Introduction
Materials and methods
Study population
Isolation of peripheral blood mononuclear cells
Flow cytometry antibody staining
Flow cytometry data analysis
Corrections and statistical analyses
Results
Patient characteristics
CRC patients | Healthy donors | ||
---|---|---|---|
(N = 71) | (N = 19) | P value | |
Age* | 0.06 | ||
Mean (years) | 65.6 | 55.6 | |
Range | 25–85 | 22–83 | |
Sex | 0.56 | ||
Female | 32 (45.1%) | 10 (52.6%) | |
Male | 39 (54.9%) | 9 (47.4%) | |
Tumor location | |||
Colon | 59 (83.1%) | ||
Rectum | 12 (16.9%) | ||
Tumor stage | |||
Stage 0 | 4 (5.6%) | ||
Stage I | 10 (14.1%) | ||
Stage II | 22 (35.2%) | ||
Stage III | 22 (33.8%) | ||
Stage IV | 8 (11.3%) | ||
Tumor differentiation grade | |||
Well/moderate | 55 (77.5%) | ||
Poor | 13 (18.3%) | ||
Unknown | 3 (4.2%) | ||
Lymph node invasion | |||
Yes | 30 (56.3%) | ||
No | 40 (42.3%) | ||
Unknown | 1 (1.4%) | ||
Neoadjuvant radiotherapy | |||
Yes | 10 (14.1%) | ||
No | 61 (85.9%) | ||
Adjuvant chemotherapy | |||
Yes | 27 (38.0%) | ||
No | 44 (62.0%) |
Increased percentage of circulating regulatory T cells in colorectal cancer patients compared to healthy donors
Healthy controls (N = 19) | CRC patients (N = 71) | |||||
---|---|---|---|---|---|---|
Mean | SD | Mean | SD | P value | Corrected P* value | |
Subset distribution | ||||||
T cells (%)* | 59.4 | 8.2 | 57.5 | 13.2 | 0.669T | 0.883 |
CD8+ T cells (%)* | 20.7 | 8.3 | 18.7 | 10.4 | 0.378U | 0.670 |
CD4+ T cells (%)* | 42.1 | 6.1 | 44.3 | 12.2 | 0.400T | 0.689 |
CD127lowCD25+ Treg (%)* | 5.5 | 0.5 | 7.7 | 2.4 | 0.000U |
0.000
|
NK cells (%) | 12.8 | 5.4 | 15.4 | 8.5 | 0.508U | 0.768 |
CD56dim NK cells (%) | 93.4 | 4.2 | 95 | 4.2 | 0.054U | 0.197 |
CD56bright NK cells (%) | 6.6 | 4.2 | 5.1 | 4.2 | 0.057U | 0.196 |
NKT-like cells (%) | 4.3 | 2.8 | 6.1 | 6.4 | 0.656U | 0.904 |
CD56dim NK cells | ||||||
CD16
+
(%)
| 85.3 | 6.1 | 83.7 | 10.2 | 0.909U | 0.989 |
CD158a
+
(%)
| 35.2 | 17.1 | 31.9 | 16.9 | 0.477U | 0.739 |
CD158b
+
(%)
| 37 | 11.4 | 37.5 | 14.7 | 0.786U | 0.937 |
NKG2A
+
(%)
| 45.6 | 19.3 | 43.4 | 19 | 0.656T | 0.884 |
NKG2A
+
(MFI)
| 6398 | 2138 | 7368 | 2459 | 0.275U | 0.533 |
NKG2C
+
(%)
| 13.7 | 13 | 16.2 | 17.9 | 0.909U | 0.972 |
NKG2C
+
(MFI)
| 2506 | 2797 | 2965 | 3094 | 0.213U | 0.440 |
CD161 (MFI)
| 3111 | 804 | 3057 | 1130 | 0.816T | 0.903 |
CD8
+
(%)
| 34.3 | 14.3 | 31.5 | 14.4 | 0.467U | 0.742 |
CD8
+
(MFI)
| 3481 | 774 | 3067 | 1159 | 0.067U | 0.208 |
DNAM-1 (MFI)
| 560 | 106 | 548 | 146 | 0.741T | 0.919 |
NKG2D
+
(%)
| 89.9 | 4.4 | 91.4 | 4.9 | 0.134U | 0.346 |
NKG2D
+
(MFI)
| 3600 | 1108 | 3429 | 1113 | 0.554T | 0.818 |
NKp30 (MFI)
| 1960 | 752 | 1630 | 900 | 0.052U | 0.202 |
NKp44
+
(%)
| 0.7 | 0.6 | 0.6 | 0.6 | 0.081U | 0.239 |
NKp44 (MFI)
| 131 | 13 | 115 | 26 | 0.002U |
0.041
|
NKp46
+
(%)
| 48.7 | 18 | 34.7 | 16.9 | 0.002T |
0.031
|
NKp46 (MFI)
| 732 | 345 | 478 | 271 | 0.003U |
0.031
|
CD56bright NK cells | ||||||
CD16
+
(%)
| 3 | 2.8 | 2.5 | 1.8 | 0.415U | 0.695 |
CD158a
+
(%)
| 6.6 | 4.2 | 7 | 4.8 | 0.793U | 0.928 |
CD158b
+
(%)
| 6.4 | 3 | 8.6 | 5.5 | 0.097U | 0.273 |
NKG2A
+
(%)
| 6.6 | 4.6 | 4.5 | 3.3 | 0.044U | 0.210 |
NKG2A
+
(MFI)
| 17,020 | 7986 | 19,052 | 5829 | 0.165 U | 0.365 |
NKG2C
+
(%)
| 1.9 | 2.2 | 1.2 | 1.1 | 0.145U | 0.333 |
NKG2C
+
(MFI)
| 1760 | 967 | 3038 | 3831 | 0.017U | 0.105 |
CD161 (MFI)
| 2128 | 568 | 1767 | 578 | 0.012U | 0.093 |
CD8
+
(%)
| 2.4 | 1.6 | 1.8 | 1.7 | 0.044U | 0.195 |
CD8
+
(MFI)
| 2951 | 886 | 3098 | 1318 | 0.945U | 0.977 |
DNAM-1 (MFI)
| 765 | 130 | 782 | 161 | 0.675T | 0.872 |
NKG2D
+
(%)
| 6.2 | 4.1 | 4.7 | 3.7 | 0.062U | 0.202 |
NKG2D
+
(MFI)
| 5321 | 1354 | 6210 | 1837 | 0.037U | 0.191 |
NKp30 (MFI)
| 2145 | 418 | 2413 | 769 | 0.048T | 0.198 |
NKp44
+
(%)
| 2 | 1.5 | 1.3 | 1.2 | 0.024U | 0.135 |
NKp44 (MFI)
| 252 | 49 | 236 | 78 | 0.099U | 0.267 |
NKp46
+
(%)
| 86.1 | 8.1 | 80.7 | 14.3 | 0.135U | 0.322 |
NKp46 (MFI)
| 2343 | 613 | 2090 | 812 | 0.211T | 0.451 |
NKT-like cells | ||||||
CD16
+
(%)
| 35.6 | 17.6 | 24.6 | 13.7 | 0.013U | 0.090 |
CD158a
+
(%)
| 12 | 12.9 | 9.4 | 9.8 | 0.957U | 0.973 |
CD158b
+
(%)
| 18.8 | 19.2 | 19.2 | 19.2 | 0.793U | 0.910 |
NKG2A
+
(%)
| 31.9 | 25.4 | 28.3 | 21 | 0.692U | 0.876 |
NKG2A
+
(MFI)
| 4529 | 4416 | 3444 | 3757 | 0.604U | 0.851 |
NKG2C
+
(%)
| 15 | 15.6 | 14.2 | 13.3 | 0.909U | 0.955 |
NKG2C
+
(MFI)
| 2308 | 2256 | 3205 | 3300 | 0.283U | 0.532 |
CD161 (MFI)
| 4509 | 4407 | 4445 | 5434 | 0.452U | 0.737 |
CD8
+
(%)
| 64.1 | 16.9 | 70.4 | 17.9 | 0.134U | 0.332 |
CD8
+
(MFI)
| 11,870 | 6011 | 11,673 | 5556 | 0.968U | 0.968 |
DNAM-1 (MFI)
| 847 | 211 | 766 | 279 | 0.238T | 0.476 |
NKG2D
+
(%)
| 85.4 | 14.5 | 88.6 | 10.7 | 0.345U | 0.629 |
NKG2D
+
(MFI)
| 4730 | 1589 | 4995 | 2041 | 0.602T | 0.868 |
NKp30 (MFI)
| 217 | 70.1 | 250 | 136 | 0.797U | 0.898 |
NKp44
+
(%)
| 1.4 | 1 | 1.4 | 1.2 | 0.774U | 0.941 |
NKp44 (MFI)
| 134 | 17 | 120 | 33 | 0.002U |
0.025
|
NKp46
+
(%)
| 5.4 | 1.9 | 4.6 | 2.8 | 0.011U | 0.097 |
NKp46 (MFI)
| 112 | 53 | 61 | 47 | 0.000U |
0.000
|
Reduced expression of natural cytotoxicity receptors on circulating CD56dim NK cells and NKT-like cells from colorectal cancer patients compared to healthy donors
No correlation between distribution of immune cell subsets and immunophenotype of circulating lymphocytes and tumor characteristics
Association between the peripheral blood immune cell profile and disease-free survival of colorectal cancer patients
Factor | Univariate analysis | Multivariate analysis** | ||||
---|---|---|---|---|---|---|
P value | HR | 95% CI | P value | HR | 95% CI | |
Age* | 0.053 | 1.036 | 0.999–1.075 | |||
Sex | ||||||
Female | Ref. | |||||
Male | 0.085 | 1.942 | 0.912–4.136 | |||
Tumor type | ||||||
Colon | Ref. | |||||
Rectum | 0.571 | 1.299 | 0.526–3.211 | |||
Tumor stage | ||||||
Stage II | Ref. | |||||
Stage III |
0.006
| 2.946 | 1.354–6.412 | |||
Tumor differentiation grade | ||||||
Well/moderate | Ref. | |||||
Poor | 0.885 | 0.924 | 0.320–2.669 | |||
Lymph node invasion | ||||||
No | Ref. | |||||
Yes |
0.010
| 2.825 | 1.289–6.193 | |||
Treg (%)*** | ||||||
≤ 6.9% | Ref. | Ref. | ||||
> 6.9% | 0.072 | 2.551 | 0.921–7.069 | 0.119 | 3.142 | 0.744–13.273 |
NKp30 CD56bright NK cells (MFI) | ||||||
≤ MFI 2327 | Ref. | Ref. | ||||
> MFI 2327 |
0.047
| 2.143 | 1.009–4.551 | 0.169 | 1.752 | 0.788–3.893 |
CD16+ NKT-like cells (%) | ||||||
≤ 20.3% | Ref. | Ref. | ||||
> 20.3% |
< 0.001
| 4.697 | 2.046–10.783 |
0.004
| 4.192 | 1.578–11.137 |
CD158a+ NKT-like cells (%) | ||||||
≤ 5.1% | Ref. | Ref. | ||||
> 5.1% | 0.074 | 1.969 | 0.937–4.136 | 0.134 | 1.803 | 0.834–3.897 |