Journal: Journal for Immunotherapy of Cancer

Article Title: Multifaceted effects of soluble human CD6 in experimental cancer models

doi: 10.1136/jitc-2019-000172

Figure Lengend Snippet: Generation and immunophenotypical characterization of shCD6LckEμTg mice. (A) Schematic representation of the transgene coding for soluble human CD6 (shCD6). (B) PCR screening of genomic DNA from homozygous shCD6LckEµTg and non-transgenic (NonTg) mice. Bands correspond to transgene (Tg) and internal PCR control (C). (C) ELISA quantification of plasma shCD6 levels from NonTg (-/-), and heterozygous (-/+) and homozygous (+/+) shCD6EμLckTg (Tg) mice. Represented are cumulative data from two independent experiments, expressed as mean±SEM. ***p<0.001; two-tailed Student’s t-test. (D) Immunophenotypical characterization of primary lymphoid organs from shCD6LckEμTg mice. Left: total lymphoid cell numbers from thymus and bone marrow of shCD6LckEμTg (n=21 and n=13, respectively) and NonTg (n=15 and n=16, respectively) mice. Middle: percentage of CD24 low BP1 - (pre-pro B cells), CD24 + BP1 - (early pro B cells), CD24 + BP1 + (late pro B cells) and CD24 high BP1 + (pre B cells) within gated CD43 + B220 + bone marrow cells from shCD6LckEμTg and NonTg mice. Right: percentage of CD4 - CD8 - (DN), CD4 + CD8 + (DP), CD4 + CD8 - (CD4SP) and CD4 - CD8 + (CD8SP) thymocytes from shCD6LckEμTg and NonTg mice. Represented are cumulative data from at least two independent experiments, expressed as mean±SEM. *p<0.05; **p<0.01; two-tailed Student’s t-test. (E) Immunophenotypical characterization of secondary lymphoid organs from shCD6LckEμTg mice. Left: total lymphoid cell numbers in spleen, and lymph node (LN) from shCD6LckEμTg (n=50 and n=23, respectively) and NonTg (n=61 and n=28, respectively) mice. Right: percentage of viable cells (7AAD - AnnexinV - ) from spleen and LN of shCD6LckEμTg and NonTg mice. One of two independent experiments is shown. (F) In vivo Bromodeoxyuridine (BrdU)-incorporation assays in secondary lymphoid organs from shCD6LckEμTg mice. Percentage of spleen (left) and LN (right) BrdU + lymphoid cells represented as total cells and as cells gated on CD19 + , CD4 + and CD8 + . Represented are cumulative data from at least two independent experiments, expressed as mean±SEM. *p<0.05; **p<0.01; ***p<0.001; two-tailed Student’s t-test.

Article Snippet: The codon-optimized cDNA sequence encoding the whole ectodomain of human CD6 (GenBank X60992.1) from the initiation M1 to R373 was synthesized at GenScript (New Jersey, USA) and cloned into the Bam HI site of the p1026x vector (kindly provided by Dr R.M.

Techniques: Transgenic Assay, Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Two Tailed Test, In Vivo, BrdU Incorporation Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Multifaceted effects of soluble human CD6 in experimental cancer models

doi: 10.1136/jitc-2019-000172

Figure Lengend Snippet: Effects of soluble human CD6 (shCD6) on the frequency and functionality of Treg cells. (A) Percentage of CD25 + FoxP3 + in CD4 + gated cells (Treg) from spleen, lymph node (LN) and thymus of shCD6LckEμTg and non-transgenic (NonTg) mice. (B) Geometric mean fluorescence intensity (GM) for latency-associated peptide (LAP) expression on spleen and LN Treg cells (defined as in a) from shCD6LckEμTg and NonTg mice. (C) Percentage of proliferation of CD4 + CD25 - (Tconv) cells from NonTg mice stimulated for 72 hours with anti-CD3 mAb plus anti-CD28 monoclonal antibody (mAb) in the absence (w/o) or presence of different ratios of CD4 + CD25 + (Treg) cells from shCD6LckEμTg or NonTg mice. (D) Sorted naïve CD4 + CD25 - CD62L hi CD44 lo splenocytes (1×10 5 ) from C57BL/6J (n=5) were activated in triplicate for 72 hours with anti-CD3 (2 µg/mL) and anti-CD28 (0.5 µg/mL) mAbs under Treg, Th1, Th2, and Th17 polarization conditions, all in the presence or absence of different amounts of shCD6 (0, 0.1, 1 and 10 µg/mL). Percentage of Treg, Th1, Th2, and Th17 in CD4 + cells as deduced from intracellular FOXP3, IFN-γ, IL-4 and IL-17 co-staining, respectively, are represented as mean±SEM. **p<0.01; ***p<0.001; unpaired t-test with Welch’s correction. (E) In vitro polarized Treg cells generated as in (D) and cultured alone (UT) or in the presence of 10 µg/mL HSA or shCD6 (left) were then subjected to flow cytometry analysis for surface LAP expression. percentage (%) and geometric mean fluorescence intensity (GM) of LAP + Treg cells is shown (middle and right, respectively). *p<0.05; **p<0.01; ***p<0.001; two-tailed Student’s t-test.

Article Snippet: The codon-optimized cDNA sequence encoding the whole ectodomain of human CD6 (GenBank X60992.1) from the initiation M1 to R373 was synthesized at GenScript (New Jersey, USA) and cloned into the Bam HI site of the p1026x vector (kindly provided by Dr R.M.

Techniques: Transgenic Assay, Fluorescence, Expressing, Staining, In Vitro, Generated, Cell Culture, Flow Cytometry, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Multifaceted effects of soluble human CD6 in experimental cancer models

doi: 10.1136/jitc-2019-000172

Figure Lengend Snippet: Immunophenotypical analysis of lymphoid organs from wild-type (WT) mice infused with recombinant soluble human CD6 (shCD6) protein. (A) Total lymphoid cell numbers in thymus, spleen, lymph node (LN) and peritoneum of WT mice after intraperitoneal administration of shCD6 (1.25 mg/kg) or vehicle (PBS 10% glycerol) every 48 hours for 14 days. (B) Percentage of CD25 + FoxP3 + in gated CD4 + T cells (Treg) from spleen and LN of same mice as in (A) is shown. Data represent cumulative results from two independent experiments expressed as mean±SEM. *p<0.05; two-tailed Student’s t-test.

Article Snippet: The codon-optimized cDNA sequence encoding the whole ectodomain of human CD6 (GenBank X60992.1) from the initiation M1 to R373 was synthesized at GenScript (New Jersey, USA) and cloned into the Bam HI site of the p1026x vector (kindly provided by Dr R.M.

Techniques: Recombinant, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Multifaceted effects of soluble human CD6 in experimental cancer models

doi: 10.1136/jitc-2019-000172

Figure Lengend Snippet: Growth curves of subcutaneously implanted syngeneic cancer cells into wild-type (WT) mice prophylactically infused with soluble human CD6 (shCD6) protein. B16.F0 (5×10 4 ), MCA205 (5×10 4 ), EL-4 (5×10 4 ), and RMA-S (5×10 4 ) cells were subcutaneously injected into WT mice receiving intraperitoneal injections of shCD6 (1.25 mg/kg) or vehicle (PBS 10% glycerol) every other day from tumor implantation. Tumor growth (area in mm 2 ) and weight (GR) were measured every other day and at sacrifice, respectively. Two-way analysis of variance and Mann-Whitney tests were used for statistical analysis of tumor growth and weight, respectively. Data are presented as mean±SE.

Article Snippet: The codon-optimized cDNA sequence encoding the whole ectodomain of human CD6 (GenBank X60992.1) from the initiation M1 to R373 was synthesized at GenScript (New Jersey, USA) and cloned into the Bam HI site of the p1026x vector (kindly provided by Dr R.M.

Techniques: Injection, Tumor Implantation, MANN-WHITNEY

Journal: Journal for Immunotherapy of Cancer

Article Title: Multifaceted effects of soluble human CD6 in experimental cancer models

doi: 10.1136/jitc-2019-000172

Figure Lengend Snippet: Inhibition of galectin-induced T-cell apoptosis and tumor cell growth and migration by soluble human CD6 (shCD6). (A) Relative Gal-1 and Gal-3 mRNA expression in the different tumor cell lines analyzed in this study, as assessed by qRT-PCR. (B) Splenocytes from wild-type (WT) C57BL/6J mice were activated for 72 hours with anti-CD3 (1 µg/mL) and anti-CD28 (5 µg/mL) monoclonal antibodies (mAbs) and then incubated for 30 min with 100 µg/mL of endotoxin-free glutathione S-transferase (GST), GST-Gal-1 or GST-Gal-3 in the presence of growing concentrations (10 and 20 µg/mL) of shCD6 protein (n=4/group). Percentage of apoptotic cells was determined by flow cytometry. (C) Indicated tumor cells were stained for surface ALCAM expression and mean fluorescence intensity (MFI) was determined by flow cytometry. (D) Tumor cells (2×10 4 ) were cultured with increasing concentrations (5, 10 and 20 µg/mL; n=4/group) of shCD6 or HSA and their numbers counted at 24-hour intervals. (E) Wound healing assays were used to measure the migratory activity of B16.F0 cells (5×10 5 ) seeded onto 12-well plates for 24 hours, followed by 2-hour exposure to mitomycin-C (50 µg/mL) and further 12-hour incubation with increasing concentrations (5 and 25 µg/mL) of shCD6 (n=4/group) or HSA control (n=3). Data represent percentage of cell-covered area. In all cases, cumulative results from two independent experiments expressed as mean±SEM are presented. *p<0.05; two-tailed Student’s t-test. ALCAM, activated leukocyte cell adhesion molecule; HSA, human serum albumin.

Article Snippet: The codon-optimized cDNA sequence encoding the whole ectodomain of human CD6 (GenBank X60992.1) from the initiation M1 to R373 was synthesized at GenScript (New Jersey, USA) and cloned into the Bam HI site of the p1026x vector (kindly provided by Dr R.M.

Techniques: Inhibition, Migration, Expressing, Quantitative RT-PCR, Bioprocessing, Incubation, Flow Cytometry, Staining, Fluorescence, Cell Culture, Activity Assay, Control, Two Tailed Test