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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2023, Vol. 43 Issue (12): 102-110    DOI: 10.13523/j.cb.2310046
    
TRBC1 and TCR Vβ Comparative Analysis of Two Flow Cytometry Methods for Detecting T Cell Clonality
SHI Wei-hua,LIU Yan-rong,CHANG Yan,YUAN Xiao-ying,HE Ling-ling,HAO Le,YIN Jia-yi,LU Jing-quan,WANG Ya-zhe()
Peking University People’s Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Beijing 100044, China
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Abstract  

Objective: To analyze the expression of TRBC1 and TCR Vβ repertoire in different subsets of T lymphocytes in healthy donors and compare two flow cytometry methods for detecting T cell clones and their judgment criteria. Methods: Peripheral blood was collected in 40 healthy individuals, in which five subsets were divided, including CD7+CD5+(T1), CD7+CD5dim+(T2), CD7+CD5dim2+(T3), CD7+CD5-(T4) and CD7-CD5+(T5) gated on total CD3+ cells and CD3+TCR γδ- T cells, respectively. The expression of CD4, CD8, TCR Vβ repertoire and TRBC1 were analyzed in these subsets. Results: Thirty-nine healthy donors were included for statistical analysis. The average proportions of T1, T2, T3, T4, and T5 in CD3+cells were 57.29%, 27.74%, 5.15%, 1.43%, and 5.99%, respectively. T1 and T5 are mainly composed of CD4+cells, while T2, T3, and T4 are mainly composed of CD8+cells. The TRBC1+% in T2, T3, and T4 subsets of CD3+ is significantly lower than the corresponding value of TCR γδ- within T cells of CD3+ (P<0.01). Abnormal TRBC1+% (<15% or >85%) was found in 22 subsets in 21 healthy subjects, mainly in the T3 and T4 subsets. Partial subsets show a single dominant Vβ class, with dominant Vβ class proportion ≥60% or Vβ sum value of <15% used as the criterion for determining abnormal clones. After revising the TRBC1 clonal diagnostic criteria to TRBC1+%<12% or >82%, the consistency between the two methods for determining clonality was 96.77% (180/186). After the effect of TCR γδ+ cells is removed, the distribution trend remains unchanged. The final confirmed incidence of monoclonal T cells was 38.46% (15/39), and 86.67% (13 cases) had a proportion of clonal T cells in lymphocytes below 5%, indicating physiological clonal proliferation. The other two cases accounted for 6.76% and 33.24%, respectively, which may be T-cell clones of uncertain significance (T-CUS) and T lymphocyte proliferative diseases. Conclusions: Many physiologic T-cell clones were found frequently in CD5dim+ or CD5- subsets after gated by CD7/CD5 pattern in healthy people. The significance and interpretation of clonal T cells must be closely integrated with clinical practice.

Key words： Flow cytometry      T cells      Clonality      TCR Vβ      TRBC1     
Received: 30 October 2023      Published: 16 January 2024
ZTFLH:  Q-33  
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Wei-hua SHI
Yan-rong LIU
Yan CHANG
Xiao-ying YUAN
Ling-ling HE
Le HAO
Jia-yi YIN
Jing-quan LU
Ya-zhe WANG
Cite this article:

Wei-hua SHI, Yan-rong LIU, Yan CHANG, Xiao-ying YUAN, Ling-ling HE, Le HAO, Jia-yi YIN, Jing-quan LU, Ya-zhe WANG. TRBC1 and TCR Vβ Comparative Analysis of Two Flow Cytometry Methods for Detecting T Cell Clonality. China Biotechnology, 2023, 43(12): 102-110.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2310046     OR     https://manu60.magtech.com.cn/biotech/Y2023/V43/I12/102

Fig.1 Gate strategy of T cell subsets CD7+CD5+ cells(T1), CD7+CD5dim+ cells(T2), CD7+CD5dim2+ cells(T3), CD7+CD5- cells(T4) and CD7-CD5+ cells (T5)
不同细胞亚群比例 CD3+细胞 CD3+TCRγδ-细胞 淋巴细胞
TCRγδ+/% 9.67±8.04 1.57±1.18
CD4+/% 49.72±11.26 55.07±11.72* 12.87±3.81
CD8+/% 40.01±11.06 42.30±11.64 10.31±4.63
CD4∶CD8 1.40±0.64 1.48±0.68
T1(CD7+CD5+)/% 57.29±13.13 61.64±13.84 35.98±11.07
T2(CD7+CD5dim+)/% 27.74±9.30 24.75±9.88 14.59±7.41
T3(CD7+CD5dim2+)/% 5.15±4.44 4.04±4.35 2.40±2.74
T4(CD7+CD5-)/% 1.43±1.24 0.69±0.92* 0.39±0.51
T5(CD7-CD5+)/% 5.99±3.26 6.39±3.20 3.75±2.07
Table 1 The proportion of different T cell subsets in normal peripheral blood
V β受体表位 CD3+ T1 T2 T3 T4 T5
vβ5.3 0.96±0.39 1.17±0.45 0.62±0.41 0.51±1.18 1.39±7.70 0.84±0.45
vβ7.1 2.06±0.71 2.35±0.65 1.82±1.67 0.74±1.02 1.84±9.48 2.09±2.51
vβ3 5.10±3.11 5.40±3.07 4.28±3.38 5.12±7.86 4.89±10.19 4.43±4.85
vβ9 4.35±2.29 5.23±2.55 2.90±3.55 2.61±5.35 0.30±0.85 3.52±2.25
vβ17 3.35±1.24 4.04±1.21 2.36±1.54 1.26±1.62 0.54±1.44 4.13±4.16
vβ16 1.95±1.48 1.75±1.13 1.83±2.28 3.47±8.77 6.14±17.40 1.92±1.60
vβ18 0.45±0.34 0.61±0.54 0.25±0.29 0.15±0.49 0.01±0.07 0.34±0.34
vβ5.1 5.12±4.24 5.40±1.22 3.96±8.23 3.54±13.17 0.91±2.70 4.29±2.21
vβ20 1.98±0.76 2.55±0.88 1.19±0.67 0.65±0.80 0.07±0.22 2.28±2.38
vβ13.1 3.48±2.03 3.87±1.91 2.62±1.83 2.25±5.37 1.31±3.32 3.20±3.42
vβ13.6 1.47±0.54 1.73±0.73 1.11±0.87 0.86±2.48 1.25±4.66 1.74±2.19
vβ8 3.71±2.28 4.16±1.64 3.34±4.65 3.58±8.02 1.40±4.22 3.39±3.19
vβ5.2 1.01±0.65 1.16±0.54 0.83±1.00 0.31±0.57 0.04±0.15 1.11±1.20
vβ2 8.80±3.80 9.92±5.22 7.51±4.56 4.53±4.35 2.05±5.24 11.17±7.76
vβ12 1.32±0.50 1.58±0.47 0.76±0.62 1.08±3.64 0.19±0.74 1.58±1.93
vβ23 1.24±1.66 1.14±1.11 1.10±2.37 2.48±8.12 1.89±5.48 0.91±1.45
vβ1 4.89±4.43 4.63±3.17 5.04±6.92 3.16±5.30 1.62±6.17 4.88±7.30
Vβ21.3 2.05±0.98 2.55±1.28 1.25±0.86 0.97±2.12 0.53±1.19 2.67±3.20
vβ11 1.15±0.64 1.21±0.59 1.06±1.09 0.89±1.63 0.52±2.63 0.82±0.72
vβ22 3.77±1.89 4.24±2.36 2.81±2.47 3.18±9.40 2.90±8.87 5.29±7.40
vβ14 2.80±0.93 3.03±0.70 2.78±1.74 1.94±3.65 1.09±4.33 2.34±1.59
vβ13.2 4.48±1.88 5.31±2.22 3.55±1.88 3.19±4.65 1.13±3.26 3.48±1.64
vβ4 1.60±1.23 1.80±0.92 1.40±2.46 0.80±1.46 0.31±1.01 1.84±1.57
vβ7.2 1.26±0.81 1.32±1.03 1.31±1.18 1.52±4.00 0.83±2.18 1.36±2.17
总TCR Vβ 68.35±7.67 76.14±6.83 55.66±14.52 48.81±22.71 33.14±27.54 69.64±14.94
Table 2 Percentages of TCR Vβ repertoire and TRBC1 in CD3+ and T1-T5 subsets in a cohort of 39 normal specimen
Fig.2 Comparison of TRBC1 expression in different subsets between CD3+ and CD3+TCRγδ- cells
细胞亚群 编号 淋巴细胞
比例/%		 TRBC1+/% TCR Vβ检测 单克隆
<15% >85% 单个优势表位/% 总和/% 优势表位占比/%
T2 10 21.98 13.65 26.65 71.66 37.19 否
38 33.24 8.16 51.81 85.77 60.41 是
T3 4 6.76 5.39 73.28 77.36 94.73 是
1 1.37 12.87 无 27.55 否
5 3.31 13.19 无 29.38 否
10 1.37 8.68 39.48 64.79 60.94 是
21 3.52 13.58 38.85 80.84 48.06 否
24 2.43 8.26 11.20 31.62 35.42 否
35 4.39 13.72 25.33 69.08 36.67 否
T4 11 1.53 95.33 46.10 50.53 91.23 是
7 0.27 94.00 12.81 15.01 92.01 是
5 0.76 3.29 45.96 52.99 86.73 是
6 0.94 0.00 48.70,33.68 88.81 92.76 是
14 0.35 13.24 69.44 80.21 86.57 否
21 1.31 11.62 74.30 98.54 75.40 是
23 1.19 10.15 无 5.74 是
26 2.15 5.63 无 10.52 是
27 0.17 10.20 无 1.19 是
33 1.77 0.54 15.40 21.44 71.83 是
36 0.43 11.29 59.35 84.66 70.10 是
37 0.38 10.43 无 11.45 是
Table 3 Comparison the results of TCR Vβ repertoire with TRBC1 in T1-T5 subsets
Fig.3 Abnormal antigen pattern of CD7/CD5 and the expression of TRBC1 in different subsets in one healthy donor
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