1. Semaphorin-3A is expressed by tumor cells and alters T-cell signal transduction and function
by Alfonso Catalano, Paola Caprari, Simona Moretti, Monica Faronato, Luca Tamagnone, and Antonio Procopio
Blood
Volume 107(8):
April 15, 2006
©2006 by American Society of Hematology

2. Expression of Sema-3A and NP-1 in tumor cells and renal cell carcinomas.
Expression of Sema-3A and NP-1 in tumor cells and renal cell carcinomas. (A) Western blot detection of secreted Sema-3A in concentrated (5 ×) TSNs (150 μg) from the indicated cells using anti-Sema-3A specific antibody. (B) Total lysates from cell lines indicated in panel A were immunoprecipitated with anti-Sema-3A antibody (IP Ab: Sema-3A). Immune complexes were immunoblotted (Blot Ab) with anti-Sema-3A antibody to detect Sema-3A precursor (top). The same lysates (50 μg) were also processed to detect NP-1 by immunoblotting. Expression of actin was used as internal control. Data are representative of 3 experiments. Sizes are shown in kilodaltons. (C) Total RNA (50 ng/μL) was isolated from the indicated cells and subjected to real-time PCR, as described in “Materials and methods,” to detect SEMA3A and NP1 transcripts. The mRNA levels of each gene were normalized by the mRNA levels of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Reported values are an average of 3 separate samples of each type analyzed in triplicate; bars, ± SE. (D) Immunoperoxidase staining of a paraffin-embedded tissue section of a representative human renal cell carcinoma specimen (Clear cell type, grade 3, stage I) using an anti-Sema-3A and anti-NP-1 antibody or the same dilution of a control IgG (magnification, × 40). Scarce amounts of Sema-3A and NP-1 were also detected in normal renal tissue (data not shown). Antibody localization was effected using a peroxidase reaction with 3,3-diaminobenzidine tetrahydrochloride as chromogen.
Alfonso Catalano et al. Blood 2006;107:
©2006 by American Society of Hematology

3. Sema-3A binds T cells and inhibits T-cell proliferation.
Sema-3A binds T cells and inhibits T-cell proliferation. (A) Resting T cells were incubated with Sema3A/Fc or hIgG and then analyzed by flow cytometry as described in “Materials and methods.” Mean fluorescence intensity (MFI) derived from 3 independent experiments is shown (left). Alternatively, cells were first incubated with the blocking anti-NP-1 antibody or a control Ab. Then, T cells were treated with Sema3A/Fc or hIgG as described (right). (B) COS-7 cells were stably transfected with a vector that allowed a high (COS-7/C1) and intermediate (COS-7/C2 and COS-7/C3) amount of Sema-3A expression, as shown by Western blot in concentrated serum-free supernatants (CM) (insert). CM (48 hours of conditioning, 1:2 of dilution) derived from COS-7 cells and Sema-3A-expressing COS-7 cells were incubated with T cells for 18 hours in the absence (Control) or in the presence of soluble anti-CD3 (300 ng/mL), either alone or with anti-CD28 (200 ng/mL) or PMA (5 ng/mL). T-cell proliferation was then measured by [3H]thymidine incorporation. *P ≤ .001 versus COS-7/-, ANOVA, n = 3. (C) T cells were treated with CM derived form COS-7/- and COS-7/C1 clones, stained with PI and analyzed by flow cytometry. (D) T cells were treated as described in panel A in the presence of CM derived form COS-7/- and COS-7/C1 clones for the indicated times, and the p21Kip1 protein expression was detected by Western blotting with anti-p27Kip1 antibody. Expression of actin was used as loading control. Data are representative of 3 experiments. Error bars indicate SEM (n = 3).
Alfonso Catalano et al. Blood 2006;107:
©2006 by American Society of Hematology

4. Sema-3A reduces cytokines production.
Sema-3A reduces cytokines production. (A) T cells were cultured for the indicated times with anti-CD3 (300 ng/mL) plus anti-CD28 (200 ng/mL) alone or with CM derived from Sema-3A-expressing COS-7 cells as described in Figure 2A. Supernatants were collected, and IL-2, IL-10, IFN-γ, and IL-4 concentrations were assessed by ELISA. (B) T cells were treated as described in panel A for 8 hours. Total RNA was extracted, and IL-2 expression was analyzed by real-time PCR. GAPDH were used as housekeeping gene. *P < .001 versus COS-7/-, ANOVA, n = 3. (C) T cells were cultured for 24 hours with anti-CD3 + anti-CD28 in the presence of CM derived from COS-7/- or COS-7/C1 clones. Recombinant human IL-2, IL-4, and IL-10 (1.0 ng/mL) were added to the culture, and T-cell proliferation was measured by [3H]thymidine incorporation. *P < .05 versus untreated cells, paired t test, n = 3. Error bars indicate SEM.
Alfonso Catalano et al. Blood 2006;107:
©2006 by American Society of Hematology

5. Sema-3A inhibited CD3 plus CD28-mediated IL-2 transcription and expression and transactivation of known IL-2 transcription factors.
Sema-3A inhibited CD3 plus CD28-mediated IL-2 transcription and expression and transactivation of known IL-2 transcription factors. (A) Jurkat T cells were transiently transfected with the reporter construct pIL-2-luc (10 μg). Forty hours later, 5 × 105 cells per sample were cultured for 8 hours with either anti-CD3 + anti-CD28 alone or with increasing concentrations of recombinant human Sema-3A (Sema3A/Fc), and luciferase activity was measured. *P < .05 versus none, ANOVA, n = 4. Error bars indicate SEM. (B) Jurkat cells were cultured for 3 hours with anti-CD3 + anti-CD28 in the presence of Sema3A/Fc or Sema6A/Fc (100 ng/mL), whole-cell extracts were prepared, and expression of transcription factors was determined by immunoblotting with antibodies specific for NFATp, c-Fos, and actin. The whole-cell extracts was also immunoblotted using anti-phospho-c-Jun and anti-c-Jun antibodies. (C) Whole-cell extracts from Jurkat cells, treated as described in panel B for the indicated times, were prepared and immunoblotted with an anti-IκBα antibody and reprobed with an anti-JNK1 antibody to control for loading. Data are representative of 2 experiments.
Alfonso Catalano et al. Blood 2006;107:
©2006 by American Society of Hematology

6. Down-regulation of Sema-3A expression in tumor cells sensitizes T-cell activation.
Down-regulation of Sema-3A expression in tumor cells sensitizes T-cell activation. (A) Caki-1 and H28 cells were transfected with C-siRNA or Sema-3A siRNA (600 nM). Then, immunoprecipitated whole-cell lysates and TSNs were prepared 72 hours after transfection and analyzed as reported in Figure 1. (B) TSNs of transfected H28 cells were collected, and IL-6, TGF-β, and VEGF concentrations were assessed by ELISA. (C-D) T cells were cultured for 24 hours with either anti-CD3 + anti-CD28 alone or with the indicated TSNs (1:2) from Caki-1 cells in the absence or presence of increasing concentrations of Sema3A/Fc or Sema6A/Fc. T-cell proliferation was then assessed by [3H]-thymidine uptake (C). In parallel, supernatants of T cells were collected, and IL-2 and IFN-γ concentrations were assessed by ELISA (D). *P < .001, ANOVA, n = 4. Error bars indicate SEM.
Alfonso Catalano et al. Blood 2006;107:
©2006 by American Society of Hematology

7. Sema-3A signaling.
Sema-3A signaling. (A) H28 cells were transfected as described in Figure 5. Seventy-two hours from transfection, cells were incubated with T cells that had been prelabeled with the fluorescent dye hydroethidine. Heterotypic cell clustering was measured by flow cytometry. The involvement of NP-1 in heterotypic cell clustering was studied by preincubating labeled allogenic resting T cells with blocking NP-1 antibodies (T cells + anti-NP-1) or with nonblocking NP-1 antibody (T cells + Control Ab). *P < .05, ANOVA, n = 3. Error bars indicate SEM. (B) T cells were stimulated for the indicated time points with anti-CD3 + anti-CD28 in the presence of Sema3A/Fc or Sema6A/Fc (100 ng/mL). Cell lysates were then immunoblotted with antibodies against phosphorylated ERK1/2 (anti-p-ERK1/2), p38 MAPK (anti-p-38), or phosphorylated MEK1 (anti-p-MEK1). The blots were stripped and reprobed for total ERK1, p38, or MEK1. (C) Jurkat cells were stimulated with anti-CD3 alone or with anti-CD28 in the presence of Sema3A/Fc (right) or Sema6A/Fc (left) (100 ng/mL) for 10 minutes and lysed. GTP-bound Rap1 and H-ras were detected with pulldown assays by using immobilized GST fusion proteins of RalGDS-RBD and Raf-RBD as described in “Materials and methods.” Western blot of total cell lysates with anti-Ras and anti-Rap1 antibody is also shown. Data are representative of 3 experiments.
Alfonso Catalano et al. Blood 2006;107:
©2006 by American Society of Hematology

8. Rap1 mediates the immunoinhibitory effects of Sema-3A.
Rap1 mediates the immunoinhibitory effects of Sema-3A. (A-B) Jurkat T cells were transfected with an empty vector (pcDNA3.1) or a vector containing cDNA of a dominant-negative Rap1 (Rap1N17). Then, stable transfectants were stimulated with anti-CD3 + anti-CD28 in the absence or presence of Sema3A/Fc (100 ng/mL) for 10 minutes and lysed. Cell lysates were electrophoresed and blotted with anti-p-ERK1/2 antibody. The blots were stripped and reprobed for total ERK1 (A). After 24 hours, the supernatants were harvested for IL-2 measurement by ELISA. *P ≤ .05, ANOVA, n = 3. Error bars indicate SEM. (C) Control and anti-CD3 and anti-CD28-stimulated T cells (107 cells per test) were either untreated or treated with Sema3A/Fc (100 ng/mL) for 5 minutes, and the lysates were then prepared. Equivalent amounts of whole-cell lysates were either immunoprecipitated with anti-Ras antibody and immunoblotted with anti-Raf-1 antibody (right) or immunoprecipitated with anti-Rap-1 antibody and immunoblotted with anti-Raf-1 antibody (left). Western blot of immunoprecipitated cell lysates with anti-Rap1 and anti-Ras antibody is also shown. Data are representative of 3 experiments.
Alfonso Catalano et al. Blood 2006;107:
©2006 by American Society of Hematology