Technical Reference #1637

1637.

Cell Surface Expression of Channel Catfish Leukocyte Immune-Type Receptors (IpLITRs) and Recruitment of Both Src Homology 2 Domain- Containing Protein Tyrosine Phosphatase (SHP)-1 SHP-2 Benjamin Montgomery; Jacqueline Mewes; Chelsea Davidson; Deborah Busrchtyn; James Stafford, University of Alberta, Developmental and Comparative Immunology, 33(1637), (2009)
Link To Paper

Abstract:
Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF)members believed to play a role in the control and coordination of cellular immune responses in teleost.Putative stimulatory and inhibitory IpLITRs are co-e

Keywords:
innate immunity; immunoglobulin superfamily; receptors; signaling; natural killer cells; tyrosine phosphorlation; cellular inhibition; phosphatases

Materials & Methods:
2.1. Cell culture HeLa and HEK 293T cells were grown at 37 8C and 5% CO2 in DMEM/High Glucose (HyClone) supplemented with 2 mM Lglutamine (Gibco) 100 Units/mL penicillin (Gibco) 100 mg/mL streptomycin (Gibco) 1 mM sodium pyruvate (Gibco) 1% MEM non-essential amino acid solution (Gibco) and 10% heat-inactivated fetal bovine serum (characterized; Hyclone). Prior to use culture media was filter sterilized using 0.22 mm filter units (Corning). 2.2. Antibodies The following antibodies were used in this study; anti-HA mAb clone HA.C5 (Cedarlane Laboratories Ltd.) HRP-conjugated goat anti-HA polyclonal antibody (GenScript Corp.) anti-phosphotyrosine mAb 2C8 (Santa Cruz Biotechnology Inc.) biotin-conjugated anti-phosphotyrosine mAb 4G10 (Upstate) anti-FLAG mAb M2 (Stratagene) anti-FLAG M2 mAb peroxidase conjugated (Sigma– Aldrich) goat anti-mouse IgG (H + L)-FITC (Cedarlane Laboratories Ltd.) goat anti-mouse IgG (H + L)-PE (Beckman Coulter) anti-KIR mAb (LIG-1) [31] and anti-KIR mAb (DX27) [32]. 2.3. Generation of ‘native’ epitope-tagged IpLITR Several putative inhibitory IpLITRs have been cloned from alloantigen-stimulated catfish cytotoxic T lymphocytes [30]. These cDNAs were cloned into the pCR4-TOPO vector (Invitrogen) and provided as a kind gift from Dr. Norman Miller (Univ. Mississippi Medical Center) as templates for generating an epitope-tagged receptor. Ten nanograms of the pCR4-TOPO vector containing the sequence for TS32.17 L1.1b (NCBI accession number: ABI16050) was amplified with pDISPLAY L1.1b/SmaI Fwd and pDISPLAY L1.1b/SalI Rvs primers (Table 1) using 0.4 U of Phusion High- Fidelity DNA polymerase (Finnzymes) in 20 mL reactions according to manufacturer’s recommended instructions. Cycling parameters were as follows; 40 s at 98 8C 30 cycles of 98 8C for 20 s 64 8C for 25 s 72 8C for 30 s and a final extension step for 7 min at 72 8C. Note: The generation of all expression constructs in this study were performed using Phusion High-Fidelity polymerase. Reactions were then separated on a 1.0% TAE-agarose gel and visualized by staining with ethidium bromide solution (50 mg/L) and the PCR product (1500 bp) was excised gel purified (Qiagen Gel Extraction Kit) and cloned into pJET1.2/blunt using the bluntend protocol (Fermentas). Cloning reactions were then transformed into NEB 10-beta E. coli (New England BioLabs) and positive clones identified by performing colony PCR with the pJET1.2 Fwd and Rvs sequencing primers (Table 1). A randomly selected positive colony was grown overnight in 8 mL of LB-ampicillin (100 mg/mL) and the plasmid isolated using the Qiagen miniprep kit according to manufacturer’s instructions. Two micrograms of the purified plasmid was double digested for 2 h with 10 U of SmaI/ SalI and the insert isolated and then ligated into the pDISPLAY vector (Invitrogen) using T4 DNA ligase (Fermentas) and a 2.5 h incubation at 25 8C followed by an overnight incubation alternating between 4 8C and 16 8C every 45 min. Positive pDISPLAY-IpLITR TS32.17 L1.1b clones were identified by restriction digestion and purified plasmids were sequenced to verify the cDNA sequence and to ensure that the construct was in-frame without any base pair changes. All sequencing was performed at the molecular biology services unit in the Department of Biological Sciences University of Alberta on an ABI 3730 DNA sequencer. 2.4. KIRED-LITRCYT chimeric constructs To characterize the signaling potential of putative inhibitory catfish LITRs we constructed ‘chimeric’ expression constructs consisting of the ED and TM segment of human KIR2DL3 fused to the tyrosine-containing CYT region of two different IpLITR receptors (Fig. 1). Briefly the full-length cDNA for KIR2DL3 (NCB accession number: U24074) in the pSC65 vector was subcloned into pSPORT-1 using SalI and NotI digests. pSPORT-1/KIR2DL3 was then amplified with forward and reverse primers (Table 1) designed to amplify around the vector in order to introduce a KpnI restriction site just after the TM segment of KIR2DL3 (i.e. bp position 795 of the open reading frame). This strategy allowed for the direct fusion of the CYT regions of IpLITR cDNAs directly after the ED and TM segment of KIR2DL3. Catfish LITRs used for the CYT amplifications (NCBI accession numbers: ABI16050 and ABI16051) were previously cloned into pCR4-TOPO [30]. The IpLITR CYT regions were amplified using specific forward primers (Table 1) with a 50 phosphate (to facilitate fusion immediately after the 795 bp of KIR2DL3) and a reverse primer (Table 1) that introduced a KpnI restriction site immediately following the native IpLITR CYT ‘stop codon; TAG’. Products were gel purified digested with KpnI and then ligated into the pSPORT-1/KIR2DL3 to create a chimeric cDNA construct [KIR2DL3ED/TM/LITRCYT]. Three different constructs were generated designated as 1.0 2.0 and 3.0 (Fig. 1). Construct 1.0 contains the entire CYT region of ABI16051 (i.e. 1170–1326 bp; amino acid 391–441) construct 2.0 contains a truncated region of the CYT of NCBI accession number: ABI16050 (i.e. 1167–1398 bp; amino acid 389–466) and construct 3.0 contains the entire CYT region of ABI16050 (i.e. 1167–1533 bp; amino acid 389–511). During the sequencing of construct 3.0 a frame-shift mutation was identified in one of the clones that introduced premature stop codon resulting in a mutated receptor that was devoid of any tyrosine residues in its short 28 amino acid CYT. Consequently this construct was chosen as a negative control termed 3 (Fig. 1). All chimeric constructs were then amplified with PCR primers (Table 1) to facilitate SalI/NotI restriction cloning into the final destination vector pBABE [33] which is a retroviral expression plasmid that facilitates stable expression of proteins in NK tumor cell lines such as human YTS cells and transient expression in HeLa and HEK 293T cells as described below. Prior to cellular transfections all KIRED-LITRCYT were sequenced to verify the cDNA sequence and to ensure that the constructs were in-frame without any base pair changes. Several of the tyrosine residues found in the IpLITR CYT regions are encoded within ITIM or ITIM-like motifs [1230]. KIRED-LITRCYT construct 1.0 encodes one ITIM and one ITIM-like motif. Tyrosine residues identified in such motifs are indicated as hatched boxes (Fig. 1). Construct 3.0 was designed to test the full CYT of the receptor TS32.15 L1.1b which to date encodes the longest CYT region of all IpLITRs identified containing six tyrosine residues. As shown in Fig. 1 the C-terminal end of this CYT contains an arrangement of tyrosines embedded in motifs that are similar to the shorter CYT of TS32.15 L1.2a (i.e. Construct 1.0). However preceding this region are three membrane proximal tyrosine residues that are not present within a consensus ITIM which is indicated by a white box (Fig. 1). We also constructed a truncated version of the TS32.17 L1.1b CYT that only included these residues designated as construct 2.0. 2.5. Cellular transfections Transfections were performed using cells (e.g. HeLa and HEK 293T) seeded in 6-well tissue culture plates (Costar) or 60 mm tissue culture dishes (Costar). For 6-well plates 5 105 cells were seeded in 2 mL DMEM/10% FBS per well and incubated overnight prior to transfection with 1 mg of plasmid DNA. For each expression construct DNA was first diluted in 190 mL of serumfree DMEM and then 4 mL of TurboFect in vitro transfection reagent (Fermentas) was added. Samples were gently mixed and incubated for 20 min at room temperature. The DMEM-plasmid-TurboFect solution was then evenly layered onto the cells which were then incubated for 24–48 h at 37 8C to allow for protein production. Cotransfections with different expression constructs were performed as described for single transfections except 1 mg of each plasmid were both diluted in 190 mL of serum-free DMEM. For transfections in 60 mm dishes the same procedure for 6-well plates was followed except initial seeding volume of medium amount of plasmid and amount of TurboFect were all doubled. Prior to seeding cells for transfection experiments cell viability was examined using Trypan blue staining to ensure that cultures had >95% viable cells prior to seeding. 2.6. Flow cytometry and microscopy Flow cytometry for the detection of KIRED-LITRCYT and HAtagged ‘native’ IpLITR surface expression was performed using transfected HEK 293T cells. After 48 h post-transfection DMEM/ transfection solution was removed from each well and the cells were gently washed with 2 mL of sterile Dulbecco’s PBS (D-PBS) containing 2 mM of EDTA. PBS was removed and 500 ml of 0.25% Trypsin-EDTA (Gibco) was added to each well and the plate gently agitated until cells detached. Two milliliters of DMEM-10% FBS was added and aliquots of 2 105 cells placed in 1.5 mL Eppendorf tubes. Cells were centrifuged at 4 8C (400 g for 8 min) supernatants aspirated and 1.4 mL of ice-cold antibody staining buffer ASB (D-PBS 0.05% NaN3 1% bovine serum albumin) added. Prior to addition of primary antibody cells were centrifuged once more and ASB aspirated. Cell pellets were gently disrupted and 1 mg of primary antibody (i.e. anti-DX27 mAb or anti-HA mAb clone HA.C5) diluted in 50 mL of ASB was added. Cells were incubated on ice for 30 min with gentle mixing every 10 min followed by the addition of 1.2 mL ice-cold ASB centrifugation and aspiration. Cells pellets were again disrupted and 0.5 mg of goat anti-mouse IgG (H + L)-PE diluted in 50 mL of ASB was added and staining/ washing repeated as above. Cells were then analyzed for fluorescence using a FACS Calibur flow cytometer; Becton Dickinson. To visualize cell surface expression of KIRED-LITRCYT constructs and HA-tagged ‘native’ LITR HeLa cells were seeded at 5 104 cells in 35 mm glass bottom dishes (MatTek) transfected and then antibody stained as described above for flow cytometry. After the final wash cells were fixed with formalin for 10 min at room temperature rinsed with D-PBS and then analyzed using a Zeiss Axiovert 2500 M fluorescence microscope.

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
HEK-293