DNA demethylating agent decitabine broadens the peripheral T cell receptor repertoire

Jing Nie, Yan Zhang, Xiang Li, Meixia Chen, Chuanjie Liu, Weidong Han, Jing Nie, Yan Zhang, Xiang Li, Meixia Chen, Chuanjie Liu, Weidong Han

Abstract

Purpose: Decitabine, a promising epi-immunotherapeutic agent has shown clinical responses in solid tumor patients, while the anti-tumor mechanisms were unclear. We aimed to investigate the immunomodulatory effect of decitabine in peripheral T cells.

Experimental design: We applied next-generation sequencing to investigate the complementarity-determining region 3 (CDR3) of the TCRβ gene, the diversity of which acts as the prerequisite for the host immune system to recognize the universal foreign antigens. We collected the peripheral blood mononuclear cells (PBMCs) from 4 patients, at baseline and after 2 cycles of low-dose decitabine therapy.

Results: An increase of the unique productive sequences of the CDR3 of TCRβ was observed in all of the 4 patients after decitabine treatment, which was characterized by a lower abundance of expanded clones and increased TCR diversity compared with before decitabine treatment. Further analysis showed a tendency for CD4 T cells with an increased CD4/CD8 ratio in response to decitabine therapy. In addition, the genome-wide expression alterations confirmed the effects of decitabine on immune reconstitution, and the increase of TCR excision circles (TRECs) was validated.

Conclusions: The low-dose DNMT inhibitor decitabine broadens the peripheral T cell repertoire, providing a novel role for the epigenetic modifying agent in anti-tumor immune enhancement.

Keywords: T cell receptor repertoire; decitabine; epigenetic therapy; pharmacometabonomics; solid tumor.

Conflict of interest statement

None.

Figures

Figure 1. Global analysis of V β…
Figure 1. Global analysis of Vβ-Jβ gene segment combinations
(A, B) The frequencies of TCRβ V (A) and J (B) gene segments in peripheral blood samples at baseline (pre) and following 2 cycles of decitabine therapy (post) from UPN1 and UPN2 are shown. (C–F) Frequencies of specific Vβ-Jβ gene segment combinations in TCRβ CDR3 sequences are expressed in UPN1 (C), UPN2 (D), UPN3 (E), and UPN4 (F) before and after decitabine therapy.
Figure 2. Analysis of CDR3 spectrums
Figure 2. Analysis of CDR3 spectrums
(A–D) A comparison of CDR3 region length and distribution between baseline and after 2 cycles of decitabine therapy in UPN1 (A), UPN2 (B), UPN3 (C), and UPN4 (D).
Figure 3. Analysis of high clonal V-J…
Figure 3. Analysis of high clonal V-J combinations
(A–D) The 10 most frequent V-J combinations of PBMCs from UPN1 (A), UPN2 (B), UPN3 (C) and UPN4 (D) at baseline and after 2 cycles of decitabine therapy are indicated in color; the remaining V-J combinations are grouped in white.
Figure 4. Analysis of changes in relative…
Figure 4. Analysis of changes in relative TCRβ V and TCRβ J
(A) PBMCs from the 4 patients were collected as detected by flow cytometry using antibodies against CD3, CD4, and CD8. The average ratios of CD3+CD4+: CD3+CD8+ from these patients are shown. (B–E) The frequencies of TCRβ V5-4 (B), TCRβ V7-2 (C), TCRβ V18 (D) and TCRβ V7-9 (E) in PBMCs from each patient before and after decitabine therapy are shown. (F) The top 10 V-J combinations in PBMCs from the 4 patients following decitabine therapy are listed.
Figure 5. Low-dose decitabine increased TCR diversity…
Figure 5. Low-dose decitabine increased TCR diversity in PBMCs
(A–D) Frequency distribution of clonotypes in plots of UPN1 (A), UPN2 (B), UPN3 (C), and UPN4 (D) at baseline (in blue) and after 2 cycles of decitabine therapy (in red). Each dot represents a distinct TCRβ clonotype. Clonotypes are presented at frequencies > 1% and the top 10 clonotypes with their cumulative frequency (percentage of reads). Values in the lower-left corner depict TCR diversity. (E) The 10 most frequent clonotypes in the 4 patients at baseline (pre) and after 2 cycles of decitabine therapy (post) are indicated in color and listed at the right; the remaining clonotypes are grouped in black. (F) TCR Simpson diversity of the 4 patients after 2 cycles of decitabine therapy compared to that of the baseline. (G) The average T cell repertoire Shannon diversity of the 4 patients before and after decitabine therapy.
Figure 6. Genome-wide expression alteration and PRKDC…
Figure 6. Genome-wide expression alteration and PRKDC induction
(A) The peripheral T cells were sorted by flow cytometry using anti-CD3 antibody from blood samples in these 4 patients (UPN1 to UPN4). The percentages of 5-mC in T cells from the 4 patients before and after decitabine therapy were detected relative to the total cytosine content. (B) Beta value changes in CpG islands in patients UPN1 and UPN2 before and after decitabine therapy. (C) Heat map and sample clustering analysis of the differentially expressed genes before and after decitabine therapy in the same patients as (B). (D) Significantly enriched up-regulated and down-regulated GO terms in biological process (BP) clusters were analyzed. The data shown are the negative log2 P-values within each category. (E) Central pathways and differentially expressed genes are shown according to the chip data. Genes in red type indicate the up-regulated genes, and genes in black type indicate the down-regulated genes. (F) The relative mRNA expression level of PRKDC was detected by quantitative RT-PCR assay. (G) TRECs and GAPDH expression levels were detected in the 4 patients before and after decitabine therapy as described in the “Method” section. UPN# (line 7 and 8) was shown as negative control, who was not received the DAC treatment.

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