Eur. J. Immunol. 2012. 42: 195–205 T-lymphocyte populations or Jurkat cell lines, 3 106 cells were loaded with the calcium indicators Fluo-3 and Fura Red (Molecular Probes) at the ﬁnal concentrations of 5 and 10 mM, respectively, in complete RPMI and incubated for 45 min at 371C. Basal calcium levels were analyzed by ﬂow cytometry for 10 s before samples were removed, the agonist (OKT3 at 1 mg/mL) was added, and the samples monitored for calcium release during 5 min. Cells were at a concentration of 3 106 cells/mL. Analysis was performed using the FlowJo software (Treestar). The ratio FL1/FL3 was derived and plotted against time. Kinetic plots are represented as mean of the FL1/FL3 ratio. CD6 knockdown using MOs Knockdown of hCD6 from T lymphocytes was achieved through the transient transfection of CD6 MOs into T cells using Amaxa nucleofection. In brief, 1 107 cells were centrifuged, resus- pended in 100 mL of prewarmed nucleofector solution, mixed with 150 mM of MOs for CD6 (MO-CD6, 50-GTCTGGAGCT GTCTCTGGCTGCTAC-30, GeneTools), or 150 mM of scramble MO-control (MO-c; 50-GTgTGCAGCTCTCTCTGCCTGGTAC-30- ﬂuorescein), and transferred into nucleofector cuvettes (Amaxa/Lonza). Nucleofection was performed in a Nucleofector II with the V-024 program. Cells were transferred to and maintained in complete RPMI 1640 medium at 371C and used for analysis 72 h post-transfection. CD6 knockdown was conﬁrmed by Western blotting, using MEM-98 [42] for the detection of hCD6. Western blotting was performed as described previously [33]. The membranes were then stripped and restained with an Ab speciﬁc to b-actin. The densities of bands were measured with a Quantity-One version 4.6.9 imaging system (BioRad) and normalized with b-actin. Cell-surface biotinylation and detection of biotinylated CD6 Cell-surface biotinylation and detection of biotinylated CD6 was performed as described previously [33]. Tetramer production and staining A cDNA fragment encoding the extracellular domain of human CD58 (residues Phe29-Arg213) was ampliﬁed by PCR from template kindly provided by M. Vuong (University of Oxford) using forward 50-TAGGGATCCACCGGTTTTTCCCAACAAATA- TATGGT-30 and reverse oligos 50-CTAAGATCTGGATCCTCTT- GAATGACCGCTGCTTGG-30 containing AgeI and BamHI restriction sites, respectively. The CD58 fragment was cloned via AgeI and BamHI sites into the lab-modiﬁed TOPO vector containing the secretion peptide sequence (SP) from pHLsec [44] followed by a HA-tag with AgeI and BamHI sites immediately afterward. Fragments containing SP, HA-tag, and CD58 insert & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Leukocyte signaling 203 were then cut out from the TOPO vector constructs using HindIII and BamHI for transfer into a lab-modiﬁed version of the pEE14 vector [45] using HindIII and BclI sites, in order to obtain CD58 chimeric cDNA encoding, in the following order, SP, HA-tag, the extracellular region of human CD58 (residues 29–213), and BirA [46] and 6 His tag sequences. The cDNA coding for the extracellular domain of hCD6 (residues Asp25-Glu398) was ampliﬁed from CD6/pBJ-neo [12] by PCR using forward 50-TAGTAGGGATCCGACCAGCTCAACACCAGCAGTG-30 and reverse 50-CTACTAGGATCCTTAGTGATGTCTAGACTCCCGA- GATTCCTTGTTC-30 oligos containing BamHI restriction sites. PCR products were puriﬁed and cloned into the BamHI-BclI sites of the lab-modiﬁed version of pEE14, in order to obtain CD6 chimeric cDNA encoding, in the following order, SP, HA-tag, the extracelluar region of hCD6 (residues 25–398), and BirA and 6 His tag sequences. The CD58 and CD6 chimera constructs were transfected into CHO-K1 cells using GeneJuice transfection reagent (Merck). Clones resistant to 25 mM methionine sulfoximine were selected [47] and screened for soluble CD58 (sCD58-BirA-His) or CD6 (sCD6-BirA-His) chimera expression using dot blots and Western blots. The best clones expressing sCD58-BirA-His or sCD6-BirA-His were selected for large-scale production of protein and grown in cell factories (Nunc). sCD58-BirA-His and sCD6-BirA-His secreted into tissue culture supernatants were harvested after approximately 4 wk and the proteins were extracted by metal-chelate chromatography using Ni-NTA agar- ose (QIAGEN). The sCD58-BirA-His and sCD6-BirA-His were eluted from the Ni-NTA agarose with 250 mM imidazole in 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0, and further puriﬁed by size- exclusion chromatography. Soluble chimeras were then biotiny- lated using the BirA enzyme (Avidity LLC), and incubated over- night at room temperature with phycoerythrin (PE)-streptavidin (Invitrogen) at a 12:1 and 6:1 molar ratio, respectively, to allow the formation of tetrameric CD58 and CD6 complexes. The resulting tetramers were then puriﬁed using size-exclusion chromatography (Superose 6 HR 10/30 column) on an AKTA FPLC. Analysis of cytokine production and cell division Jurkat E6.1 cells expressing hCD6 isoforms were plated at 105 cells/well in complete RPMI, in the presence or absence of stimulation with OKT3 at 1 mg/mL. Supernatants were collected after 24 h and assayed for IL-2 production by ELISA (Abcam) according to the manufacturer’s protocol. Ag-induced cell proliferation was assessed via CFSE ﬂuores- cence. Human PBLs at 0.5 106/mL were incubated with 1 mM CFSE (Molecular Probes) at 371C for 15 min in PBS. For blocking the CD6–CD166 interaction, PBLs were then washed twice with PBS 20% FCS, and incubated with OX126 for 30 min at 41C. During the same period of time, Raji cells (1 105) were incu- bated with 3A6. The negative control Ab used was mouse IgG (BD Pharmingen). PBLs were stimulated by incubation with Raji cells www.eji-journal.eu