Flow cytometry evaluation of the T-cell receptor Vbeta repertoire among human T-cell lymphotropic virus type-1 (HTLV-1) infected individuals: effect of interferon alpha therapy in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP)

Human T-cell lymphotropic virus type-1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is chronic inflammatory disease of the spinal cord characterized by perivascular lymphocytic cuffing and parenchymal lymphocytic infiltration. In this study using flow cytometry, we have investigated the T-cell receptor (TCR) Vbeta repertoire of peripheral blood T lymphocytes in 8 HAM/TSP patients, 10 HTLV-1 infected healthy carriers, and 11 uninfected healthy controls to determine if there is a biased usage of TCR Vbeta. We found that TCR Vbeta7.2 was under-utilized and Vbeta12 was over-utilized in CD4+ T cells of HTLV-1 infected individuals compared with healthy uninfected controls, whereas there were no such differences in CD8+ T cells. Comparison of Vbeta repertoire changes before and after interferon-alpha (IFN-alpha) treatment for HAM/TSP revealed that one out of five patients showed dramatic decrease of specific Vbeta in CD8+ T cells. Our results suggest that dominant Vbeta subpopulations in CD4+ T cells evolved associated with chronic HTLV-1 infection, and IFN-alpha treatment for HAM/TSP does not induce a specific pattern of TCR Vbeta changes.     

Neuropathology of human T lymphotropic virus type I-associated myelopathy

In order to clarify pathogenesis of human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) a detailed neuropathological analysis of eight autopsy patients with HAM/TSP was performed. Inflammatory infiltrates of mononuclear cells and degeneration of myelin and axons were noted in the middle to lower thoracic spinal cords and were extended continuously to the entire spinal cord. Horizontal distribution of inflammatory lesions was symmetric at any spinal levels. Immunohistochemical analysis demonstrated T cell dominance. The numbers of CD4+ T cells and CD8+ T cells were present in equal numbers in patients with shorter clinical course. Apoptosis of helper/inducer T cells were observed in the presence of TIA1+ cytotoxic T cells in these active inflammatory lesions. Inflammatory infiltrates were markedly decreased and CD8+/TIA1- T cells were predominated over CD4+ cells in patients with prolonged clinical course. HTLV-I proviral deoxyribonucleic acid (DNA) amounts in the freshly frozen spinal cord measured by quantitative polymerase chain reaction (PCR) were well correlated with the numbers of infiltrated CD4+ cells. In situ PCR of HTLV-I provial DNA using multi-primar pairs demonstrated the presence of HTLV-I infected cells exclusively in the mononuclear infiltrates of perivascular areas. From these findings, it is suggested that the target of the inflammatory process seen in HAM/TSP lesions may be HTLV-I infected CD4+ T cells infiltrating the spinal cord.     

Activation of macrophages/microglia with the calcium-binding proteins MRP14 and MRP8 is related to the lesional activities in the spinal cord of HTLV-I associated myelopathy

Macrophages and microglia may play an important role in the pathogenesis of chronic inflammatory process in HTLV-I associated myelopathy (HAM) and tropical spastic paraparesis (TSP). However, the etiology and cellular mechanism of chronic inflammation are poorly understood in HAM/TSP. To help to define the roles of macrophages and microglia we analyzed the various patterns of macrophage and microglia activation in the central nervous system (CNS) of HAM/TSP using several monoclonal antibodies recognizing the different states of activation. The results indicate that a large number of macrophages and microglia express both MRP14 and MRP8 in active-chronic inflammatory lesions of the patients with a short duration of illness (2.5 years). In the patient whose duration of illness was 4.5 years, perivascular and parenchymal macrophages and microglia were reactive for MRP8 but not for MRP14. In contrast, MRP14 and MRP8 were negative on the perivascular and parenchymal macrophages and microglia in inactive-chronic lesions and in controls. This study suggests that (a) activated macrophages and microglia as well as CD4+ T lymphocytes and CD8+ cytotoxic T lymphocytes are main components of the inflammatory process in the CNS in HAM/TSP, (b) activation of macrophages and microglia is related to the amount of HTLV-I proviral DNA in situ.     

Increased Fas-mediated cytotoxicity of CD4-positive T cells in patients with human T-lymphotropic virus type I-associated myelopathy

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release assay, we investigated Fas-mediated cytotoxicity of peripheral blood CD4⁺ T cells of patients with human T-lymphotropic virus type-I (HTLV-I)-associated myelopathy (HAM) against T98G, a glioblastoma cell line which expresses Fas. Cytotoxic activity of CD4⁺ T cells against T98G was significantly higher in HAM patients than in controls. Moreover, when CD4⁺ T cells of HAM patients were preincubated with a monoclonal antibody to human Fas ligand (FasL), cytotoxic activity against T98G was significantly suppressed. These results suggest that damage to nervous tissues by the Fas/FasL system is involved in the pathogenesis of HAM.     

Pathogenesis of chronic progressive myelopathy associated with human T-cell lymphotropic virus type I

Human T-cell lymphotropic virus type I (HTLV-I) induces a chronic demyelinating disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). While only 0.25% of HTLV-I-infected individuals develop HAM/TSP, the mechanisms responsible for the progression of an HTLV-I carrier state to clinical disease are not clear. In particular, no specific sequence differences have been found between HTLV-I recovered from HAM patients and HTLV-I-infected carriers. Since CD4 T cells are the major reservoir of the virus, at least three hypotheses implicating CD4 T cells directly or indirectly have been proposed: 1) The cytotoxic hypothesis predicts that activated and HTLV-I-infected CD4 T cells migrate to the CNS and infect resident cells. Cytotoxic CD8 T cells may then recognize viral antigens on HTLV-I-infected CNS cells causing a cellularly mediated cytotoxic demyelination. 2) The autoimmune hypothesis predicts that either (a) virally reactive T cells cross-react with a CNS antigen, or (b) random infection of CD4 T cells eventually results in the infection of CNS-autoreactive CD4 T cells that, by virtue of the productive HTLV-I infection, become activated, expand and migrate to the CNS, where they encounter their antigen. This results in a specific immune response and demyelination, as is known to occur in experimental autoimmune encephalomyelitis. 3) The bystander damage hypothesis does not implicate a specific response against CNS cells. Instead this hypothesis suggests that the presence of IFN-γ-secreting HTLV-I-infected CD4 T cells and their recognition by virally specific CD8 T cells in the CNS induce microglia to secrete cytokines, such as TNF-α, which may be toxic for the myelin.     

Expression of adhesion molecules and monocyte chemoattractant protein-1 (MCP-1) in the spinal cord lesions in HTLV-I-associated myelopathy

Leukocyte adhesion molecules to endothelium plays an important role in the pathogenesis of inflammatory diseases, including HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP). To help define the role of adhesion molecules in HAM/TSP, we studied the expression of lymphocyte function-associated antigen-1 (LFA-1), Mac-1, very late antigen-4 (VLA-4), Sialyl Lewis^x (SLex), intercelluar adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial leukocyte adhesion molecule-1 (ELAM-1) and monocyte chemoattractant protein-1 (MCP-1) in the spinal cord lesions of HAM/TSP. The results indicate that spinal cord lesions of HAM/TSP have greater VCAM-1 expression on endothelium compared with those of controls. Infiltrating mononuclear cells, especially perivascular lesions, expressed VLA-4. Although the expression of ICAM-1 in the spinal cords was not distinctive between HAM/TSP and controls, infiltrating mononulcear cells in the spinal cords of HAM/TSP strongly expressed LFA-1 and Mac-1. ELAM-1 was expressed on endothelium in the inactive-chronic lesions from three of five HAM/TSP, but was not detectable in the spinal cords of controls. SLex reaction was detectable on occasional perivascular cells in the spinal cord of HAM/TSP, but not in those of controls. MCP-1 was detectable on perivascular infiltrating cells and vascular endothelium in active-chronic lesions. This study suggests that VLA-4/VCAM-1 interaction may play an important role for lymphocyte migration into the central nervous system (CNS), and MCP-1 may also be involved in inflammatory cell recruitment to the CNS in HAM/TSP. Received: 4 September 1995 / Revised, accepted: 23 October 1995     

Reduction in HTLV-I proviral load and spontaneous lymphoproliferation in HTLV-I–associated myelopathy/tropical spastic paraparesis patients treated with humanized anti-tac

Human T-lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological disease that results from an interaction of retroviral infection and immune activation. In this study, five doses (1 mg/kg) of humanized anti-Tac antibody were administered to 9 HAM/TSP patients at weeks 0,2,6,10, and 14. Preliminary immunological studies on HAM/TSP patients treated with humanized anti-Tac indicate that there is a selective down-regulation of activated T cells and a decrease in the HTLV-I viral load in peripheral blood lymphocytes, most likely through the selective removal of HTLV-I–infected, activated CD4⁺ lymphocytes.     

Familial erythrophagocytic lymphohistiocytosis

Summary The neuropathologic study of a 7-monthold female patient affected by familial erythrophagocytic lymphohistiocytosis (FEL) reveals three main features: (1) a lymphohistiocytic leptomeningitis with erythrophagocytosis; (2) perivascular lymphohistiocytic cuffs in the cerebral and cerebellar white matter and, to a lesser extent, in the thalamus, the reticular formation of the brain stem and the griseum pontis; (3) perifocal gliosis and demyelination, especially in the cerebellar white matter.The lesions remind of the perivenous post-in-fectious encephalomyelitides. Perifocal demyelination has been reported in FEL very seldom. Although its pathogenesis is not known, immune mechanisms could play a role by analogy with perivenous encephalomyelitis.Supported by grant no. 3.0004.81 from the Fonds voor Geneeskundig Wetenschappelijk Onderszoek     

Activated T lymphocytes in cerebrospinal fluid of patients with HTLV-I-associated myelopathy (HAM/TSP)

In order to detect activated T lymphocytes in the cerebrospinal fluid (CSF) of patients with human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), we studied CSF lymphocytes in untreated patients with HAM/TSP and other neurological diseases (OND). Dual-immunofluorescence staining technique was performed using fluorescence microscopy. No significant difference in the CD4+/CD8+ ratio of CSF lymphocytes was observed between HAM/TSP patients and patients with OND. However, both CD4+ and CD8+ CSF lymphocytes of HAM/TSP patients contained higher percentages of HLA-DR-positive cells than those of patients with OND (P less than 0.05), suggesting that the activated CSF T lymphocytes were composed of both CD4+ and CD8+ subsets in patients with HAM/TSP.     

Enhanced adherence of endothelial cells to blood mononuclear cells in HAM/TSP]

We investigated the adhesion of blood mononuclear cells (MNC) isolated from patients with HTLV-1-associated myelopathy (HAM/TSP). MNC from HAM/TSP patients were significantly more adherent to activated endothelial monolayers than MNC from non-HAM/TSP (controls and HTLV-1 carriers) subjects. Blocking studies demonstrated that the adhesion molecules VLA-4 (CD49d), ICAM-1 (CD54), and L-selectin (CD62L) all contributed to increased binding. However, anti-ICAM-1 antibody was the most efficient in inhibiting binding HAM/TSP patients MNC to activated endothelial cells. Expression on MNC of molecules involved in adhesion was also studied by flow cytometry in HAM/TSP patients, HTLV-1 carriers, and healthy control subjects after two days culture without any mitogen. In HAM/TSP patients, L-selectin expression on CD4+ and CD8+ subsets was lower than in controls; interestingly, HAM/TSP patients had lower percentage of CD4+ subset expressing L-selectin than HTLV-1 carriers. The percentage of CD4+ and CD8+ cells expressing VLA-4 was found to be similar to controls in both HAM/TSP patients and HTLV-1 carriers. Following two days in culture without mitogen, the percentage of T cells expressing ICAM-1 increased in HAM/TSP and carriers, but not in controls. This study provides information regarding trans-endothelial migration of MNC across the blood brain barrier in HAM/TSP and suggests ICAM-1 and its counterpart molecule LAF-1 are involved in massive infiltration of lymphocytes observed in the spinal cord.     

Limited T cell receptor usage by HTLV-I tax-specific, HLA class I restricted cytotoxic T lymphocytes from patients with HTLV-I associated neurological disease

T cell receptor (TCR) Vα and Vβ chain usage of HTLV-I tax-specific, HLA class I restricted CD8⁺ cytotoxic T cells (CTL) was determined from lymphocytes obtained from peripheral blood of patients with HTLV-I associated neurological disease. To characterize TCR repertoire, CD8⁺ lymphocytes from peripheral blood were cloned in limiting dilution, and the resulting wells were screened for HTLV-I-specific precursor CTL activity. RNA was isolated from HLA-A2 restricted HTLV-I tax peptide-specific (tax 11–19; LLFGYPVYV) CD8⁺ CTL lines and cDNA was analyzed by PCR amplification using Vα and Vβ chain family-specific oligonucleotide primers. The results indicate that CD8⁺ cytotoxic T cell lines from HLA-A2 HAM/TSP patients express a limited repertoire of T cell receptor chains which may correlate with duration and severity of disease. The restricted use of TCR genes expressed by antigen-specific CTL may play a critical role in the pathogenesis of HAM/TSP and may be of value in developing immunotherapeutic strategies that focus on eliminating these cells or inhibiting their activity.     

The clinical and pathological features of peripheral neuropathy accompanied with HTLV-I associated myelopathy

We describe the clinical and pathological studies in HTLV-I associated myelopathy (HAM)/tropical spastic paraparesis (TSP) patients with peripheral neuropathy as proven by sural nerve biopsy. Sural nerve pathology in HAM/TSP patients revealed that the most common type of pathologic change is a combination of both demyelination and remyelination and axonal degeneration and regeneration, and this change is modified by the complications. The pathologic changes were correlated with neither the duration of disease nor human T lymphotropic virus type I (HTLV-I) proviral load. This study suggests that peripheral nerves could be involved in HAM/TSP.     

Neuropathology of HTLV‐1‐associated myelopathy (HAM/TSP)

A series of our neuropathological studies was reviewed in order to clarify pathogenesis of human T lymphotropic virus type 1(HTLV‐1)‐associated myelopathy (HAM)/tropical spastic paraparesis (TSP). The essential histopathologic finding was chronic inflammation in which inflammatory infiltrates of mononuclear cells and degeneration of myelin and axons were noted in the entire spinal cord. Immunohistochemical analysis demonstrated T‐cell dominance, and the numbers of CD4+ cells and CD8+ cells were equally present in patients with shorter clinical courses. Apoptosis of helper/inducer T‐cells were observed in these active inflammatory lesions. Horizontal distribution of inflammatory lesions was symmetric at all spinal levels and was accentuated at sites with slow blood flow in the middle to lower thoracic levels. HTLV‐1 proviral DNA amounts were well correlated with the numbers of infiltrated CD4+ cells. In situ PCR of HTLV‐1 proviral DNA and in situ hybridization of HTLV‐1 Tax gene demonstrated the presence of HTLV‐1‐infected cells exclusively in the mononuclear infiltrates of perivascular areas. From these findings, it is suggested that T‐cell mediated chronic inflammatory processes targeting the HTLV‐1 infected T‐cells is the primary pathogenic mechanism of HAM/TSP. Anatomically determined hemodynamic conditions may contribute to the localization of infected T‐cells and the formation of main lesions in the middle to lower thoracic spinal cord.     

Human T-cell lymphotropic virus type I and neurological diseases

Human T-cell lymphotropic virus type I (HTLV-I) infection is associated with a variety of human diseases. In particular, there are two major diseases caused by HTLV-I infection. One is an aggressive neoplastic disease called adult T-cell leukemia (ATL), and another is a chronic progressive inflammatory neurological disease called HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is still unknown why one virus causes these different diseases. With regard to HAM/TSP, virus-host immunological interactions are an considered to be important cause of this disease. Coexisting high HTLV-I proviral load and HTLV-I-specific T cells (CD4+ T cells and CD8+ T cells) is an important feature of HAM/TSP. Histopathological studies indicate the existence of an inflammatory reaction and HTLV-I-infected cells in the affected lesions of HAM/TSP. Therefore, the immune response to HTLV-I probably contributes to the inflammatory process of the central nervous system lesions in HAM/TSP patients.     

HTLV-I specific IFN-gamma+ CD8+ lymphocytes correlate with the proviral load in peripheral blood of infected individuals

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurological disease caused by HTLV-I infection. It has been shown that HAM/TSP patients have high proviral loads and an extraordinarily high frequency of circulating CD8 + cytotoxic T lymphocytes specific for HTLV-I in their peripheral blood when compared to asymptomatic HTLV-I carriers (AC). We have previously described an intracellular cytokine detection assay, in which interferon-gamma (IFN-gamma) + CD8 + lymphocytes are specific for HTLV-I in infected individuals. Here, we have established a competitive polymerase chain reaction assay to measure the proviral load of patients and investigate a potential relationship between proviral load and virus-specific CD8 + lymphocytes. Genomic DNA was extracted from peripheral blood lymphocytes (PBL) from eight HAM/TSP patients and seven AC for the measurement of HTLV-I measuring proviral loads. The same PBL were analyzed for intracellular IFN-gamma expression by flow cytometry. In the HAM/TSP patients and AC, the average proviral loads were 34,482 and 9784 copy/microg DNA (P = 0.021), and the average of IFN-gamma + CD8 + lymphocytes in total PBL were 1.47 and 0.08% (P = 0.001), respectively. It was confirmed that HAM/TSP patients have both high proviral loads and increased HTLV-I-specific CD8 + lymphocytes. Furthermore, we found a positive correlation between both factors in the patients with HAM/TSP (P = 0.044) but not in the AC (P = 0.508). These findings suggest that the high number of HTLV-I-specific lymphocytes may result from the increased proviral load in HAM/TSP patients.     

T cell transformation with Herpesvirus saimiri: a tool for neuroimmunological research

Splice variants of CD44 molecule-harboring exon 10 (v6), often called v6 variants (v6v), are shown to confer tumor progressive, metastatic or invasive capacities. Furthermore, CD44 molecule on activated T-cells are shown to be required for infiltration of these cells into the inflammatory site and for accelerated immune response. Human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is caused by HTLV-I infection and characterized by spastic paraparesis and urinary disturbance with perivascular HTLV-I-infected and activated CD4+ T-cell infiltration. In order to explore the underlying mechanism causing the disease after HTLV-I infection, we analyzed CD44 variant expression on peripheral blood mononuclear cells (PBMC) and in the spinal cord specimens from patients with HAM/TSP, and compared them with those from other HTLV-I-infected individuals and controls. We found that v6v expression with special direct link of exons 10 (v6) and 14(v10) was highly expressed in PBMC from patients with HAM/TSP and that v6v and CD4 double positive T-cell infiltration into the spinal cord lesion of HAM/TSP. This combination of CD44 splice variant has not been previously reported in the study of chronic inflammatory disorders and may be a marker molecule for T-cells infiltrating into the central nervous system (CNS), especially the spinal cord.