This is a prospective case-control study, conducted in King Fahad Specialist Hospital-Dammam (KFSHD), in the Eastern Province of Saudi Arabia. The appropriate ethical approval was obtained from the local research ethics committee under Institutional Review Board Number (EXT0328) during 2016-2018. A total number of 89 subjects were recruited, 46 of them represented the patients who were diagnosed with colorectal cancer during the study. A consultant oncologist at KFSHD oncology center completed staging and detailed therapy plan among these 43 patients. The remaining 43 subjects represented normal healthy control. The cases in this group were blood bank donors who visited the same medical center and did not have a known personal or family history of cancer. All subjects were informed and consented before proceeding with the study.
Isolation of peripheral blood mononuclear cells (PBMCs) and the identification of MAIT cells
Peripheral blood samples were collected from each colorectal cancer subject and healthy control using a standard venipuncture procedure and collecting in heparin-containing tubes. PBMC were isolated by Ficoll-paque Plus (GE Healthcare) and density-gradient centrifugation. MAIT cells were identified phenotypically as CD3+, CD161+/TCR7.2+, TCR γδ- [6,26].
Monoclonal antibodies (mAbs) and flow cytometry
The monoclonal antibodies used were CD3-PerCP (Cat No:130-094-965), CD161-peVio 770 (Cat No:130-099-965), Anti-TCR Vα7.2-APC-Vio700 (Cat No:130-100-179), Anti-TCRγ/δ-APC, human, (Cat No:130-096-866), CD45RA-PE, human, (Cat No:130-098-184), CD45RO-PE, human, (Cat No:130-109-508), CD62L-PE, human, (Cat No:130-099-717), CD11a-PE, human, (Cat No:130-105-479), CD158a (KIR2DL1)-PE, human, (Cat No:130-103-967), CD158b-PE, human, (Cat No:130-099-397), D158e(KIR3DL1)-PE, human, (Cat No:130-092-473), CD158f (KIR2DL5)-PE, human, (Cat No:130-115-911). The antibodies were obtained from Miltenyi Biotec, Bergisch Gladbach, Germany.
Data Collection and Analysis of Flow Cytometry
Immunophenotyping was performed on a FACS Canto II instrument (Becton Dickinson, San Jose, CA, USA). Collection and analysis were performed using FACSDiva v8.0 software. The forward scatter light (FSC) and the side scatter light (SSC) parameters were adjusted to each sample in order to observe all cell types. The cell collection rate was adjusted to 500-1000 events/second. Small particles and debris were excluded by FSC gate on lymphocytes. A fluorescence threshold was set at 5000 in order not to not exceed 0.5% of background fluorescence.
MAIT cell analysis was completed using a sequential gating strategy. Staring from gating on CD3 positive population to target T-lymphocytes, then T-cells expressing TCR gd were filtered by gating on the CD3+/TCR gd + population. Subsequently, the MAIT cells were gated as a population CD161 +/TCR7-2 +, of which MAIT associated phenotyping antigens (i.e. CD45RA, CD45RO, CD62L, CD11a, CD158a, CD158b, CD158e and CD158f) were analyzed for percentage of expression and mean fluorescent intensity.
HLA-C1 and C2 Genotyping:
For each reaction, 50-100 ng of DNA was used in a 15 μL final volume. For 16 KIR genes, HLA-C1 and HLA-C2 group typing, the same primers were used as reported before . For each reaction, positive, negative, and internal controls were used. All PCR reactions were performed with the thermocycler apparatus T100TM (Thermal Cycler from Bio-Rad Laboratories, Inc. Life Science Research, California 94547, United States, and Veriti® Thermal Cycler from Thermo Fisher Scientific, United States).
Different types of tests were used for the phenotype and functional studies. For example, unpaired t-test and paired t-test were used when the data had assumed a Gaussian distribution. In contrast, for the data that did not assume a Gaussian distribution a Mann-Whitney U test was used. Groups with a p value of < 0.05 were considered to be statistically significant difference. Statistical analysis was conducted using software Graph pad prism version 7 for the phenotyping and functional studies.