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Article Open Access

Analysis of the TCRβ repertoire characteristics when combining radiotherapy and immunotherapy in cancer

  • Authors:
    • Xueli Yang
    • Wujun Wei
    • Zhihui Feng
    • Biao Zhang
    • Yi Liu
    • Fei Yang
    • Chong Zhang
    • Minglin Ou
    • Songbai Liao
    • Xianliang Hou
  • View Affiliations / Copyright

    Affiliations: Scientific Research Department, The Second Affiliated Hospital of Guilin Medical University, Guilin, Guangxi Zhuang Autonomous Region 541199, P.R. China, Center for Clinical Laboratory Diagnosis and Research, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, Guangxi Zhuang Autonomous Region 533000, P.R. China, Guangxi Key Laboratory of Metabolic Reprogramming and Intelligent Medical Engineering for Chronic Diseases, The Second Affiliated Hospital of Guilin Medical University, Guilin, Guangxi Zhuang Autonomous Region 541199, P.R. China, Department of Neurology, The Second Affiliated Hospital of Guilin Medical University, Guilin, Guangxi Zhuang Autonomous Region 541199, P.R. China, Department of Urology, The Nanxishan Hospital of Guangxi Zhuang Autonomous Region, Guilin, Guangxi Zhuang Autonomous Region 541002, P.R. China
    Copyright: © Yang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 67
    |
    Published online on: December 3, 2025
       https://doi.org/10.3892/ol.2025.15420
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Abstract

Cancer is a global public health concern. Radio­therapy (RT) and immunotherapy are increasingly used in cancer treatment, but the specific immune mechanisms underlying their effects remain to be elucidated. The present study analyzed publicly available datasets, in which the T‑cell receptor β chain (TCRβ) repertoire in tumor‑bearing mice treated with RT and immunotherapy, as well as the TCR repertoire of T cells derived from hematopoietic stem cells (HSCs) of mice exposed to radiation doses of 0, 10 and 100 mGy were comprehensively evaluated. The results of the present study indicated that the diversity of the TCRβ complementarity‑determining region 3 (CDR3) in tumor‑bearing mice decreased after RT, anti‑cytotoxic T‑lymphocyte‑associated protein‑4 (CTLA‑4) and anti‑programmed cell death protein‑1 (PD‑1) treatment, especially in the RT and RT + anti‑CTLA‑4 or anti‑PD‑1 treatment groups. Furthermore, the CDR3 length markedly decreased in the RT group and markedly increased in the anti‑CTLA‑4 and anti‑PD‑1 groups. Next, the TCR profile of T cells differentiated from HSCs exposed to different radiation doses was examined. As the radiation dose increased, the TCRβ diversity in mice gradually decreased and the degree of clonal expansion markedly increased, the percentage of private sequences (unique sequences detected in only a single mouse) was markedly increased and the degree of overlap was markedly decreased. Furthermore, there was a notable difference in the usage frequency of TCRβ chain variable/joining gene segments among the different radiation dose groups. The present study demonstrated that RT and immunotherapy may alter the TCR repertoire owing to their effects on HSCs.
View Figures

Figure 1

CDR3 nucleotide length distribution
and diversity analysis of the TCRβ repertoire in mice with RT and
anti-CTLA-4 therapy, and the control group. The TCRβ CDR3 diversity
in the post-selection repertoire (in-frame sequences) was estimated
by (A) the D50 index, (B) Gini index, (C) Inverse Simpson index,
(D) Shannon index and (E) Simpson index. (F-J) The TCRβ CDR3
diversity in the pre-selection repertoire (out-of-frame sequences)
was estimated by the aforementioned five diversity indices. Data
are presented as the mean ± SD; comparisons were made using one-way
ANOVA or Kruskal-Wallis test and corrected for multiple comparisons
using Bonferroni. *P<0.05, **P<0.01 and ***P<0.001. (K-N)
The comparison of CDR3 length between the different groups. All of
the CDR3 lengths that differed significantly (P<0.05) between
treatment group mice and controls are presented. Data are presented
as the mean ± SD; the overall differences of the CDR3 length
distribution (ten length categories) between groups were compared
using two-way ANOVA, and Tukey's test was performed after the
two-way ANOVA to compare the differences in each CDR3 length
between groups. *P<0.05, **P<0.01, ***P<0.001 and
****P<0.0001. CDR3, complementarity-determining region 3; TCRβ,
T-cell receptor β-chain; CTLA-4, cytotoxic T lymphocyte-associated
antigen-4; RT, radiotherapy; NS, not significant.

Figure 2

Diversity indexes of the
post-selection repertoire in mice with RT and anti-PD-1 therapy,
and the control group. The diversity of the treatment area was
measured by (A) the D50 index, (B) Gini index, (C) Inverse Simpson
index, (D) Shannon index and (E) Simpson index. (F) The D50 index,
(G) Gini index, (H) Inverse Simpson index, (I) Shannon index and
(J) Simpson index were all used to evaluate the diversity of the
outside the RT field. Data are presented as the mean ± SD;
comparisons were made using one-way ANOVA corrected for multiple
comparisons using Bonferroni. *P<0.05, **P<0.01 and
***P<0.001 (two-tailed). PD-1, programmed cell death-1; NS, not
significant; RT, radiotherapy.

Figure 3

Distribution of TCRβ repertoire CDR3
length within and outside the treatment area in mice receiving RT
and anti-PD-1 therapy, as well as in the control group. (A) The
CDR3 length distribution map compared between the control and
anti-PD-1 groups. (B) The CDR3 length distribution map compared
between the control and RT groups. (C) The CDR3 length distribution
map compared between the control and RT + anti-PD-1 groups. (D) The
CDR3 length distribution map compared between the RT and the
anti-PD-1 groups. (E) The CDR3 length distribution map compared
between the control and anti-PD-1 groups. (F) The CDR3 length
distribution map compared between the control and RT groups. (G)
The CDR3 length distribution map compared between the control and
RT + anti-PD-1 groups. (H) The CDR3 length distribution map
compared between the RT and the anti-PD-1 groups. Data are
presented as the mean ± SD; the overall differences of the CDR3
length distribution (ten length categories) between groups were
compared using two-way ANOVA, and Tukey's test was performed after
the two-way ANOVA to compare the differences in each CDR3 length
between groups. *P<0.05, **P<0.01 and ****P<0.0001. TCRβ,
T-cell receptor β-chain; CDR3, complementarity-determining region
3; PD-1, programmed cell death-1; RT, radiotherapy.

Figure 4

Degree of expansion and diversity
indices of the TCRβ repertoire in the HC and RT groups. The
diversity of the post-selection repertoire was measured by (A) the
D50 index, (B) Gini index, (C) Inverse Simpson index, (D) Shannon
index and (E) Simpson index. (F) Distribution in the clonal
abundance of the post-selection repertoire. Five diversity indices
were used to assess the diversity of pre-selection repertoire: (G)
D50 index, (H) Gini index, (I) Inverse Simpson index, (J) Shannon
index and (K) Simpson index. (L) Distribution in the clonal
abundance of the pre-selection repertoire. Data are presented as
the mean ± SD; comparisons were made using the one-way ANOVA or
Kruskal-Wallis test and corrected for multiple comparisons using
Bonferroni. *P<0.05 and **P<0.01. TCRβ, T-cell receptor
β-chain; HC, healthy control; ns, not significant.

Figure 5

Altered CDR3 length distribution,
InDel patterns and public degree in the RT group. (A) The changes
in the CDR3 length in the radiation group mice from the 10 and 100
mGy groups were compared with those of the HC group. (B-G) TCRβ
CDR3 sequences from the radiotherapy and HC groups in the
post-selection repertoire were analyzed for the frequency of
clonotypes with a specific number of InDels at each of the six
rearrangement sites: (B) vDdel, (C) d3Del, (D) d5Del, (E) jDel, (F)
n1Ins and (G) n2Ins. (H-J) Overlap indices were calculated from 9
HC, 9 mice with 10 mGy and 9 mice with 100 mGy, where (H) is the
comparison between HC group mice and mice receiving 10 mGy
radiation, (I) is the comparison between HC group mice and mice
receiving 100 mGy radiation and (J) is the comparison between mice
receiving 10 mGy radiation and mice receiving 100 mGy radiation.
Data are presented as the mean ± SD and the means between the two
groups were compared using the unpaired t-test or the Mann-Whitney
U test. One-way ANOVA or Kruskal-Wallis test was used to compare
group means across three or more groups and corrected for multiple
comparisons using Bonferroni. *P<0.05 and **P<0.01. TCRβ,
T-cell receptor β-chain; CDR3, complementarity-determining region
3; InDel, insertions and deletions; NS, not significant; HC,
healthy control.

Figure 6

Usage frequency of TRBV/J segments is
different following three different radiation doses in the
post-selection repertoire. Significant differences in TRBV segment
usage in the (A) 10 mGy and (B) 100 mGy groups, compared with the
HC group. Differential usage of the TRBJ gene segment in the (D) 10
mGy and (E) 100 mGy groups, compared with the HC group. Use of TRBV
(C) and TRBJ (F) gene fragments in the 100 mGy group compared with
the 10 mGy group. Significant differences were analyzed by unpaired
t-test and false discovery rate correction. All of the TRBVs and
TRBJs that were found to differ significantly (P<0.05) between
radiation group mice and HC are presented. Data are presented as
the mean ± SD. Three-dimensional landscapes depicting the mean
utilization frequency of specific Vβ-Jβ gene segment combinations
in the in-frame TCRβ chains expressed in the (G) HC group and mice
with radiation from the (H) 10 mGy and (I) 100 mGy groups. (J) PCA
based on the TRBV segment out-of-frame usage demonstrated notable
differences between the radiation and HC groups. *P<0.05,
**P<0.01. TCRβ, T-cell receptor β-chain; TRBV, TCR β chain
variable gene; TRBJ, TCR β chain joining gene; PCA, principal
coordinate analysis; HC, healthy control.
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Copy and paste a formatted citation
Spandidos Publications style
Yang X, Wei W, Feng Z, Zhang B, Liu Y, Yang F, Zhang C, Ou M, Liao S, Hou X, Hou X, et al: Analysis of the TCR&beta; repertoire characteristics when combining radiotherapy and immunotherapy in cancer. Oncol Lett 31: 67, 2026.
APA
Yang, X., Wei, W., Feng, Z., Zhang, B., Liu, Y., Yang, F. ... Hou, X. (2026). Analysis of the TCR&beta; repertoire characteristics when combining radiotherapy and immunotherapy in cancer. Oncology Letters, 31, 67. https://doi.org/10.3892/ol.2025.15420
MLA
Yang, X., Wei, W., Feng, Z., Zhang, B., Liu, Y., Yang, F., Zhang, C., Ou, M., Liao, S., Hou, X."Analysis of the TCR&beta; repertoire characteristics when combining radiotherapy and immunotherapy in cancer". Oncology Letters 31.2 (2026): 67.
Chicago
Yang, X., Wei, W., Feng, Z., Zhang, B., Liu, Y., Yang, F., Zhang, C., Ou, M., Liao, S., Hou, X."Analysis of the TCR&beta; repertoire characteristics when combining radiotherapy and immunotherapy in cancer". Oncology Letters 31, no. 2 (2026): 67. https://doi.org/10.3892/ol.2025.15420
Copy and paste a formatted citation
x
Spandidos Publications style
Yang X, Wei W, Feng Z, Zhang B, Liu Y, Yang F, Zhang C, Ou M, Liao S, Hou X, Hou X, et al: Analysis of the TCR&beta; repertoire characteristics when combining radiotherapy and immunotherapy in cancer. Oncol Lett 31: 67, 2026.
APA
Yang, X., Wei, W., Feng, Z., Zhang, B., Liu, Y., Yang, F. ... Hou, X. (2026). Analysis of the TCR&beta; repertoire characteristics when combining radiotherapy and immunotherapy in cancer. Oncology Letters, 31, 67. https://doi.org/10.3892/ol.2025.15420
MLA
Yang, X., Wei, W., Feng, Z., Zhang, B., Liu, Y., Yang, F., Zhang, C., Ou, M., Liao, S., Hou, X."Analysis of the TCR&beta; repertoire characteristics when combining radiotherapy and immunotherapy in cancer". Oncology Letters 31.2 (2026): 67.
Chicago
Yang, X., Wei, W., Feng, Z., Zhang, B., Liu, Y., Yang, F., Zhang, C., Ou, M., Liao, S., Hou, X."Analysis of the TCR&beta; repertoire characteristics when combining radiotherapy and immunotherapy in cancer". Oncology Letters 31, no. 2 (2026): 67. https://doi.org/10.3892/ol.2025.15420
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