Common خ³-chain blocking peptide reduces in vitro immune 2015-08-31آ  Common خ³-chain blocking...

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  • Common γ-chain blocking peptide reduces in vitro immune activation markers in HTLV-1-associated myelopathy/tropical spastic paraparesis Raya Massouda, Yoshimi Enose-Akahataa, Yutaka Tagayab, Nazli Azimic, Asjad Basheerc, and Steven Jacobsona,1

    aViral Immunology Section, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892; bCell Biology Laboratory, Division of Basic Science and Vaccine Research, University of Maryland School of Medicine, Baltimore, MD 21201; and cBioniz, LLC, Irvine, CA 92618

    Edited* by Robert C. Gallo, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, and approved July 20, 2015 (received for review July 21, 2014)

    Human T-cell lymphotropic virus type 1 (HTLV-1)-associated mye- lopathy/tropical spastic paraparesis (HAM/TSP) is a progressive inflammatory myelopathy occurring in a subset of HTLV-1-infected individuals. Despite advances in understanding its immunopatho- genesis, an effective treatment remains to be found. IL-2 and IL-15, members of the gamma chain (γc) family of cytokines, are prom- inently deregulated in HAM/TSP and underlie many of the charac- teristic immune abnormalities, such as spontaneous lymphocyte proliferation (SP), increased STAT5 phosphorylation in the lympho- cytes, and increased frequency and cytotoxicity of virus-specific cytotoxic CD8+ T lymphocytes (CTLs). In this study, we describe a novel immunomodulatory strategy consisting of selective block- ade of certain γc family cytokines, including IL-2 and IL-15, with a γc antagonistic peptide. In vitro, a PEGylated form of the pep- tide, named BNZ132-1-40, reduced multiple immune activation markers such as SP, STAT5 phosphorylation, spontaneous degran- ulation of CD8+ T cells, and the frequency of transactivator protein (Tax)-specific CD8+ CTLs, thought to be major players in the immu- nopathogenesis of the disease. This strategy is thus a promising therapeutic approach to HAM/TSP with the potential of being more effective than single monoclonal antibodies targeting either IL-2 or IL-15 receptors and safer than inhibitors of downstream signal- ing molecules such as JAK1 inhibitors. Finally, selective cytokine blockade with antagonistic peptides might be applicable to multi- ple other conditions in which cytokines are pathogenic.

    human T-cell lymphotropic virus type 1 | HAM/TSP | gamma chain | IL-15

    Human T-cell lymphotropic virus type 1 (HTLV-1)-associatedmyelopathy/tropical spastic paraparesis (HAM/TSP) is a central nervous system (CNS) disease occurring in up to 3% of HTLV-1-infected individuals (1). Clinically, it is a spontaneous lymphocyte proliferation (SP) with sphincter dysfunction, pointing to predominant involvement of the lower thoracic spinal cord. However, many other syndromes [such as cognitive and auto- nomic dysfunction, amyotrophic lateral sclerosis (ALS)-like syn- drome, peripheral neuropathy, and myositis] have been reported, suggesting more widespread neurologic damage (2). Typically, the onset is insidious, and significant disability occurs within years of symptom manifestation. However, a rapidly progressive form with acute onset and complete paraplegia in less than 2 y can also occur (3). There is substantial evidence that HAM/TSP is an immune-

    mediated disorder, the pathogenesis of which is associated with deregulated immune activation. Pathologically, there is peri- vascular and parenchymal inflammatory infiltration in the water- shed areas of the CNS with associated axonal loss, demyelination, reactive astrocytosis, and fibrillary gliosis (4). Early in the course of the disease, the inflammatory infiltrate contains equal numbers of CD4+, CD8+ T cells, and foamy macrophages; over time, the lesions become less inflammatory with a predominance of CD8+ T cells and high levels of IFN-γ (2, 5). Immunologically, ex vivo

    spontaneous proliferation of the peripheral blood mononuclear cells (PBMCs) is a hallmark of ongoing lymphocyte activation in HAM/TSP. Both CD4+ and CD8+ T cells are involved in SP, but the CD8+ T cells, and in particular, HTLV-1-specific CD8+ cy- totoxic T lymphocytes (CTLs), proliferate more vigorously (6). These virus-specific CTLs infiltrate CNS lesions, where they could function to eliminate HTLV-1-infected CD4+ T cells, resulting in the secretion of damaging proinflammatory factors (such as TNF-α and IFN-γ) and bystander CNS damage (7). Notably, transactivator protein (Tax)-specific CTLs are found at higher frequency in the peripheral blood and cerebrospinal fluid of patients with HAM/TSP compared with in asymptomatic carriers (8, 9); these cells are predominantly of the memory ef- fector phenotype and have increased cytotoxicity (10).The phe- nomenon of SP has been suggested to be a surrogate marker for the expansion and persistence of such virus-specific pathogenic CTLs in the CNS (6). IL-2 and IL-15 are pleiotropic cytokines that play pivotal roles

    in inflammation and immune responses to infections. Both are glycoprotein members of the four α-helix bundle-containing cytokines and signal through receptor complexes that use the gamma chain (γc; CD132) and the β chain [CD122; IL-2/IL-15

    Significance

    IL-2 and IL-15, members of the gamma chain family of cyto- kines, are prominently deregulated in human T-cell lympho- tropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and underlie many of the char- acteristic immune abnormalities such as spontaneous lympho- cyte proliferation, increased STAT5 phosphorylation in the lymphocytes, and increased frequency and cytotoxicity of virus-specific CD8+ T lymphocytes (CTLs). In this in vitro study, we demonstrate that selective and simultaneous blockade of IL-2 and IL-15, with a γ-chain antagonistic peptide, reduces spontaneous lymphocyte proliferation (SP), STAT5 phosphory- lation, and more important, the degranulation of CD8+ T cells and the frequency of HTLV-1-specific CTLs. Thus, selective cy- tokine blockade with antagonistic peptides might be a thera- peutic approach in HAM/TSP and is potentially applicable to multiple other conditions in which cytokines are pathogenic.

    Author contributions: R.M., Y.E.-A., Y.T., N.A., and S.J. designed research; R.M. and A.B. performed research; Y.T. and N.A. contributed new reagents/analytic tools; R.M. and Y.E.-A. analyzed data; and R.M., Y.E.-A., Y.T., N.A., and S.J. wrote the paper.

    Conflict of interest statement: N.A. and A.B. are employees of Bioniz, the developer of the peptide used in this study.

    *This Direct Submission article had a prearranged editor.

    Freely available online through the PNAS open access option. 1To whom correspondence should be addressed. Email: jacobsons@ninds.nih.gov.

    This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1412626112/-/DCSupplemental.

    11030–11035 | PNAS | September 1, 2015 | vol. 112 | no. 35 www.pnas.org/cgi/doi/10.1073/pnas.1412626112

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    http://crossmark.crossref.org/dialog/?doi=10.1073/pnas.1412626112&domain=pdf mailto:jacobsons@ninds.nih.gov http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1412626112/-/DCSupplemental http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1412626112/-/DCSupplemental www.pnas.org/cgi/doi/10.1073/pnas.1412626112

  • receptor (R)]. γc is additionally shared with IL-4, IL-7, IL-9, and IL-21, all of which are members of the common γc family of cytokines (11). Cytokine binding to the β/γc complex results in heterodimerization of their cytoplasmic domains with activation of the Janus family tyrosine kinases, JAK1 (in association with β chain) and JAK3 (associating with the γc). Activated JAK1 and JAK3 then phosphorylate signal transducer and activator of transcription proteins STAT3 and STAT5, respectively, to me- diate IL-2 and IL-15 effects in immune cells. IL-2 is primarily produced by activated CD4+ T cells and, at

    lower amounts, by other immune cells (CD8+ T cells, NK cells, activated dendritic cells, and mast cells). It signals via combi- nations of IL-2 R subunits: IL-2 Rα (CD25), IL-2/IL-15 Rβ, and the γc. The combination IL-2 Rβ with γc forms intermediate- affinity IL-2 R; all three subunits together form the high-affinity IL-2 R. Both these classes of receptors are functional and are differentially expressed on immune cell subsets. In addition to being a potent T-cell growth factor, IL-2 is also an important modulator of the effector cell response. In particular, it is in- volved in the differentiation of Th1 and Th2, promotes antibody production and proliferation of B cells, and is essential for activation-induced cell death and for the development of CD4+

    FOXP3+ regulatory T cells (T-reg) (12). IL-15 shares not only structural and signaling components with

    IL-2 but also many functional redundancies. In particular, both induce the proliferation and increase the cytolytic activity of NK and CD8+ T cells, and both can induce the proliferation and dif- ferentiation of stimulated human B cells. However, certain im- portant immune functions are exclusively mediated by IL-15. In particular, IL-15 is critical for the development of NK cells and antigen-specific memory CD8+ T cells and is best characterized for its role in maintaining memory pools of CD8+ T cells (13). The IL-2/IL-2 Rα and IL-15/IL-15 Rα cytokine loops are

    deregulated in HAM/TSP and contribute to a variety of immu- nological abnormalities characteristic of this disorder, providing a strong rationale for anticytokine therapy in this condition (14). Such therapy could potentially reduce clonal expansion and activ