Samples were first classified according to their BMI based on height and weight correlation.
We have examined Saudi patients with CRC, of which 30 males and 16 females, and Saudi control subjects consisted of 27 males and 16 females. The mean ages of participants were 56.37 ± 12.93 years in males, and 55.69 ± 13.05 years in females. In both groups the height, weight and BMI did not show significant differences: in CRC patients, the mean height was 1.7 ± 0.08 in males and 1.55 ± 0.07 in females. Subsequently, the mean weight was 84.85 ± 20.13 in males and 81.94 ± 23.95 in females. BMI was 29.30 ± 5.95 and 34.04 ± 9.27, respectively. The mean height was 1.69 ± 0.05 in males and 1.57 ± 0.05 in females among control subjects’ group. The mean weight was 83.59 ± 11.73 in males and 75.44 ± 22.59 in females. The mean BMI was 27.92 ± 6.93 and 30.32 ± 7.29, respectively.
3.1. Distribution of the KIR ligand C1/C2 in patients and control groups:
The distribution of HLA-C groups and genotypes are shown in (Table 1). Statistical analysis showed a high frequency of HLA - C2 among patients with CRC (87%) compared with control subjects (74.4%). The frequency of HLA-C1 was higher in control subjects (72.1%) compared with CRC patients (65.2%). In addition, genotype distribution showed that the frequencies of HLA-C1C2 was higher in CRC patients (52.2%) compared with control subjects (48.8%).
Table 1
The characteristics of CRC patients and Control subjects of the study.
Variables | Control Subjects | CRC Patients |
men | women | men | women |
Number of Subjects | 27 | 16 | 30 | 16 |
Age | 54.74 ± 11.55 | 54.94 ± 11.53 | 56.37 ± 12.93 | 55.69 ± 13.05 |
Height | 1.69 ± 0.05 | 1.57 ± 0.05 | 1.70 ± 0.08 | 1.55 ± 0.07 |
Weight | 83.59 ± 11.73 | 75.44 ± 22.59 | 84.85 ± 20.13 | 81.94 ± 23.95 |
BMI | 27.92 ± 6.93 | 30.32 ± 7.29 | 29.30 ± 5.95 | 34.04 ± 9.27 |
Table 2
The frequencies and percentage of the KIR ligand C1/C2 in CRC patients and control.
Genotype | Patient | Control | OR | CI 95% | Chi square & P - value |
C1C1 | 6 (13.04) | 10 (23.3) | 0.495 | 0.163–1.505 | Χ2 = 1.57; P-value = 0.21 |
C2C2 | 16 (34.8) | 11 (25.6) | 1.552 | 0.62–3.87 | Χ2 = 0.89; P-value = 0.35 |
C1C2 | 24 (52.2) | 21 (48.8) | 1.14 | 0.50–2.63 | Χ2 = 0.1; P-value = 0.75 |
C1 | 30 (65.2) | 31 (72.1) | 0.73 | 0.29–1.79 | Χ2 = 0.49; P-value = 0.48 |
C2 | 40 (87) | 32 (74.4) | 2.29 | 0.76–6.87 | Χ2 = 2.26; P-value = 0.13 |
3.2. MAIT cells frequencies in blood of CRC patients and healthy subjects
The presence of MAIT cells in the peripheral blood of CRC patients and control subjects were evaluated by identifying the expression of CD3+, CD161+/TCR7.2 + on their surface. Four-color flow cytometry was used to determine the percentage of MAIT cells. The lymphocytes populations were gated on forward and side scatter (Fig. 1).
The percentage of MAIT cells were higher in CRC patients compared to control subjects. However, the difference between the two groups did not reach statistically significant differences (Fig. 2). The median value was higher in CRC patients (3.265) compared to control subjects (2.6).
MAIT cells were stratified in patients with CRC by disease stage. The result showed that the percentage of MAIT cells was higher in patients with CRC in stage III and IV, but lower in stage II compared with control subjects (Fig. 3). However, the difference between the four groups did not reach the statistical significant differences.
3.3. MAIT cells phenotype antigen and KIR expression
The protein expression of MAIT cells associated phenotyping antigens and some KIR receptors such as (CD45RA, CD45RO, CD62L, CD11a, CD158a, CD158b, CD158e and CD158f) were analyzed for percentage of expression. A relatively lower percentage of CD45RA expression was seen in CRC patients compared with control subjects (Fig. 4). There was a significant reduction in CD45RO expression in CRC patients in comparison with control subjects (Fig. 4). CD11a expression indicated similarity in both patients and control with no statistically significant differences (Fig. 4). However, CD62L analysis indicated a lower expression on MAIT cells in CRC patients compared with control subjects with significant difference (Fig. 4). In CRC patients CD158a, CD158e and CD158f were expressed at lower percentage on MAIT cells in CRC patients and higher percentage was observed in control subjects with significant difference (P value = 0.01, 0.01, 0.03) (Fig. 5), also similar result was obtained for CD158b, however the results did not reach statistically significant differences (Fig. 5).
Stratification analysis revealed that the percentage of MAIT cells that express CD158a, cd158e and CD158f were lower in CRC patients in all stages compared with control subjects. While CD158b was lower in CRC patients in stages III and IV and higher in stage II compared with control subjects. However, the difference between the four groups did not reach statistically significant differences (Fig. 6).
KIR2DL2 and 2DL3 recognize HLA-C group 1 alleles, while HLA-C2 alleles are bound by KIR2DL1. Therefore, frequencies of KIR + MAIT cells were measured in CRC patients and control subjects with or without the gene for the cognate ligand.
The percentage of MAIT cells expressing CD158a in the presence of HLA-C2 was lower in CRC patients compared with control subjects with significant differences (P value = 0.004). However, in the absence of HLA-C2, CD158a expression was higher in CRC patients (Fig. 7a and b).
The percentage of MAIT cells expressing CD158b in the presence of HLA-C1 was lower in CRC patients compared with control subjects with no significant differences. In the absence of HLA-C1, CD158b expression was higher in CRC patients (Fig. 8a and b).
KIR2DL2 and 2DL3 recognize HLA-C group 1 alleles while HLA-C2 alleles are bound by KIR2DL1. Therefore, frequencies of KIR + MAIT cells were measured in CRC patients with the gene for the cognate ligand according to the stage. The percentage of MAIT cells expressing CD158a in the presence of HLA-C2 was lower in CRC patients in stages II, III and IV compared with control subjects with statistically significant differences (P value = 0.03) (Fig. 9). Median levels of CD158a + MAIT cells indicate positive correlation with advancing cancer stages.
The percentage of MAIT cells expressing CD158b in the presence of HLA-C1 were quite similar in CRC patients in all stages and control subjects with no significant differences (Fig. 10).