HTLV-1 Triggers HIAP-1 and cFLIP Expression in CD8+ T-Cells While Impairing DNA Repair in CD4+ T-Cells.

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATLL), a CD4+ mature lymphoproliferation that is preceded by the persistent clonal expansion of CD4+ and CD8+ infected lymphocytes. It depends on the expression of the viral oncogene tax that is restricted to the preleukemic phase of the infection. The virus prevents cell death in CD8+ lymphocytes whereas recruiting CD4+ lymphocytes into the cell cycle (J Clin Invest. 2006; 116:974–983). In vitro, Tax+ cell lines overexpress the antiapoptotic genes cFLIP (Genes to Cells 2006; 11:177–191; Blood 2006; 107:4491–4499), HIAP-1 (Blood 2006; 107:3933–3939), and TRAIL (J Virol. 2005; 79:1367–1378). Furthermore HTLV-1 causes defects on DNA repair in cell lines while cloned HTLV- 1 positive tax-expressing CD4+ lymphocytes cumulate morphologic cellular defects characteristic of genetic instability. Here we explored certain mechanisms underlying apoptosis inhibition and genetic instability in CD4+ and CD8+ cells. 56 clones harboring distinct and unique TCRs, including 40 uninfected (27 CD4+, 13 CD8+) and 16 infected clones (10 CD4+, 6 CD8+), were generated by limiting dilution cloning of PBMCs deriving from an infected individual without malignancy. To investigate cell death inhibition, we first analyzed the expression of the genes involved in apoptosis. Microarrays confirmed by real-time RT-PCR revealed that upon infection, CD8+ cells specifically underexpressed FasL while overexpressing cFLIP-l (p=0.012) and HIAP-1 (p=0.0063). cFLIP-s expression was not influenced by HTLV-1 infection in CD8+ and CD4+ cells. In contrast to CD8+ cells, infected and uninfected CD4+ lymphocytes displayed the same amount of cFLIP-l and HIAP-1 transcripts. HIAP-1 and tax expression correlated in both CD4+ (p=0.002) and CD8+ cells (p=0.001) whereas a negative correlation between tax and cFLIP-s characterized CD8+ cells (p=0.043). Anti-Fas antibody and recombinant Fas ligand triggered cell death in all CD4+ clones, in uninfected CD8+ clones and in 4/6 infected CD8+ clones. Thus the defect in FasL expression, that is restricted to infected CD8+ clones, seems accounting for preventing cell death in ~67% of HTLV-1+-CD8+ clones whereas an additional defect downstream the Fas pathway is involved for the remaining HTLV-1+-CD8+ clones. We investigated DNA repair mechanisms through comet assays in the same cloned lymphocytes. We analyzed 4945 cells before and after 10 Gy irradiation. Infected and uninfected CD8+ clones displayed the same proportion of cells with repaired DNA. CD4+ clones displayed 2 significantly distinct patterns of time-dependent DNA repair. Clones with no or moderate tax expression displayed no significant difference in time-dependent DNA repair when compared to uninfected CD4+ clones. Before irradiation, the pattern of DNA repair of tax-expressing CD4+ clones was indistinguishable from that of control cells but at 2, 6, and 25 hours after irradiation, the mean proportion of infected CD4+ cells with repaired DNA was 9, 2, and 1.2 fold lower than that of uninfected cells. A significant correlation was observed between tax expression and the tail moment for the 1454 cells deriving from HTLV-1+CD4+ clones (p<10–4); in contrast, no significant correlation could be observed for the 1000 cells deriving from CD8+HTLV-1+ clones. These data indicate that tax expression impairs DNA repair in naturally infected CD4+Tax+ cells whereas HTLV-1 infection does not influence DNA repair in CD8+ cells. We next investigated the expression of the genes involved in DNA repair in cloned lymphoid cells from infected patients. Microarray analysis revealed that upon infection, there was no significant transcriptional deregulation of these genes in CD8+ cells; in contrast, the expression of 17 genes directly or indirectly involved in DNA repair was found to be significantly deregulated in CD4+HTLV-1+Tax+ clones. The deregulation of some of these genes was consistent with present comet findings. In conclusion, present results suggest that 1°) the CD8+-restricted anti-apoptotic effect of HTLV-1 relies on a tax-dependent overexpression of HIAP-1, a tax-independent overexpression cFLIP-l, together with a defect in the Fas pathway with evidence for clonal heterogeneity; 2°) upon tax expression, CD4+ but not CD8+ HTLV-1 naturally infected cells are phenotypically and genotypically impaired for DNA repair.