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OBJECTIVE: Human T lymphotropic virus type I (HTLV-I) may be associated with some connective tissue autoimmune diseases, including systemic lupus erythematosus (SLE). To determine the relationship between HTLV-I infection and SLE, we examined the clinical manifestations of SLE patients with HTLV-I infection. METHODS: Eighty-nine patients with SLE were screened for antibodies to HTLV-I by electrochemiluminescence immunoassay. The presence of HTLV-I proviral sequences in peripheral blood mononuclear cells (PBMC) was determined by real-time polymerase chain reaction (PCR) quantification and Southern blotting analysis. The differences in clinical manifestations between HTLV-I-seropositive and seronegative patients with SLE were analyzed statistically. RESULTS: Fourteen of 89 (15.7%) patients were HTLV-I seropositive. All PBMC samples from 11 patients tested by PCR and 3 samples from 10 patients tested by Southern blotting analysis were positive for HTLV-I-related sequences. The age of HTLV-I-seropositive patients with SLE was significantly higher than that of seronegative patients (median 60 vs 42 yrs; p < 0.0005). The age at onset of SLE in HTLV-I-seropositive patients was also significantly higher than that of seronegative patients (median 45.5 vs 30 yrs; p <0.0005). The lymphocyte count in HTLV-I-seropositive SLE patients was significantly higher than that of seronegative patients (median 1740 vs 1066/µl; p = 0.027). The maintenance dose of prednisolone in HTLV-I-seropositive patients with SLE was significantly lower than that in seronegative patients (median 5 vs 9 mg/day; p = 0.012). CONCLUSION: This is the first report of the differences in clinical manifestations between SLE patients with and without HTLV-I infection. Our results suggest some involvement of HTLV-I in the pathogenesis of SLE.

A rapid, simple and reliable high-performance liquid chromatography (HPLC) column-switching method with UV detection (270 nm) for the simultaneous determination of propentofylline and its metabolites in human and rat sera was developed. The method involves direct injection of serum onto an HPLC column, which contains a shielded hydrophobic stationary phase for the separation of analytes from proteins in serum, and then loading the analytes onto a short octadecylsilylated silica gel (ODS) column using a switching valve. Propentofylline and its three metabolites in serum were separated from the serum components within 30 min after the injection. The detection limits (S/N = 3) of analytes spiked in human and rat sera ranged from 0.08 to 0.57 nmol/mL, and the net volume of serum used was 20 µL. The relative standard deviations for within- and between-day variations using rat serum were less than 4.3 and 5.6%, respectively. The method was used to determine propentofylline and its main metabolites in rat serum after a single intravenous dose of propentofylline (5 mg/kg). Copyright © 1999 John Wiley & Sons, Ltd.

A rapid, simple and reliable high‐performance liquid chromatography (HPLC) column‐switching method with UV detection (270 nm) for the simultaneous determination of propentofylline and its metabolites in human and rat sera was developed. The method involves direct injection of serum onto an HPLC column, which contains a shielded hydrophobic stationary phase for the separation of analytes from proteins in serum, and then loading the analytes onto a short octadecylsilylated silica gel (ODS) column using a switching valve. Propentofylline and its three metabolites in serum were separated from the serum components within 30 min after the injection. The detection limits (S/N= 3) of analytes spiked in human and rat sera ranged from 0.08 to 0.57 nmol/mL, and the net volume of serum used was 20 µL. The relative standard deviations for within‐ and between‐day variations using rat serum were less than 4.3 and 5.6%, respectively. The method was used to determine propentofylline and its main metabolites in rat serum after a single intravenous dose of propentofylline (5 mg/kg). Copyright © 1999 John Wiley & Sons, Ltd.

Adult T-cell leukemia/lymphoma (ATLL) develops after infection with human T-cell leukemia virus-1 (HTLV-1) with a long latent period. The negative regulatory programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) pathway has been implicated in the induction of CTL exhaustion during chronic viral infection and tumor escape from host immunity. We have previously reported decreased frequency and function of HTLV-1 Tax-specific CD8+ T-cell in ATLL patients due to insufficient cytolytic effector molecule. To determine whether the PD-1/PD-L1 pathway is involved in the immunosuppressive state in HTLV-1-infected individuals and immune evasion in ATLL patients, we examined PD-1/PD-L1 expression in asymptomatic HTLV-1 carriers (ACs) and ATLL patients. PD-1 expression was significantly up-regulated on lymphocytes in ACs and ATLL patients compared to non-HTLV-1-infected individuals. The rates of PD-1 expression on HTLV-1-specific CTL in ACs and ATLL patients were similarly high (p = 0.32) although PD-1 expression was significantly up-regulated on total lymphocytes, cytomegalovirus- and EBV-specific CTL in ATLL patients compared to ACs and non-HTLV-1-infected individuals (Figure1, p < 0.01). This may be one reason why HTLV-1-infected cells are not eradicated even in the presence of anti-HTLV-1 antigen-specific CTL in the carrier state. Primary ATLL cells in 15.0% of ATLL patients and an HTLV-1-infected cell line expressed PD-L1, while few lymphocytes from ACs were positive for this ligand. Anti-PD-L1 antagonistic antibody restored HTLV-1-specific CTL functions (IFN-γ, TNF-α, CD107a) ex vivo (Figure2). These observations suggest that the immunoregulatory PD-1/PD-L1 pathway is operative during persistent viral infection, and the pathway may induce the exhausted phenotype of CTL in HTLV-1 infected individuals, which may lead to immunodeficiency and immune evasion in ACs and ATLL patients. Furthermore, reactivation of exhausted T cells by blocking the PD-1/PD-L1 may be an effective immunological strategy for the clearance of persistent HTLV-1 infection and the treatment of ATLL. FIGURE 1. PD-1 expression on virus-specific CD8+ T lymphocytes in ACs and ATLL patients. Percentage of PD-1 expression on HTLV-1-, CMV-, and EBV-specific CD8+ lymphocytes in AC, ATLL, and HD. Horizontal bars indicate the mean percentage of PD-1 positive cells. *P < 0.01; **P < 0.05 (Significant differences by Mann-Whitney U test). Numbers are means ± SD. FIGURE 1. PD-1 expression on virus-specific CD8+ T lymphocytes in ACs and ATLL patients. Percentage of PD-1 expression on HTLV-1-, CMV-, and EBV-specific CD8+ lymphocytes in AC, ATLL, and HD. Horizontal bars indicate the mean percentage of PD-1 positive cells. *P < 0.01; **P < 0.05 (Significant differences by Mann-Whitney U test). Numbers are means ± SD. Figure 2. Blocking the PD-1/PD-L1 pathway increases INF-γ production in HTLV-1-specific cytolytic cell activation. Percentage of intracellular IFN-γ on HTLV-1-specific CD8+ T lymphocytes without peptide, in the presence of peptide and with anti-PD-L1 treatment. *P < 0.05 (Significant differences by Wilcoxon matched pairs tests). Figure 2. Blocking the PD-1/PD-L1 pathway increases INF-γ production in HTLV-1-specific cytolytic cell activation. Percentage of intracellular IFN-γ on HTLV-1-specific CD8+ T lymphocytes without peptide, in the presence of peptide and with anti-PD-L1 treatment. *P < 0.05 (Significant differences by Wilcoxon matched pairs tests).

Adult T-cell leukemia/lymphoma (ATLL) develops after infection with human T-cell leukemia virus-1 (HTLV-1) with a long latent period. The negative regulatory programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) pathway has been implicated in the induction of CTL exhaustion during chronic viral infection and tumor escape from host immunity. We have previously reported decreased frequency and function of HTLV-1 Tax-specific CD8+ T-cell in ATLL patients due to insufficient cytolytic effector molecule. To determine whether the PD-1/PD-L1 pathway is involved in the immunosuppressive state in HTLV-1-infected individuals and immune evasion in ATLL patients, we examined PD-1/PD-L1 expression in asymptomatic HTLV-1 carriers (ACs) and ATLL patients. PD-1 expression was significantly up-regulated on lymphocytes in ACs and ATLL patients compared to non-HTLV-1-infected individuals. The rates of PD-1 expression on HTLV-1-specific CTL in ACs and ATLL patients were similarly high (p= 0.32) although PD-1 expression was significantly up-regulated on total lymphocytes, cytomegalovirus- and EBV-specific CTL in ATLL patients compared to ACs and non-HTLV-1-infected individuals (Figure1, p< 0.01). This may be one reason why HTLV-1-infected cells are not eradicated even in the presence of anti-HTLV-1 antigen-specific CTL in the carrier state. Primary ATLL cells in 15.0% of ATLL patients and an HTLV-1-infected cell line expressed PD-L1, while few lymphocytes from ACs were positive for this ligand. Anti-PD-L1 antagonistic antibody restored HTLV-1-specific CTL functions (IFN-γ, TNF-α, CD107a) ex vivo(Figure2). These observations suggest that the immunoregulatory PD-1/PD-L1 pathway is operative during persistent viral infection, and the pathway may induce the exhausted phenotype of CTL in HTLV-1 infected individuals, which may lead to immunodeficiency and immune evasion in ACs and ATLL patients. Furthermore, reactivation of exhausted T cells by blocking the PD-1/PD-L1 may be an effective immunological strategy for the clearance of persistent HTLV-1 infection and the treatment of ATLL.

At present, allogeneic hematopoietic stem cell transplantation (HCST) has been an only curative modality for the treatment of adult T cell leukemia/lymphoma (ATLL). Disease relapse and GVHD are the major causes of mortality after allo-HSCT for ATLL. To achieve better outcome, early detection of disease relapse after allo-HSCT is absolutely crucial for decision-making of interventions such as donor lymphocyte infusion and/or adjuvant chemotherapy, while tumor burden still remains in the level of minimal residual disease (MRD). Serum soluble interleukin-2 receptor alpha (sIL-2R) has been known as a marker of ATLL tumor burden in affected patients, however it increases in patients with acute and chronic GVHD as well. Here we propose a new strategy to distinguish disease progression of ATLL and GVHD at an early stage by measuring serum levels of soluble CD30 antigen (sCD30) and sIL-2R. The surface CD30 is normally expressed by activated B or T cells. It has been known that Hodgkin cells, anaplastic large cell lymphoma and ATLL cells also express surface CD30. The soluble form of CD30 is produced by metalloprotease cleavage. Acute type ATLL patients whose tumor cells express surface CD30 (CD30+ATLL), who underwent allo-HSCT, were included in this study. As control, the serum levels of sCD30 were measured in a healthy donor, an asymptomatic HTLV-I carrier and acute type ATLL patients whose tumor cell were negative for surface CD30 (CD30−ATLL), all were below measurable range. In addition, the level of sCD30 was undetectable in a CD30− ATLL patient with grade II gut acute GVHD as well. Confirming these low backgrounds, both of sCD30 and sIL2R levels were sequentially measured from induction chemotherapy and throughout allo-HSCT. During chemotherapy, the levels of sCD30 and sIL2R are well-correlated along with tumor burden as previously reported, and both markers increase at disease relapse after allo-HSCT as well. In contrast, during GVHD, sIL-2R levels increase along with GVHD status, while sCD30 remained lower or undetectable. In conclusion, serum levels of sCD30 and sIL-2R may predict not only disease progression at an early stage after allo-HSCT, but also distinguish that from GVHD. These findings may help to introduce an early intervention for ATLL progression with DLI and/or adjuvant chemotherapy at the level of minimal residual disease. Figure Figure

At present, allogeneic hematopoietic stem cell transplantation (HCST) has been an only curative modality for the treatment of adult T cell leukemia/lymphoma (ATLL). Disease relapse and GVHD are the major causes of mortality after allo-HSCT for ATLL. To achieve better outcome, early detection of disease relapse after allo-HSCT is absolutely crucial for decision-making of interventions such as donor lymphocyte infusion and/or adjuvant chemotherapy, while tumor burden still remains in the level of minimal residual disease (MRD). Serum soluble interleukin-2 receptor alpha (sIL-2R) has been known as a marker of ATLL tumor burden in affected patients, however it increases in patients with acute and chronic GVHD as well. Here we propose a new strategy to distinguish disease progression of ATLL and GVHD at an early stage by measuring serum levels of soluble CD30 antigen (sCD30) and sIL-2R. The surface CD30 is normally expressed by activated B or T cells. It has been known that Hodgkin cells, anaplastic large cell lymphoma and ATLL cells also express surface CD30. The soluble form of CD30 is produced by metalloprotease cleavage. Acute type ATLL patients whose tumor cells express surface CD30 (CD30+ATLL), who underwent allo-HSCT, were included in this study. As control, the serum levels of sCD30 were measured in a healthy donor, an asymptomatic HTLV-I carrier and acute type ATLL patients whose tumor cell were negative for surface CD30 (CD30−ATLL), all were below measurable range. In addition, the level of sCD30 was undetectable in a CD30−ATLL patient with grade II gut acute GVHD as well. Confirming these low backgrounds, both of sCD30 and sIL2R levels were sequentially measured from induction chemotherapy and throughout allo-HSCT. During chemotherapy, the levels of sCD30 and sIL2R are well-correlated along with tumor burden as previously reported, and both markers increase at disease relapse after allo-HSCT as well. In contrast, during GVHD, sIL-2R levels increase along with GVHD status, while sCD30 remained lower or undetectable. In conclusion, serum levels of sCD30 and sIL-2R may predict not only disease progression at an early stage after allo-HSCT, but also distinguish that from GVHD. These findings may help to introduce an early intervention for ATLL progression with DLI and/or adjuvant chemotherapy at the level of minimal residual disease.

[Background] Although the anti-HTLV-I Tax protein-specific CTL (anti-Tax-CTL) have been reported to play a crucial role for the Graft-v.s.-ATL effect (GvATL) in allo-SCT for ATLL, whether or not anti-Tax-CTL is the real weapon of the GvATL still remains undetermined, because large part of primary ATLL cells do not express Tax protein via several gene alternations, and chronic GVHD seems highly correlated to the longer survival.

[Background] Although the anti-HTLV-I Tax protein-specific CTL (anti-Tax-CTL) have been reported to play a crucial role for the Graft-v.s.-ATL effect (GvATL) in allo-SCT for ATLL, whether or not anti-Tax-CTL is the real weapon of the GvATL still remains undetermined, because large part of primary ATLL cells do not express Tax protein via several gene alternations, and chronic GVHD seems highly correlated to the longer survival. [Study design] To explore how significantly anti-Tax CTL contribute to GvATL, we investigated the correlation between anti-Tax-CTL and other immunological parameters after RIST without T-cell depletion (TCD) from HLA-mismatched relative donors for patients with refractory ATLL and who lack suitable donors. All 4 cases did not have KIR incompatibility in the direction of GVHD. Three cases of 4 received PBSC from HTLV-I negative donors, and survived longer than 7 months. About these long survivors, we longitudinally monitored immunological parameters including Tax-protein derived peptides-specific CD8+ T-cells by tetramer assay in accordance with HTLV-I proviral load in the periphery determined by quantitative-PCR as a surrogate marker of residual ATLL. [Case report] A 54-year-old woman with ATLL received non-TCD RIST from HTLV-I negative and HLA-haploidentical her son (recipient;HLA-A;*0201,-, B;*54,*40*11, Cw;*0102,-,DRB1;*04,*11, donor; HLA-A;*11, *2402,B;*54,-,Cw;*0102,-,DRB1;*04,-). She achieved complete donor chimerism on day 15. By day 30 HTLV-I proviral load became undetectable (less than 0.5 copies/1x10e3 PBMC), and she achieved CR. 6 months later, her ATLL relapsed with several skin nodules. At relapse, donor-derived HLA-A*2402 restricted HTLV-I Tax 301-309-specific CTL were documented (0.11% of CD8) for the first time. Three weeks after 1x10e6/kg of CD3 cells of DLI, she suffered from GVHD (skin; stage 3), and those anti-Tax CTL further increased upto 0.38% of CD8 in number. These CTL bore cytoplasmic granzyme B, and produced IFN-γ in response to identical peptide in vitro. Interestingly those donor-derived anti-Tax CTL were unable to kill recipient ATLL cells, because recipient cells were negative for HLA-A*2402. Eventually her relapsed ATLL was well controlled mainly by single course of DLI. Additionally the other 2 of 3 long survivors (who had either HLA-A*0201 or A*2402 molecule) did not show any-anti-Tax-CTL throughout their clinical courses. One patient surviving longer than 20 months who had chronic GVHD (extended) and shared HLA-A*2402 molecule with his donor, that theoretically suggests the more feasible condition for the induction of anti-Tax CTL. [Discussion] In the setting of HLA-mismatched SCT, our observation clearly demonstrated that anti-Tax-specific CTL unable to kill recipient ATLL cells could be induced. Alloantigens and undetermined ATLL-related tumor antigens other than HTLV-I Tax protein should be equally considered for the better understanding of GvATL and improve the outcome of allo-SCT against ATLL.