Unsong Oh

Regulatory T Cells are Reduced During Anti-CD25 Antibody Treatment of Multiple Sclerosis

OBJECTIVE Maintenance therapy with anti-CD25 antibody has emerged as a potentially useful treatment for multiple sclerosis (MS). Constitutive CD25 expression on CD4+CD25+ regulatory T cells (Treg) suggests that anti-CD25 antibody treatment may potentially target a subset of T cells that exhibit immune suppressive properties. We examined changes to CD4+CD25+ Treg in patients with MS receiving maintenance anti-CD25 monoclonal antibody treatment to determine the effect of treatment on Treg and, consequently, on immunological tolerance. DESIGN Peripheral blood and cerebrospinal fluid samples obtained from a before-and-after trial of anti-CD25 antibody monotherapy were examined to compare baseline and treatment differences in CD4+CD25+ Treg. SUBJECTS A total of 15 subjects with MS. One subject was withdrawn owing to an adverse effect. RESULTS Sustained reduction of the frequency of CD4+CD25+ Treg was observed during treatment. Anti-CD25 antibody treatment led to evidence of impaired in vivo Treg proliferation and impaired ex vivo Treg suppression. Inflammatory MS activity was substantially reduced with treatment despite reduction of circulating Treg, and there was no correlation between changes in the frequency of Treg and changes in brain inflammatory activity. However, new-onset inflammatory disease, notably dermatitis, was also observed in a number of subjects during treatment. CONCLUSION The reduction in Treg did not negatively affect maintenance of central nervous system tolerance during anti-CD25 antibody treatment. The incidence of new-onset inflammatory disease outside of the central nervous system in a subset of patients, however, warrants further studies to examine the possibility of compartmental differences in the capacity to maintain tolerance in the setting of reduced CD4+CD25+ Treg.

Detection of Active Human Herpesvirus-6 Infection in the Brain: Correlation with Polymerase Chain Reaction Detection in Cerebrospinal Fluid

One-half of bone-marrow transplant (BMT) and stem-cell transplant recipients have reactivation of latent human herpesvirus (HHV)-6 2-4 weeks after transplant. Although the detection of viral DNA, RNA, and antigen in brain material confirmed active HHV-6 variant B infection, peak viral loads in cerebrospinal fluid (CSF) and serum occurred 2-4 weeks before death and decreased to low levels before or at autopsy. All autopsy samples consistently demonstrated HHV-6 active infection in the hippocampus. Astrocytic cells positive for viral antigen provided support for an HHV-6-specific tropism for hippocampal astrocytes. HHV-6 DNA in CSF and serum may not reflect the level of active viral infection in the brain after BMT.

Foxp3 Represses Retroviral Transcription by Targeting Both NF-κB and CREB Pathways

Forkhead box (Fox)/winged-helix transcription factors regulate multiple aspects of immune responsiveness and Foxp3 is recognized as an essential functional marker of regulatory T cells. Herein we describe downstream signaling pathways targeted by Foxp3 that may negatively impact retroviral pathogenesis. Overexpression of Foxp3 in HEK 293T and purified CD4+ T cells resulted in a dose-dependent and time-dependent decrease in basal levels of nuclear factor-κB (NF-κB) activation. Deletion of the carboxyl-terminal forkhead (FKH) domain, critical for nuclear localization and DNA-binding activity, abrogated the ability of Foxp3 to suppress NF-κB activity in HEK 293T cells, but not in Jurkat or primary human CD4+ T cells. We further demonstrate that Foxp3 suppressed the transcription of two human retroviral promoters (HIV-1 and human T cell lymphotropic virus type I [HTLV-I]) utilizing NF-κB-dependent and NF-κB-independent mechanisms. Examination of the latter identified the cAMP-responsive element binding protein (CREB) pathway as a target of Foxp3. Finally, comparison of the percent Foxp3+CD4+CD25+ T cells to the HTLV-I proviral load in HTLV-I-infected asymptomatic carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis suggested that high Foxp3 expression is associated with low proviral load and absence of disease. These results suggest an expanded role for Foxp3 in regulating NF-κB- and CREB-dependent cellular and viral gene expression.

Reduced Foxp3 Protein Expression Is Associated with Inflammatory Disease during Human T Lymphotropic Virus Type 1 Infection

The Foxp3 protein is a specific marker of CD4(+)CD25(+) regulatory T (T(reg)) cells, and its expression is critical to their development and function. Several studies have demonstrated the dysregulation of Foxp3 expression during human inflammatory diseases. Infection with human T lymphotropic virus type 1 (HTLV-1) is associated with the development of a number of inflammatory conditions, including myelopathy, although the majority of individuals who are infected with HTLV-1 remain asymptomatic. To examine the role played by T(reg) cells in the development of inflammatory disease during HTLV-1 infection, we examined Foxp3 expression by flow cytometry. Our analysis showed that HTLV-1-associated myelopathy/tropical spastic paraparesis was associated with a lower expression (compared with that in asymptomatic HTLV-1 carriers and healthy donors) of Foxp3 in peripheral-blood leukocytes. In individuals infected with HTLV-1, Foxp3 expression was inversely correlated with HTLV-1 tax proviral DNA load. These results suggest that impaired Foxp3 expression may contribute to the development of inflammatory disease during HTLV-1 infection.

Interferon-β1a therapy in human T-lymphotropic virus type I–associated neurologic disease

Human T‐lymphotropic virus type I (HTLV‐I)–associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an immune‐mediated inflammatory disorder of the central nervous system. Immune activation in the host, which results from high levels of persistent antigenic stimulation and from transactivation of host immunoregulatory genes by HTLV‐I, appears important in the pathogenesis of HAM/TSP. In a single‐center, open‐label trial, 12 patients with HAM/TSP were treated with doses of interferon‐β1a of up to 60μg twice weekly, based on its antiviral and immunomodulatory effects. Primary end points were immunological and virological measures that are potential biomarkers for HAM/TSP. Interferon‐β1a therapy reduced the HTLV‐I tax messenger RNA load and the frequency of potentially pathogenic HTLV‐I–specific CD8+ cells. The HTLV‐I proviral DNA load remained unchanged. Spontaneous lymphoproliferation, a marker of T‐cell activation in HAM/TSP, also was reduced. Some measures of motor function were improved, and no significant clinical progression occurred during therapy. These results indicate that interferon‐β1a may beneficially affect the immune mechanisms central to the pathogenesis of HAM/TSP. Ann Neurol 2005;57:526–534

CP-690,550, a therapeutic agent, inhibits cytokine-mediated Jak3 activation and proliferation of T cells from patients with ATL and HAM/TSP.

The retrovirus, human T-cell-lymphotrophic virus-1 (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL) and the neurological disorder HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-I-encoded protein tax constitutively activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems that in turn activate the Jak3 (Janus kinase 3)/STAT5 (signal transducers and activators of transcription 5) pathway, suggesting a therapeutic strategy that involves targeting Jak3. We evaluated the action of the Jak3 inhibitor CP-690,550 on cytokine dependent ex vivo proliferation that is characteristic of peripheral blood mononuclear cells (PBMCs) from select patients with smoldering or chronic subtypes of ATL, or from those with HAM/TSP whose PBMCs are associated with autocrine/paracrine pathways that involve the production of IL-2, IL-9, IL-15, and their receptors. CP-690,550 at 50 nM inhibited the 6-day ex vivo spontaneous proliferation of PBMCs from ATL and HAM/TSP patients by 67.1% and 86.4%, respectively. Furthermore, CP-690,550 inhibited STAT5 phosphorylation in isolated ATL T cells ex vivo. Finally, in an in vivo test of biological activity, CP-690,550 treatment of mice with a CD8 T-cell IL-15-transgenic leukemia that manifests an autocrine IL-15/IL-15Rα pathway prolonged the survival duration of these tumor-bearing mice. These studies support further evaluation of the Jak3 inhibitor CP-690,550 in the treatment of select patients with HTLV-I-associated ATL and HAM/TSP.

Treatment of HTLV-I-Associated Myelopathy/Tropical Spastic Paraparesis: Toward Rational Targeted Therapy

The treatment of HAM/TSP is a challenge. No agent has shown to significantly modify the long-term disability associated with HAM/TSP. Advances in our understanding of the pathogenesis of HAM/TSP have led to the identification of several biomarkers and therapeutic targets. Clinical trials in HAM/TSP continue to be opportunities for further qualification and refinement of biomarkers and therapeutic targets. The validation of HAM/TSP relevant biomarkers and the identification of new targets remain key challenges in the development of effective targeted therapy in HAM/TSP.

Anti-HTLV antibody profiling reveals an antibody signature for HTLV-I-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP)

BackgroundHTLV-I is the causal agent of adult T cell leukemia (ATLL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Biomarkers are needed to diagnose and/or predict patients who are at risk for HAM/TSP or ATLL. Therefore, we investigated using luciferase immunoprecipitation technology (LIPS) antibody responses to seven HTLV-I proteins in non-infected controls, asymptomatic HTLV-I-carriers, ATLL and HAM/TSP sera samples. Antibody profiles were correlated with viral load and examined in longitudinal samples.ResultsAnti-GAG antibody titers detected by LIPS differentiated HTLV-infected subjects from uninfected controls with 100% sensitivity and 100% specificity, but did not differ between HTLV-I infected subgroups. However, anti-Env antibody titers were over 4-fold higher in HAM/TSP compared to both asymptomatic HTLV-I (P < 0.0001) and ATLL patients (P < 0.0005). Anti-Env antibody titers above 100,000 LU had 75% positive predictive value and 79% negative predictive value for identifying the HAM/TSP sub-type. Anti-Tax antibody titers were also higher (P < 0.0005) in the HAM/TSP compared to the asymptomatic HTLV-I carriers. Proviral load correlated with anti-Env antibodies in asymptomatic carriers (R = 0.76), but not in HAM/TSP.ConclusionThese studies indicate that anti-HTLV-I antibody responses detected by LIPS are useful for diagnosis and suggest that elevated anti-Env antibodies are a common feature found in HAM/TSP patients.

Dysregulation of TGF-β signaling and regulatory and effector T-cell function in virus-induced neuroinflammatory disease

We previously demonstrated that CD4(+)CD25(+) T regulatory cells (Tregs), important for the maintenance of immune tolerance and prevention of autoimmune disease, from patients with human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) exhibit reduced Foxp3 expression and Treg suppressor function compared with healthy donors. Since TGF-beta signaling has been previously reported to be critical for both Foxp3 expression and Treg function, we examined whether this signaling pathway was dysregulated in patients with HAM/TSP. Levels of TGF-beta receptor II (TGF-betaRII) as well as Smad7 (a TGF-beta-inducible gene) were significantly reduced in CD4(+) T cells in patients with HAM/TSP compared with healthy donors, and the expression of TGF-betaRII inversely correlated with the HTLV-I tax proviral load. Importantly, both CD4(+)CD25(+) and CD4(+)CD25(-) T cells from HAM/TSP patients exhibited reduced TGF-betaRII expression compared with healthy donors, which was associated with functional deficits in vitro, including a block in TGF-beta-inducible Foxp3 expression that inversely correlated with the HTLV-I tax proviral load, loss of Treg suppressor function, and escape of effector T cells from Treg-mediated control. This evidence suggests that a virus-induced breakdown of immune tolerance affecting both regulatory and effector T cells contributes to the pathogenesis of HAM/TSP.

Retrovirally induced CTL degranulation mediated by IL-15 expression and infection of mononuclear phagocytes in patients with HTLV-I–associated neurologic disease

CD8(+) T cells contribute to central nervous system inflammation in human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We analyzed CD8(+) T-cell dysfunction (degranulation and IFN-gamma production) and have demonstrated that CD8(+) T cells of patients with HAM/TSP (HAM/TSP patients) spontaneously degranulate and express IFN-gamma in ex vivo unstimulated culture. CD8(+) T cells of HTLV-I asymptomatic carriers and healthy donors did not. Spontaneous degranulation was detected in Tax11-19/HLA-A*201 tetramer(+) cells, but not in CMV pp65 tetramer(+) cells. Interestingly, degranulation and IFN-gamma production in CD8(+) T cells was induced by coculture with autologous CD14(+) cells, but not CD4(+) T cells, of HAM/TSP patients, which correlated with proviral DNA load in CD14(+) cells of infected patients. Moreover, the expression of IL-15, which induced degranulation and IFN-gamma production in infected patients, was enhanced on surface of CD14(+) cells in HAM/TSP patients. Blockade of MHC class I and IL-15 confirmed these results. Thus, CD8(+) T-cell dysregulation was mediated by both virus infection and enhanced IL-15 on CD14(+) cells in HAM/TSP patients. Despite lower viral expression than in CD4(+) T cells, HTLV-I-infected or -activated CD14(+) cells may be a heretofore important but under recognized reservoir particularly in HAM/TSP patients.