Quantification of ALKBH2 mRNA expression and HTLV-1 viral factors in PBMCs derived from patients infected with HTLV-1
To assess the gene expression of ALKBH2, we used total RNA extracted from PBMCs collected from 16 HC, 11 ACs, and 10 patients with acute-type ATL. The mRNA expression of ALKBH2 in the AC group was 2.55 and 2.25 times lower than that in the HC and ATL groups, respectively, and the difference between the HC and AC groups was statistically significant (Fig. 1a and Table 1).
Table 1
Detection efficiency of ALKBH2 transcripts
| mRNA | pre-mRNA |
| HC | AC | ATL | HC | AC | ATL |
Number of detected / analyzed | 16/16 | 10/11 | 9/10 | 15/16 | 7/11 | 5/10 |
Detection rate (%) | 100 | 90.9 | 90.0 | 93.8 | 63.6 | 50.0 |
HBZ is an HTLV-1 viral protein constantly detected in HTLV-1–infected cells. Consistent with general knowledge, HBZ mRNA was only detected in the AC and ATL groups, and HBZ mRNA expression was significantly higher in the ATL group (by a factor of 38.36; Fig. 1b and Table 2).
Table 2
Detection efficiency of Tax and HBZ mRNA
| | Tax | | | HBZ | |
| HC | AC | ATL | HC | AC | ATL |
Number of detected / analyzed | 0/16 | 0/11 | 0/10 | 0/16 | 8/11 | 9/10 |
Detection rate (%) | 0 | 0 | 0 | 0 | 72.7 | 90.0 |
We also attempted to evaluate Tax mRNA expression, but it could not be detected in any of the analyzed specimens (Table 2). The certainty of the assay methods to detect HBZ and Tax mRNA was confirmed in the cultured T-cell lines (Supplementary Fig. 2). To evaluate the propagation of HTLV-1–infected cells, we analyzed a dataset of HTLV-1 proviral load (PVL) that was previously reported by our research group 32. Consistent with HBZ mRNA expression, the HTLV-1 PVL in the ATL group was significantly higher than that in the AC group, by a factor of 7.14 (Fig. 1c).
We performed a correlation analysis between the mRNA expression of ALKBH2 and HTLV-1 viral factors. In each analysis, incomplete data that contained an undetected result for either parameter were omitted (Tables 1 and 2). The mRNA expression levels of ALKBH2 were moderately, but significantly, correlated with those of HBZ (Fig. 1d). In contrast, the mRNA expression levels of ALKBH2 were not correlated with the HTLV-1 PVL (Fig. 1e). It was also confirmed there was no significant correlation between HBZ mRNA and PVL (Fig. 1f). These observations suggested that HTLV-1 infection, which is possibly associated with the expression of HBZ mRNA, is a key factor in the regulation of ALKBH2 mRNA.
Investigation of mRNA and pre-mRNA expression of ALKBH2 in HBZ-Tg
To explore the role of HBZ in the mRNA expression of ALKBH2 in immune cells, we analyzed spleen cells from HBZ-Tg mice, which artificially expressed HBZ in CD4+ T-cells and established as an animal model for ATL 33. Whole spleen cells and negatively isolated CD4+ T-cells were analyzed after determining the population of CD4+ T-cells (Table 3).
Table 3
States of the analyzed mice
| | | | Population of CD4+ cells (%) |
Genotype | ID | Gender | Killed (weeks) | Whole SP cells | Isolated CD4+ cells |
Wild type | #1 | F | 29 | 15.9 | 86.5 |
| #2 | F | 31 | 13.7 | 85.1 |
| #3 | F | 38 | 11.7 | 72.8 |
| #4 | F | 53 | 11.1 | 74.2 |
HBZ-Tg | #5 | F | 49 | 26.9 | 95.5 |
| #6 | F | 49 | 34.6 | 97.5 |
| #7 | F | 64 | 50.3 | 99.1 |
| #8 | F | 64 | 58.1 | 99.3 |
We also confirmed that HBZ mRNA was constantly expressed, and a significant increase was observed in the isolated CD4+ T-cells in HBZ-Tg mice (Fig. 2a).
The mRNA expression of ALKBH2 in whole spleen cells and isolated CD4+ T-cells from HBZ-Tg mice was 2.11 and 2.43 times lower than in wildtype mice, respectively, although the differences were not statistically significant (Fig. 2b). Constant expression of ALKBH2 protein was confirmed by western blotting in both wildtype and HBZ-Tg mice, indicating functional mRNA expression of ALKBH2 (Fig. 2c, Supplementary Fig. 3).
We further examined possible relationship between HBZ and ALKBH2 gene regulation in HBZ-Tg mice. As a result, there was no statistical significance but characteristic reduction of pre-mRNA expression of ALKBH2 was discovered in whole spleen cells and isolated CD4+ T-cells from HBZ-Tg mice (by factors of 17.51 and 10.79, respectively, Fig. 2b, right panel). Furthermore, the expression ratio of ALKBH2 mRNA to pre-mRNA was significantly higher in whole spleen cells from HBZ-Tg mice in comparison to that in CD4+ T-cells from HBZ-Tg mice, and in both specimens from wildtype mice, indicating the unbalanced mRNA and pre-mRNA expression of ALKBH2 (Fig. 2d). These observations also suggested that the gene expression of ALKBH2 in HBZ-Tg mice would be regulated by cell-to-cell communication in whole spleen cells including HBZ-expressing CD4+ T-cells.
Analysis of the pre-mRNA expression levels of ALKBH2 in patients infected with HTLV-1 and cultured T-cell lines
We additionally analyzed the pre-mRNA expression of ALKBH2 using same set of clinical specimens with the quantification of ALKBH2 mRNA expression. The pre-mRNA expression of ALKBH2 in the AC and ATL groups was significantly decreased compared to that in the HC group (by factors of 24.18 and 110.46, respectively, Fig. 3a). Notably, the unbalanced mRNA and pre-mRNA expression of ALKBH2 was prominent in the ATL group, because their ALKBH2 mRNA expression was similar levels with the HC group in the ALKBH2 pre-mRNA decreased condition (Fig. 1a and 3a). A decrease in the detection rates of ALKBH2 pre-mRNA was also observed in the AC and ATL groups, whereas ALKBH2 mRNA was mostly detectable (Table 1). Correlation analysis revealed that the mRNA and pre-mRNA expression of ALKBH2 was significantly correlated in the HC group (Fig. 3b). Due to a number of undetected pre-mRNAs, it was difficult to perform a similar correlation analysis in the AC and ATL groups (Table 1).
Finally, we examined the mRNA and pre-mRNA expression of ALKBH2 in three intact T-cell lines (Jurkat, CEM, and Molt4), and five HTLV-1–infected or ATL based T-cell lines (MT1, MT2, MT4, HUT102, and TLOm1). The mRNA and pre-mRNA expressions of ALKBH2 in the HTLV-1–related T-cell lines was significantly decreased compared to that in the intact T-cell lines (by factors of 2.72 and 2.74, respectively, Fig. 3c and 3d). The expression ratio of ALKBH2 mRNA to pre-mRNA was consistent in the examined T-cell lines, and statistical significance was not observed (Fig. 3e). These observations revealed that the unbalanced mRNA and pre-mRNA expression of ALKBH2 was not reproducible in HTLV-1–related T-cell lines. Consistent with the observations in the analysis of HBZ-Tg mice, it was also suggested that cell-to-cell communication in various immune cell populations would be essential to induce the unbalanced mRNA and pre-mRNA expression of ALKBH2.