B. Dose response of CTLA-4 expression in CTCL cells co-incubated with CD3-treated apoptotic CTCL cells alone or pulsed onto DCs (PDF, 53 KB) -
CTCL cells recovered after treatment with doses of CD3 tested in Figure S2A were incubated overnight alone or pulsed onto autologous DCs; induction of cytoplasmic CTLA-4 expression was measured in freshly isolated V
 8+ tumor cells by flow cytometry. (i) CTCL cells cultured alone without CD3. (ii) CTCL cells treated with 33 µg/mL CD3 and cultured alone. (iii) 3 µg/mL CD3. (iv) 0.3 µg/mL CD3. (v) Purified CTCL cells were not treated and were re-added to autologous DCs overnight. (vi) CTCL cells treated with 33 µg/mL CD3 were pulsed onto autologous DCs overnight and then loaded DCs were used to stimulate responding V8+ CTCL cells. (vii) As in panel vi, a 3 µg/mL dose of CD3. (viii) As in panels vi and vii, 0.3 µg/mL CD3.
- Figure S3A. Comparison of mediators of apoptosis for induction of a Treg phenotype in CTCL (PDF, 49 KB) -
CTCL cells were purified from a 1-month culture and treated with CD3 (3.5 µg/mL),
 -irradiation (3000 rad, cesium source), and anti-Fas antibody (CD95-purified 250 ng/mL, Coulter). CTCL cells purified from a 1-month culture of a second patient were treated with staurosporine (chemically mediated apoptosis, 2 µM, Sigma). The cells were washed and pulsed onto autologous DCs overnight, and freshly purified CTCL cells added and stained for membrane CD3/cytoplasmic CTLA-4. (i) DCs fed CD3-treated CTCL cells. (ii) DC fed -irradiated CTCL cells. (iii) DCs fed CD95, anti-Fas antibody–treated CTCL cells. (iv) Baseline, non-loaded DCs. (v) DCs from a second patient fed CD3-treated CTCL cells. (vi) DCs fed staurosporine-treated CTCL cells. (vii) Baseline, non-loaded DCs.
- Figure S3B. CTLA-4 expression in CTCL cells exposed to DCs pulsed with CTCL cells rendered apoptotic by psoralen and UVA treatment (PDF, 42 KB) -
CTCL cells were purified from a 2-month culture of a third patient and treated with 8-methoxypsoralen (8-MOP, 100 ng/mL) and ultraviolet A light (UVA, 3 joules/cm2), a protocol known to induce gradual apoptosis in CTCL cells.27 Treated CTCL cells were pulsed onto DCs and used to stimulate fresh CTCL cells that were stained as described in Figure S3A. (i) Baseline, non-loaded DCs. (ii) DCs fed CD3-treated CTCL cells. (iii) DCs were fed -irradiated CTCL cells. (iv) DCs fed 8-MOP/UVA–treated CTCL cells.
- Figure S4A. Effect of neutralization of IL10 or TGF-β (PDF, 14 KB) -
Normal lymphocytes were stimulated with tetanus toxoid and co-cultured at a 1:1 ratio with CTCL cells that were induced to assume a Treg profile (incubated with tumor-loaded DCs) or control CTCL cells (cocultured with autologous DCs pulsed with CD4-treated CTCL cells) in the presence of 1-10 µg/mL neutralizing antibody to IL10 (Figure S4A) or TGF- (Figure S4B) or isotype-matched control antibodies for 5 days. The dose of neutralizing antibody used was confirmed to inhibit 95% ± 2% of the cytokine produced by the Treg CTCL cells by ELISA. The production of IFN- was measured by ELISA assay.
- Figure S4B. Effect of neutralization of IL10 or TGF-β (PDF, 15 KB) -
Normal lymphocytes were stimulated with tetanus toxoid and co-cultured at a 1:1 ratio with CTCL cells that were induced to assume a Treg profile (incubated with tumor-loaded DCs) or control CTCL cells (cocultured with autologous DCs pulsed with CD4-treated CTCL cells) in the presence of 1-10 µg/mL neutralizing antibody to IL10 (Figure S4A) or TGF- (Figure S4B) or isotype-matched control antibodies for 5 days. The dose of neutralizing antibody used was confirmed to inhibit 95% ± 2% of the cytokine produced by the Treg CTCL cells by ELISA. The production of IFN- was measured by ELISA assay.
- Figure S4C. Separation of Treg CTCL cells by a transwell membrane does not impede suppression of the normal T-cell antigen response (PDF, 15 KB) -
Treg CTCL cells or control CTCL cells (prepared as in Figure S4A), were added to the top of a transwell and compared for inhibition of the normal T-cell IFN- response to tetanus toxoid in the bottom wells. In addition, no cells (0) were added to the top of a transwell. After 5 days, the production of IFN- was measured by ELISA in supernatant obtained from the bottom wells.
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