Masakazu Tanaka

Spontaneous lymphoproliferation (SP) is an ex vivo phenomenon where lymphocytes proliferate without exogenous stimulation in peripheral blood mononuclear cells (PBMCs) from patients with human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). SP is thought to reflect spinal cord pathology in HAM/TSP, but its cellular mechanisms remain unclear. PBMCs from 30 patients with HAM/TSP, 12 asymptomatic HTLV-1 carriers, and 8 healthy controls (HCs) were labeled with a proliferation tracer and cultured for 6 days without exogenous stimulation. Flow cytometry was used to identify T-cell subsets, HTLV-1-infected cells, and HTLV-1-specific cytotoxic T lymphocytes (CTLs). Additional analyses included CTL antigen specificity, proviral load (PVL), phenotypic profiling (differentiation, activation, exhaustion markers), and cytokine/chemokine quantification in culture supernatants. CD8⁺ T cells exhibited greater proliferation than CD4⁺ T cells during SP. Among proliferating CD4⁺ T cells, 38.15% were CADM1+, 61.84% were Tax+, and PVL analysis indicated that nearly all proliferating CD4⁺ T cells were HTLV-1 infected. The frequency of HTLV-1 Tax 301-309-specific CTLs among CD8⁺ T cells increased from 3.81% to 17.67% during SP. After a subsequent resting phase, IFN-γ responses were detected in 37.38% of CD8⁺ T cells to a Tax peptide mixture and in 33.42% to an HTLV-1 bZIP factor peptide mixture, together accounting for over 70% of proliferating CD8⁺ T cells. Sorted CD4⁺ T cells proliferated independently, whereas sorted CD8⁺ T cells did not; however, coculture with CD4⁺ T cells restored CD8⁺ T-cell proliferation. Proliferating infected cells exhibited a Th1 phenotype, and most proliferating T cells coexpressed activation and exhaustion markers with memory phenotypes. IL-6 and IFN-γ levels were significantly elevated in culture supernatants from patients with HAM/TSP compared with HCs. IFN-γ levels in SP supernatants correlated with CSF PVL. SP responses in HTLV-1 carriers were attenuated, with reduced proliferation compared with patients with HAM/TSP. SP is driven by HTLV-1-infected CD4⁺ T cells, which are essential for sustaining HTLV-1-specific CD8⁺ CTL expansion. This phenomenon appears to recapitulate certain immunologic features of neuroinflammation in HAM/TSP. This ex vivo model may help evaluate therapies targeting HTLV-1-infected cells and immune-mediated pathology.

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neuroinflammatory disease with no effective treatment. In this study, we investigated whether dimethyl fumarate (DMF), an immunomodulatory agent approved for treating multiple sclerosis, exerts therapeutic effects relevant to HAM/TSP. Peripheral blood mononuclear cells (PBMCs) from 16 people living with HAM/TSP were used to evaluate the effects of DMF on cell viability, spontaneous proliferation, inflammatory cytokine production and HTLV-1 proviral load (PVL). DMF significantly inhibited lymphocyte proliferation in a concentration-dependent manner, with reductions of 42.1% at 10 µM, 56.3% at 25 µM, 60.6% at 50 µM and 69.9% at 100 µM. This suppressive effect was particularly evident in CD8+ T cells, CD4+ T cells and HTLV-1-infected CD4+ T cells. Furthermore, DMF reduced the production of interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) released from these proliferating cells. A reduction in PVL was also observed in a subset of ex vivo PBMC cultures derived from individuals with HAM/TSP exhibiting high viral proliferative activity. These results suggest that DMF suppresses pathogenic immune activation in HAM/TSP and may therefore represent a promising therapeutic candidate for this disabling neuroinflammatory disorder.

HTLV‐1 is a retrovirus associated with adult T cell leukemia/lymphoma (ATL) and inflammatory diseases, including HTLV‐1‐associated myelopathy (HAM) and HTLV‐1‐associated bronchopneumonopathy (HAB). Although pulmonary complications are common in HTLV‐1‐associated diseases, the underlying mechanisms remain unclear. We compared HTLV‐1 proviral load (PVL) and chemokine receptor expression in bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMCs) from asymptomatic carriers (ACs) and patients with HAB, HAM, or ATL. T cell subsets were analyzed by flow cytometry, and the expression of CXCR3 and CXCL10 in lung tissue was assessed by immunohistochemistry. HTLV‐1 proviral DNA was detectable in BAL cells not only from HAB and ATL cases with pulmonary involvement, but also from some ACs and HAM cases without clinical respiratory symptoms, suggesting subclinical pulmonary infiltration. BAL samples from HAB and ATL showed increased CD8 + T cell frequency. Both CD4+ and CD8 + T cells in BAL expressed higher CXCR3 than their PBMC counterparts, whereas CCR4 and CXCR5 were not elevated. CADM1 + CD4 + T cells in BAL also exhibited higher CXCR3 than in PBMCs, and CXCR3+ frequencies were similar between CADM1+ and CADM1‐ CD4 + T cells within BAL, indicating that enhanced CXCR3 expression was largely independent of infection status. Histology revealed CD3+ mononuclear cell infiltration in alveolar septa and peribronchiolar regions in HAB and HAM, consistent with T cell–mediated alveolitis and bronchiolitis, and CXCL10 expression was elevated in infiltrated lesions. Collectively, these findings implicate the CXCR3/CXCL10 axis as a common pathway for pulmonary T cell recruitment in HTLV‐1‐associated diseases.

The transcription factor E2F is the principal target of the tumor suppressor pRB. In almost all cancers, pRB function is disabled due to oncogenic changes, leading to enhanced E2F activity, thereby facilitating aberrant cell proliferation. Enhanced E2F activity has been utilized to drive gene expression preferentially in cancer cells using E2F target promoters, such as the E2F1 promoter. However, these promoters are also activated by physiological E2F activity in normal proliferating cells, resulting in gene expression in normal proliferating cells. In contrast, promoters of tumor suppressor genes, such ARF and TAp73, are activated by deregulated E2F activity, induced by loss of pRB control, but not by physiological E2F activity, induced by growth stimulation, thereby providing a mechanism to drive expression specifically in cancer cells. Here we show artificial promoters, in which E2F-responsive elements of the TAp73 gene are tandemly connected to the ARF core promoter, exhibited higher cancer cell specificity than E2F1, hTERT, or ARF promoters. Moreover, adenoviruses driving a cytotoxic gene using these artificial promoters showed cancer cell-specific cytotoxicity and inhibited tumor growth in a xenograft mouse model. These results indicate utility of tumor suppressor gene promoter elements to drive gene expression specifically in cancer cells.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with adult T-cell leukemia (ATL), a hematological malignancy, and HTLV-1-associated myelopathy (HAM), a progressive neurological disorder. HTLV-1 predominantly infects CD4+ T cells in vivo. The T-cell receptor (TCR)/CD3 complex on CD4+ helper T cells plays a pivotal role in immune responses by recognizing antigens and facilitating coordination with other immune cells. Dysfunction of the TCR/CD3 complex may impair immune function. Although CD3 downregulation has been identified as a characteristic of ATL cells, it remains uncertain whether a similar downregulation occurs in HTLV-1-infected cells from HAM patients. We hypothesized that HTLV-1 infection leads to TCR and CD3 downregulation, contributing to immune dysfunction in HAM patients. To test this hypothesis, we analyzed TCR/CD3 expression, TCR signaling, and immune responses in HTLV-1-infected cells from HAM patients. Intracellular HTLV-1 Tax detection revealed that HTLV-1 preferentially targets CD4+ over CD8+ T cells. CD3 and TCR expression levels were significantly lower in CD4+ T cells from HAM patients compared to healthy controls. Furthermore, HTLV-1-infected cells exhibited markedly reduced CD3 and TCR expression compared to uninfected cells. Impairments in TCR signaling, assessed through Lck and ZAP70 phosphorylation upon CD3 stimulation, were observed in CD4+ T cells from HAM patients compared to those from healthy controls. Notably, this reduction in TCR signaling was more pronounced in HTLV-1-infected CD4+ T cells than in uninfected CD4+ T cells in HAM patients. Additionally, cytomegalovirus (CMV)-specific CD4+ T cells detected by an addition of CMV antigens demonstrated reduced interferon-γ production in HTLV-1-infected cells compared to their uninfected counterparts. These findings suggest that TCR/CD3 downregulation and impaired TCR signaling contribute to immune dysfunction in HTLV-1-infected CD4+ T cells. As CD4+ T cells play a central role in immune responses, this mechanism may partially explain the cellular immune dysfunction to other pathogens observed in HAM patients.

Increased human T-cell leukemia virus type 1 (HTLV-1) proviral load (PVL) is a significant risk factor for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is controversy surrounding whether HTLV-1-specific cytotoxic T lymphocytes (CTLs) are beneficial or harmful to HAM/TSP patients. Recently, HTLV-1 Tax 301–309 has been identified as an immunodominant epitope restricted to HLA-A*2402. We investigated whether HLA-A*24 reduces HTLV-1 PVL and the risk of HAM/TSP using blood samples from 152 HAM/TSP patients and 155 asymptomatic HTLV-1 carriers. The allele frequency of HLA-A*24 was higher in HAM/TSP patients than in asymptomatic HTLV-1 carriers (72.4% vs. 58.7%, odds ratio 1.84), and HLA-A*24-positive patients showed a 42% reduction in HTLV-1 PVL compared to negative patients. Furthermore, the PVL negatively correlated with the frequency of Tax 301–309-specific CTLs. These findings are opposite to the effects of HLA-A*02, which reduces HTLV-1 PVL and the risk of HAM/TSP. Therefore, we compared the functions of CTLs specific to Tax 11–19 or Tax 301–309, which are immunodominant epitopes restricted to HLA-A*0201 or HLA-A*2402, respectively. The maximum responses of these CTLs were not different in the production of IFN-γ and MIP-1β or in the expression of CD107a—a marker for the degranulation of cytotoxic molecules. However, Tax 301–309-specific CTLs demonstrated 50-fold higher T-cell avidity than Tax 11–19-specific CTLs, suggesting better antigen recognition at low expression levels of the antigens. These findings suggest that HLA-A*24, which induces sensitive HTLV-1-specific CTLs, increases the risk of HAM/TSP despite reducing HTLV-1 PVL.

The centrosome is involved in cytoplasmic microtubule organization during interphase and in mitotic spindle assembly during cell division. Centrosome amplification (abnormal proliferation of centrosome number) has been observed in several types of cancer and in precancerous conditions. Therefore, it is important to elucidate the mechanism of centrosome amplification in order to understand the early stage of carcinogenesis. Primary cells could be used to better understand the early stage of carcinogenesis rather than immortalized cells, which tend to have various genetic and epigenetic changes. Previously, we demonstrated that a poly(ADP-ribose) polymerase (PARP) inhibitor, 3-aminobenzamide (3AB), which is known to be nontoxic and nonmutagenic, could induce centrosome amplification and chromosomal aneuploidy in CHO-K1 cells. In this study, we compared primary mouse embryonic fibroblasts (MEF) and immortalized MEF using 3AB. Although centrosome amplification was induced with 3AB treatment in immortalized MEF, a more potent PARP inhibitor, AG14361, was required for primary MEF. However, after centrosome amplification, neither 3AB in immortalized MEF nor AG14361 in primary MEF caused chromosomal aneuploidy, suggesting that further genetic and/or epigenetic change(s) are required to exhibit aneuploidy. The DNA-damaging agents doxorubicin and γ-irradiation can cause cancer and centrosome amplification in experimental animals. Although doxorubicin and γ-irradiation induced centrosome amplification and led to decreased p27Kip protein levels in immortalized MEF and primary MEF, the phosphorylation ratio of nucleophosmin (Thr199) increased in immortalized MEF, whereas it decreased in primary MEF. These results suggest that there exists a yet unidentified pathway, different from the nucleophosmin phosphorylation pathway, which can cause centrosome amplification in primary MEF.

Human T-cell leukemia virus-1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a slowly progressive neurological disease that arises from HTLV-1 infection. Pathologically, the condition is characterized by diffuse myelitis, which is most evident in the thoracic spinal cord. Clinical manifestations of the infectious disease, HAM/TSP, are empirically known to include weakness of the proximal muscles of the lower extremities and atrophy of the paraspinal muscles, which is characteristic of the distribution of disturbed muscles usually seen in muscular diseases, except that the upper extremities are almost normal. This unique clinical presentation is useful information for physicians and physical therapists involved in diagnosing and rehabilitating patients with HAM/TSP, as well as critical information for understanding the pathogenesis of HAM/TSP. However, the precise pattern of muscle involvement in this condition has yet to be reported. The purpose of this study was to identify the muscles affected by HAM/TSP in order to understand the pathogenesis of HAM/TSP as well as to aid in the diagnosis and rehabilitation of HAM/TSP. A retrospective review of medical records was conducted on 101 consecutively admitted patients with HAM/TSP at Kagoshima University Hospital. Among 101 patients with HAM/TSP, all but three had muscle weakness in the lower extremities. Specifically, the hamstrings and iliopsoas muscle were the most frequently affected in over 90% of the patients. Manual muscle testing (MMT) revealed that the iliopsoas was the weakest of the muscles assessed, a consistent feature from the early to advanced stages of the disease. Our findings demonstrate a unique distribution of muscle weakness in HAM/TSP, with the proximal muscles of the lower extremities, particularly the iliopsoas muscle, being the most frequently and severely affected.

Human T lymphotropic virus type 1–assoicated (HTLV-1–associated) myelopathy/tropical spastic paraparesis (HAM/TSP) is a neuroinflammatory disease caused by the persistent proliferation of HTLV-1–infected T cells. Here, we performed a T cell receptor (TCR) repertoire analysis focused on HTLV-1–infected cells to identify and track the infected T cell clones that are preserved in patients with HAM/TSP and migrate to the CNS. TCRβ repertoire analysis revealed higher clonal expansion in HTLV-1–infected cells compared with noninfected cells from patients with HAM/TSP and asymptomatic carriers (ACs). TCR clonality in HTLV-1–infected cells was similar in patients with HAM/TSP and ACs. Longitudinal analysis showed that the TCR repertoire signature in HTLV-1–infected cells remained stable, and highly expanded infected clones were preserved within each patient with HAM/TSP over years. Expanded HTLV-1–infected clones revealed different distributions between cerebrospinal fluid (CSF) and peripheral blood and were enriched in the CSF of patients with HAM/TSP. Cluster analysis showed similarity in TCRβ sequences in HTLV-1–infected cells, suggesting that they proliferate after common antigen stimulation. Our results indicate that exploring TCR repertoires of HTLV-1–infected cells can elucidate individual clonal dynamics and identify potential pathogenic clones expanded in the CNS.

Japan is one of the world's highly endemic areas for human T cell leukemia virus type 1 (HTLV-1), and it is known that the infection rate of HTLV-1 increases with age. The infection rate among the elderly has been estimated based on data from blood donors under the age of 65, and the actual number and rate of infection among the elderly are unknown. Data of 26,090 preoperative HTLV-1 screening tests conducted at Kagoshima University Hospital from 2001 to 2020, including 2726 HTLV-1-positive patients, were used for calculating the decadal infection rates for the year of birth. Estimated infection rates by birth year and demographic tables were used to estimate the current number of infected people in Kagoshima. The estimated total numbers of people infected with HTLV-1 in Kagoshima prefecture were 139,436 in 2005 and 80,975 in 2019. The infection rate increased with age for both men and women, reaching 17.3% for women born before the 1920s. Next, we tried to clarify whether the increase in infection rates with age was due to post-school age infections. The age of birth with the greatest increase in infection rate after 10 years was women born in the 1970s, and the increase in infection rate was only 0.98%, which is not a statistically significant increase. The number of infected people in Kagoshima was >80,000 in 2019. No data were available in this study to point to the involvement of horizontal transmission after school age in the high infection rate among the elderly. The high infection rate among the elderly is thought to have been high even when they were infants.