HTLV-1 propels untransformed CD4 lymphocytes into the cell cycle while protecting CD8 cells from death

Abstract

Human T cell leukemia virus type 1 (HTLV-1) infects both CD4+ and CD8+ lymphocytes, yet it induces adult T cell leukemia/lymphoma (ATLL) that is regularly of the CD4+ phenotype. Here we show that in vivo infected CD4+ and CD8+ T cells displayed similar patterns of clonal expansion in carriers without malignancy. Cloned infected cells from individuals without malignancy had a dramatic increase in spontaneous proliferation, which predominated in CD8+ lymphocytes and depended on the amount of tax mRNA. In fact, the clonal expansion of HTLV-1-positive CD8+ and CD4+ lymphocytes relied on 2 distinct mechanisms--infection prevented cell death in the former while recruiting the latter into the cell cycle. Cell cycling, but not apoptosis, depended on the level of viral-encoded tax expression. Infected tax-expressing CD4+ lymphocytes accumulated cellular defects characteristic of genetic instability. Therefore, HTLV-1 infection establishes a preleukemic phenotype that is restricted to CD4+ infected clones.

Figure 1. Proviral loads (A ) and clonality (B ) of HTLV-1–positive cells in highly purified CD4⁺ CD8^– and CD4^– CD8⁺ lymphocytes from HAM/TSP patients DO45 and SI88 (see Table 1).

The clonal distribution of integrated HTLV-1 sequences within the DNA of CD4⁺ and CD8⁺ cells was assessed by quadruplicate IPCR experiments as described in Methods. Numbers in parentheses represent the proportion of infected cells as evidenced by real-time quantitative PCR. M, molecular weight marker. C91PL is an HTLV-1 cell line used as positive control.

Figure 2. Multiplex PCR-γ denaturing gradient gel electrophoresis analysis of TCR rearrangement in human T cell clones.

Each lane represents the migration of the PCR product of 1 clone. Clones are identified by their unique clone number (bottom). Samples with 1 (lanes 8 and 9) or 2 (lanes 2–4, 6, and 7) signals correspond with mono- or biallelic rearrangements, respectively. Sample with 3 bands (lane 5) corresponds to a biallelic rearrangement with a heteroduplex. A polyclonal sample is shown on lane 1; lane 10 correspond to a negative control (NC).

Figure 3. Infected CD4⁺ lymphocytes that express tax display both nuclear abnormalities and cytokinesis defects.

Clones 13 (A), 15 (C), 57 (B), 67 (F–H), and 68 (D and E) were cytospun, fixed, and stained with May-Grünwald and Giemsa. Label notation indicates the unique clone number, the phenotype, the infected or uninfected nature of the cells, and the corresponding amount of tax mRNA, measured in arbitrary units. Compared with uninfected CD8⁺ clones (A), HTLV-1–positive CD8⁺ clones (B) displayed enlarged cells with enlarged cytoplasm. Compared with uninfected CD4⁺ clones (C), HTLV-1–positive CD4⁺ clones (D and E) frequently display giant cells, some harboring enlarged nucleus (arrows). Multinucleated cells frequently displayed nuclei of heterogeneous sizes, as identified by arrowheads in E and G and shown at higher magnification in F. Chromatin bridges connecting nuclei in multinucleated cells are identified by smaller arrows in G and H. The distribution of cellular nuclearity according to phenotype and infectious status is shown in I. *P < 0.001; **P < 0.0001, infected vs. uninfected cells. Magnification, ×40 (A–E), ×100 –H).

Figure 4. Altered growth kinetics of CD4⁺ and CD8⁺ HTLV-1–infected clones.

Sixty-six clones were analyzed. (A) Increased growth of HTLV-1–infected clones. (B) Increased growth of CD4⁺ and CD8⁺ HTLV-1–infected clones. (C) Kinetics of IL-2–dependent clonal proliferation of infected and uninfected clones. (D) Kinetics of IL-2–dependent clonal proliferation of infected and uninfected CD4⁺ and CD8⁺ clones. (E) Spontaneous clonal proliferation of infected and uninfected CD4⁺ and CD8⁺ clones.

Figure 5. HTLV-1 recruits untransformed CD4⁺ lymphocytes into the cell cycle while preventing CD8⁺ cells from cell death.

CD4⁺ and CD8⁺ clones (66 clones) were analyzed at day 6 from PHA stimulation for cell cycle (A and B) and apoptosis (A and C). ^#P = 0.001, ^##P = 0.04 versus uninfected cells of the same phenote.