Download - Common γ-chain blocking peptide reduces in vitro immune … · 2015-08-31 · Common γ-chain blocking peptide reduces in vitro immune activation markers in HTLV-1-associated myelopathy/tropical

Transcript
Page 1: Common γ-chain blocking peptide reduces in vitro immune … · 2015-08-31 · Common γ-chain blocking peptide reduces in vitro immune activation markers in HTLV-1-associated myelopathy/tropical

Common γ-chain blocking peptide reduces in vitroimmune activation markers in HTLV-1-associatedmyelopathy/tropical spastic paraparesisRaya 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, MD20892; bCell Biology Laboratory, Division of Basic Science and Vaccine Research, University of Maryland School of Medicine, Baltimore, MD 21201; andcBioniz, 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 forreview July 21, 2014)

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated mye-lopathy/tropical spastic paraparesis (HAM/TSP) is a progressiveinflammatory myelopathy occurring in a subset of HTLV-1-infectedindividuals. 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 lymphocyteproliferation (SP), increased STAT5 phosphorylation in the lympho-cytes, and increased frequency and cytotoxicity of virus-specificcytotoxic CD8+ T lymphocytes (CTLs). In this study, we describe anovel immunomodulatory strategy consisting of selective block-ade of certain γc family cytokines, including IL-2 and IL-15, witha γc antagonistic peptide. In vitro, a PEGylated form of the pep-tide, named BNZ132-1-40, reduced multiple immune activationmarkers 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 promisingtherapeutic approach to HAM/TSP with the potential of being moreeffective than single monoclonal antibodies targeting either IL-2or IL-15 receptors and safer than inhibitors of downstream signal-ing molecules such as JAK1 inhibitors. Finally, selective cytokineblockade 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% ofHTLV-1-infected individuals (1). Clinically, it is a spontaneouslymphocyte proliferation (SP) with sphincter dysfunction, pointingto 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 yearsof symptom manifestation. However, a rapidly progressive formwith acute onset and complete paraplegia in less than 2 y can alsooccur (3).There is substantial evidence that HAM/TSP is an immune-

mediated disorder, the pathogenesis of which is associated withderegulated 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 courseof the disease, the inflammatory infiltrate contains equal numbersof CD4+, CD8+ T cells, and foamy macrophages; over time, thelesions become less inflammatory with a predominance of CD8+ Tcells and high levels of IFN-γ (2, 5). Immunologically, ex vivo

spontaneous proliferation of the peripheral blood mononuclearcells (PBMCs) is a hallmark of ongoing lymphocyte activation inHAM/TSP. Both CD4+ and CD8+ T cells are involved in SP, butthe 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 theycould 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 athigher frequency in the peripheral blood and cerebrospinal fluidof patients with HAM/TSP compared with in asymptomaticcarriers (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 forthe expansion and persistence of such virus-specific pathogenicCTLs in the CNS (6).IL-2 and IL-15 are pleiotropic cytokines that play pivotal roles

in inflammation and immune responses to infections. Bothare glycoprotein members of the four α-helix bundle-containingcytokines and signal through receptor complexes that use thegamma 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/tropicalspastic paraparesis (HAM/TSP) and underlie many of the char-acteristic immune abnormalities such as spontaneous lympho-cyte proliferation, increased STAT5 phosphorylation in thelymphocytes, and increased frequency and cytotoxicity ofvirus-specific CD8+ T lymphocytes (CTLs). In this in vitro study,we demonstrate that selective and simultaneous blockade ofIL-2 and IL-15, with a γ-chain antagonistic peptide, reducesspontaneous lymphocyte proliferation (SP), STAT5 phosphory-lation, and more important, the degranulation of CD8+ T cellsand 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 tomultiple 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 thepeptide 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: [email protected].

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

Dow

nloa

ded

by g

uest

on

Oct

ober

4, 2

020

Page 2: Common γ-chain blocking peptide reduces in vitro immune … · 2015-08-31 · Common γ-chain blocking peptide reduces in vitro immune activation markers in HTLV-1-associated myelopathy/tropical

receptor (R)]. γc is additionally shared with IL-4, IL-7, IL-9, andIL-21, all of which are members of the common γc family ofcytokines (11). Cytokine binding to the β/γc complex results inheterodimerization of their cytoplasmic domains with activationof the Janus family tyrosine kinases, JAK1 (in association with βchain) and JAK3 (associating with the γc). Activated JAK1 andJAK3 then phosphorylate signal transducer and activator oftranscription 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, andthe γc. The combination IL-2 Rβ with γc forms intermediate-affinity IL-2 R; all three subunits together form the high-affinityIL-2 R. Both these classes of receptors are functional and aredifferentially expressed on immune cell subsets. In addition tobeing a potent T-cell growth factor, IL-2 is also an importantmodulator of the effector cell response. In particular, it is in-volved in the differentiation of Th1 and Th2, promotes antibodyproduction and proliferation of B cells, and is essential foractivation-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, bothinduce the proliferation and increase the cytolytic activity of NKand 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. Inparticular, IL-15 is critical for the development of NK cells andantigen-specific memory CD8+ T cells and is best characterizedfor 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, providinga strong rationale for anticytokine therapy in this condition (14).Such therapy could potentially reduce clonal expansion andactivation of the infected cells in the peripheral circulation, andsubsequently decrease their infiltration and expansion in the CNS.On the basis of this rationale, monoclonal antibody therapy inHAM/TSP using humanized anti-Tac antibody (against IL-2 Rα)was conducted with encouraging results (i.e., reduction in theproviral load and spontaneous proliferation), and a clinical trialof humanized Mik β1, monoclonal antibody against IL-2/IL-15 Rβ,is currently underway, with promising early results (15).However, we reasoned that simultaneous inhibition of the two

IL-2/IL-15 cytokine loops is potentially a more effective anti-cytokine therapy than antibody monotherapy. Signaling moleculesthat are shared by the two loops, namely, the JAK3 moleculeand the common γc, provide reasonable targets for this strategy.CP-690, 550, which was originally developed as a JAK3 inhibitormolecule, was shown to reduce in vitro immune activation inHAM/TSP PBMCs (16) and is currently approved for the treatmentof psoriasis and rheumatoid arthritis. However, this agent alsoinhibits JAK1 and lacks cytokine specificity, which probably ac-counts for the high incidence of adverse effects observed with itsclinical use; namely, bone marrow suppression, lymphocytopenia,and high incidence of serious infections (17).A novel and potentially more effective and safe targeted

therapeutic approach to HAM/TSP might consist of γc blockadewith an antagonizing peptide that selectively inhibits IL-2, IL-9,and IL-15, but not other γc cytokines. BNZ132-1 is an anticytokinepeptide that was designed on the basis of the presence of amoderately conserved region among all of the cytokines sharingthe γc receptor and overlapping with the D-helix. It is a 19-merpeptide that, according to proliferation assays in cytokine-dependenthuman and murine cells lines, was found to selectively block signalingof IL-2, IL-15, and IL-9.

In this study, we examined the effects of BNZ132-1 on mul-tiple in vitro markers of immune activation in HAM/TSP. Thepeptide significantly suppressed SP of the PBMCs and reducedthe frequency of Tax-specific CD8+ T cells, the degree of spon-taneous CD8+ T-cell degranulation, and the degree of STAT5phosphorylation. Compared with a combination of monoclonalantibodies against CD25 and CD122, targeting the IL-2 andIL-15 cytokine loops, this novel antagonist peptide was found tobe as effective in reducing STAT5 phosphorylation and moreeffective in inhibiting SP. Therefore, BNZ132-1 is a potentialtherapy for HAM/TSP.

ResultsBNZ132-1 Inhibits HAM/TSP PBMC SP in a Dose-Dependent Manner.Given the important role of IL-2 and IL-15 in SP of HAM/TSPPBMCs, the effects of the γc-blocking peptide BNZ132-1 on thisphenomenon were examined. PBMCs from six patients withHAM/TSP were cultured either in complete RPMI alone or withBNZ132-1 at 1 and 10 μM. At day 5 of culture, the peptide re-duced the degree of SP, as quantified by radioactive thymidineincorporation, in five of six samples (Fig. 1A) in a dose-dependentmanner. No decrease in cell viability was observed at these con-centrations. A PEGylated form of the peptide, BNZ132-1-40, wasthen developed and used in the subsequent experiments. At aconcentration as low as 0.01 μM, BNZ132-1-40 inhibited theproliferation of CTLL-2, an IL2-dependent T-cell line that alsoresponds to IL-15, and the inhibition was almost complete at a10 μM concentration of BNZ132-1-40 (Fig. S1). Subsequently, adose-titration experiment in one patient with HAM/TSP, HAM1,known to have IL-2/IL-15-dependent SP (based on the inhibitionby BNZ132-1; Fig. 1A), showed that at 10 μM, BNZ132-1-40inhibited SP by 60% (Fig. 1B, summarized in Fig. 1D). On thebasis of these results, additional studies were conducted with moreHAM/TSP PBMC samples. As shown in Fig. 1C, at 10 μM,BNZ132-1-40 inhibited the SP in all six patients with HAM/TSPtested. Again, no decrease in cell viability was observed with thePEGylated peptide. The percentage inhibition of SP by BNZ132-1-40 is summarized in Fig. 1D and ranged from 20% to 85%.

BNZ132-1-40 Inhibits the Proliferation of Both CD4+ and CD8+ T Cells.Because CD4+ and CD8+ T cells are both involved in HTLV-1-induced SP, the effects of BNZ132-1-40 on the proliferation ofeach of the two subsets were examined by flow cytometry, using acarboxyfluorescein diacetate succinimidyl ester dilution assay inthree patients. Fig. 2A is a representative histogram of the in-hibitory effects of BNZ132-1-40 at 10 μM on the proliferation ofCD4+ and CD8+ T-cell subsets from a patient with HAM/TSP(HAM5). In all three patients with HAM/TSP tested, BNZ132-1-40reduced the SP of both CD4+ and CD8+ T cells (Fig. 2 A and B).Notably, there was no significant difference in the levels of in-hibition of SP between CD4+ and CD8+ T cells (Fig. 2B), althoughthe degree of proliferation of CD4+ T cells in the untreated sampleswas minimal compared with CD8+ cells (e.g., Fig. 2A).Similar results were found when the differential effects on T-cell

subset proliferation were examined by Ki-67 expression, anothermarker of proliferation. As summarized in Fig. 2C, BNZ132-1-40at 10 μM reduced Ki-67 expression in both CD4+ and CD8+ T-cellsubsets in all six HAM/TSP PBMCs. Again, although there was nostatistically significant difference in the inhibition between CD8+

and CD4+ T cells, in four of six studied samples, the reduction ofKi-67 was more prominent in the CD8+ T-cell subset (Fig. 2C).Moreover, additional markers of activation such as pSTAT5 andCD25 expression were significantly more reduced in the CD8+

compared with the CD4+ T-cell subset (Fig. 3 A and B). Thedegree of inhibition of STAT5 phosphorylation in the CD8+ Tcells at day 5 of culture strongly correlated with the inhibition ofKi-67 expression in this subset (Fig. 3C).

Massoud et al. PNAS | September 1, 2015 | vol. 112 | no. 35 | 11031

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Oct

ober

4, 2

020

Page 3: Common γ-chain blocking peptide reduces in vitro immune … · 2015-08-31 · Common γ-chain blocking peptide reduces in vitro immune activation markers in HTLV-1-associated myelopathy/tropical

BNZ132-1-40 Reduces the Expression of CD132, the Common γc, inHAM/TSP PBMC. In addition to sharing the common γc for signaltransduction, IL-2 and IL-15 share the cell-surface receptorsubunit IL-2/IL-15 Rβ (CD122) known to be critical for IL-15-mediated CD8+ T-cell activation, memory cell survival, and cy-totoxic activity (18). In addition, CD25, the IL-2 private receptorchain α, is up-regulated in HAM/TSP and mediates many of theeffects of IL-2 in the CD4+ T cells. Thus, we examined the ef-fects of γc blockade by BNZ132-1-40 on the expression of thesethree receptor subunits (CD25, CD122, and CD132). HAM/TSPPBMCs from three subjects were cultured for 48 h with orwithout BNZ132-1-40, and the expression of the receptor chainswas characterized at 48 h. BNZ132-1-40 significantly reduced theexpression of CD132, both in CD4+ and CD8+ T cells (Fig. 4A),whereas there was no change in the expression of CD25 orCD122 on either of the cell subsets. In addition, the degree ofreduction in CD132 expression in the CD8+ T cells stronglycorrelated with the percentage inhibition of day 5 SP by BNZ132-1-40 in the same samples (Fig. 4B).

BNZ132-1-40 Is More Effective Than a Combination of Anti-Tac andMikβ1 Antibodies in Inhibiting HAM/TSP Spontaneous Proliferation.Murine anti-CD25 (anti-Tac) and anti-CD122 (Mikβ1) antibodies areknown to inhibit HAM/TSP SP and have been important tools inelucidating the roles of IL-2 and IL-15 in this phenomenon. Anti-Tactargets the CD25 (IL-2 Rα) and inhibits IL-2 action. Mikβ1 binds tothe IL-2/15 β chain, but only inhibits IL-15 actions that are mediatedby transpresentation through the high-affinity receptor complex (IL-15Rα, IL-15 Rβ, and IL-15 Rγ). Because each antibody inhibits eitherthe IL-2 or IL-15 pathway, we compared the effects of BNZ132-1-40peptide on pSTAT5 expression and SP with those of a combination ofanti-Tac and MiKβ1. At 10 μΜ, BNZ132-1-40 was as effective as thecombination of anti-Tac and MiK β1 (each 5 μM) in inhibitingSTAT5 phosphorylation in HAM/TSP lymphocytes (Fig. 5A) and wassignificantly more effective in inhibiting SP (P = 0.0325). (Fig. 5B).

BNZ132-1-40 Reduces the Frequency of Tax-Tetramer-Positive CD8+ TCells. IL-15 increases the proliferation, reduces the apoptosis,and enhances the cytotoxicity of HTLV-1-specific CD8+ CTLs

A B

C D Fig. 1. BNZ132-1 and its PEGylated form BNZ132-1-40 inhibit HAM/TSP PBMC spontaneous proliferationin a dose-dependent manner. (A) Spontaneous pro-liferation of PBMCs from six patients with HAM/TSPcultured with or without BNZ132-1 at 1 and 10 μM.(B) SP of PBMCs from a representative patient withHAM/TSP with and without BNZ132-1-40 at threedifferent concentrations (1, 3, and 10 μM). (C) SP ofPBMC from six patients with HAM/TSP with andwithout BNZ132-1-40 at 10 μM. (D) Percentage in-hibition of SP by BNZ132-1-40 at 10 μM in six pa-tients with HAM/TSP.

A B

C

Fig. 2. BNZ132-1-40 (10 μM) inhibits SP in CD4+

and CD8+ T cells. (A) Representative histograms ofcarboxyfluorescein diacetate succinimidyl ester inCD4+ and CD8+ T cells of one patient with HAM/TSP(HAM5) cultured with or without BNZ132-1-40.(B) Percentage inhibition of SP of CD4+ and CD8+ Tcells in three HAM/TSP samples by BNZ132-1-40.(C) Percentage inhibition of Ki-67 expression in CD4+

and CD8+ T-cell subsets from six patients with HAM/TSP.

11032 | www.pnas.org/cgi/doi/10.1073/pnas.1412626112 Massoud et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

4, 2

020

Page 4: Common γ-chain blocking peptide reduces in vitro immune … · 2015-08-31 · Common γ-chain blocking peptide reduces in vitro immune activation markers in HTLV-1-associated myelopathy/tropical

(18). The effects of BNZ132-1-40 on the frequency of HTLV-1Tax-specific CD8+ T cells were therefore examined in two HLA-A*0201 HAM/TSP individuals after culture. As shown in Fig. 6,both HAM/TSP patient 9 and patient 10 had Tax11-19 tetramerreactive cells after 6 d of culture that were reduced in the presenceof 10 μM BNZ132-1-40. The frequency of Tax-specific CD8+ Tcells was reduced from 14.4% to 4%, and from 4.68% to 1.9%,respectively (Fig. 6).

BNZ132-1-40 Reduces HTLV-1-Induced Cytolytic T-Cell Degranulation.It has been reported that patients with HAM/TSP demonstrateincreased spontaneous degranulation (CD107a) and IFN-γ se-cretion by CD8+ CTLs in unstimulated ex vivo culture comparedwith asymptomatic carriers and healthy donors (19). This responseis HTLV-1-specific, as it requires antigen presentation and IL-15transpresentation to effector CD8+ T cells by infected CD14+

cells. Notably, in HAM/TSP there is increased IL-15 expression onthe surface of CD14+ antigen presenting cells (APC) and in-creased responsiveness of CD8+ CTLs to IL-15 (19).Therefore, it was of interest to examine the effects of BNZ132-

1-40 on the frequency of CD107a (a marker of the mobilization ofcytolytic granules to the cell surface) and the production of IFN-γby CD8+ T cells from patients with HAM/TSP during short-termculture. Fig. 7A is a flow-cytometric dot plot illustrating the inhib-itory effect of BNZ132-1-40 on the spontaneous degranulation andIFN-γ expression by CD8+ CTLs from a representative HAM/TSPsubject. As shown in Fig. 7 B and C, patients with HAM/TSP dis-played variable frequencies of spontaneously degranulating/IFN-γ-expressing CTLs, which were reduced by BNZ132-1-40 by morethan 50%, in three of four tested subjects.Collectively, these results demonstrate that common γc block-

ade by BNZ132-1-40 is effective in reducing a number of IL-2- andIL-15-mediated immunological effects associated with HAM/TSPpathogenesis.

DiscussionIn this study, a new strategy to reduce the excessive immuneactivation that occurs in HAM/TSP was used by simultaneouslyinhibiting the effects of multiple proinflammatory autocrine andparacrine cytokine loops involved in the pathogenesis of thisdisease. It has long been known that HAM/TSP PBMCs spon-taneously proliferate in ex vivo cultures and that this immuno-logical finding might reflect naturally occurring events in vivo inpatients with HAM/TSP, particularly at the site of injury in theCNS. Thus, ex vivo SP is an important tool when evaluatingpotential interventional strategies and specific therapeutic agentsin HAM/TSP.Many of the immune abnormalities observed in HAM/TSP

(SP, differentiation, increased cytotoxicity, and prolonged sur-vival of HTLV-1-specific CTLs) have been linked to the HTLV-1Tax. HTLV-1 Tax, directly and through activation of NF-κB,induces the expression of IL-2, IL-15, and their specific receptors

(IL-2 Rα and IL-15 Rα), leading to excessive signaling throughproinflammatory autocrine and paracrine loops (20).It was previously demonstrated that IL-2/IL-2 Rα and IL-15/

IL-15 Rα are overexpressed in HAM/TSP PBMCs and haveadditive effects on SP, which can be inhibited by the addition ofantibodies to the cytokines or their receptors (IL-2 Rα and IL-15Rβ). IL-9, another Tax-induced γ-cytokine, plays a role in theproliferation of adult T-cell leukemia (ATL) cells. Unlike IL-2and IL-15, which act through autocrine loops on the T cells, IL-9stimulates the IL-9 Rα-expressing CD14+ monocytes, whichthen support the proliferation of ATL cells via a paracrine loop(21). In vitro studies have shown that in addition to its role in SP,IL-15 also contributes to the survival of Tax-specific CD8+ Tcells (supporting their proliferation and inhibiting apoptosis) andtheir cytotoxic function, both of which are increased in HAM/TSP and are thought to be critical mediators of CNS injury, asdiscussed previously.Therefore, the IL-2/IL-15 cytokine loops constitute a reason-

able therapeutic target in HAM/TSP. This is supported by theclinical trial of humanized anti-Tac antibody (against IL-2 Rα),showing a reduction in the proviral load and SP, and is currentlybeing explored in a phase 1/2 clinical trial of humanized Mikβ1Abdirected against IL-2/IL-15 Rβ. As an alternative approach, weinvestigated the effects of a novel immunomodulatory γc block-ing peptide, BNZ132-1, on HAM/TSP PBMCs. BNZ132-1 wasdesigned to selectively inhibit the functions of some of the γc cy-tokines (IL-2, IL-9, and IL-15), but not others (IL-4, IL-7, andIL-21). BNZ132-1 was demonstrated to be a potent inhibitor ofHAM/TSP SP. To improve the pharmacokinetic properties ofthe peptide, increase its bioavailability, and reduce its immuno-genicity for potential future clinical studies, a PEGylated form ofBNZ132-1, BZN132-1-40, was developed. BNZ-132-40 was alsoshown to inhibit SP, using a variety of immunological assays.Notably, the peptide suppressed the proliferation of both CD4+

A B C

Fig. 3. BNZ132-1-40 (10 μm) reduces CD25 expression and STAT5 phosphorylation in the CD8+ T cells. (A) Representative dot plots illustrating the expressionof pSTAT5 in CD8+ T cells from one patient with HAM/TSP when PBMCs were cultured with and without BNZ132-1-40. (B) Percentage inhibition of CD25 andpSTAT5 expression in CD8+ and CD4+ T cells of six different patients with HAM/TSP at day 5 of SP by BNZ132-1-40. (C) The inhibition of pSTAT5 in the CD8+ Tcells strongly correlates with the reduction in SP of this T-cell subset.

A B

Fig. 4. BNZ132-1-40 significantly reduces the expression of CD132 in cul-tured HAM/TSP PBMC. (A) Inhibition of CD132 expression in CD4+ andCD8+ T cells with BNZ132-1-40 (10 μM) at 48 h of culture. (B) The inhibitionof CD132 expression in CD8+ T cells correlates with inhibition of SP byBNZ132-1-40.

Massoud et al. PNAS | September 1, 2015 | vol. 112 | no. 35 | 11033

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Oct

ober

4, 2

020

Page 5: Common γ-chain blocking peptide reduces in vitro immune … · 2015-08-31 · Common γ-chain blocking peptide reduces in vitro immune activation markers in HTLV-1-associated myelopathy/tropical

and CD8+ T cells. In addition, in short-term culture of HAM/TSP PBMCs, the peptide reduced STAT5 phosphorylation, adownstream event of γc signaling, significantly more in CD8+

compared with CD4+ T cells (P = 0.028), suggesting preferentialinhibitory effects on this subset. The inhibition of pSTAT5 ex-pression and SP by BNZ132-1-40 peptide was secondary to thereduction in CD132 expression, as this was significantly reducedin both CD4+ and CD8+ T cells, whereas there were no changesin the expression of CD25 or CD122 molecules. It remains un-clear, however, whether this reflects down-regulation of the γc orits saturation by the peptide. Remarkably, compared with a com-bination of anti-Tac and Mikß1 antibodies, BNZ132-1-40 was aseffective in inhibiting STAT5 phosphorylation, but significantlymore effective in reducing PBMC SP. This is possibly mediatedby blocking the effects of another γc cytokine, IL-9, which is alsosuggested to play a role in HAM/TSP SP. It has been previouslyshown that IL-9 contributes to the proliferation of malignantHTLV-1-related ATL cells, but its importance in HAM/TSPremains to be established.Virus-specific cytotoxic CD8+ T cells are thought to be major

players in the pathogenesis of HAM/TSP. They are enriched inthe peripheral circulation and cerebrospinal fluid of affectedpatients and demonstrate increased cytotoxicity, which is at leastpartially mediated by their interaction with IL-15 overexpressingCD14+ cells. BNZ132-1-40 also was found to reduce the SP ofTax-specific CD8+ T cells in two patients with HAM/TSP, whichis consistent with the role of IL-15 in the differentiation andprolonged survival of these cells. In addition, the peptide reducedthe degree of spontaneous degranulation and IFN-γ secretion byCD8+ CTLs in short-term culture, illustrating the efficacy of thepeptide in reducing the stimulation of virus-specific CTL responseby antigen-presenting CD14+ cells.This approach highlights a promising therapeutic strategy for

HAM/TSP and overcomes the challenge posed by the deregula-tion of multiple proinflammatory cytokines. The understandingand potential reversal of immunologic mechanisms implicated ina rare, retrovirus-associated human neurologic condition, such asHAM/TSP, could also help develop treatments for inflammatoryconditions of unknown etiology. Similarly, understanding thepathogenic roles of the CD8+ CTLs and other inflammatorycytokines in HAM/TSP can be translated to other conditions inwhich similar immune abnormalities might be pathogenic. Inparticular, the anticytokine strategy described in this study is apromising therapeutic approach for many inflammatory disor-ders related to the γc cytokines; such conditions include but arenot limited to myelofibrosis, rheumatoid arthritis, psoriasis, andceliac disease (22–24).

MethodsSamples. Blood was collected from patients with HAM/TSP seen at the Na-tional Institute of Neurologic Disorders and Stroke. PBMCs were isolated bydensity centrifugation and cryopreserved before use. Written, informedconsent was obtained in compliance with the Declaration of Helsinki. Thestudy was reviewed and approved by a National Institute of NeurologicDisorders and Stroke institutional review board.

Cell Culture. PBMCs were suspended in RPMI supplemented with 1% peni-cillin/streptomycin, 1% L-glutamine, and 5% (vol/vol) Human AB serum andplaced in 96-well round-bottom plates at 3 × 105 cells per well, with or withoutBNZ132-1 or its pegylated form (BNZ132-1-40), at the indicated concentra-tion. In some experiments, HAM/TSP PBMCs were cultured in the presence ofthe antibodies murine anti-Tac (5 μM) and Mik-β1 (5 μM) (kind gifts fromThomas A. Waldmann, NIH, Bethesda). Cell viability was measured usingGuavaViaCount assay (Millipore) according to the manufacturer’s instructions.

Proliferation Assay. HAM/TSP PBMCs were cultured as described earlier intriplicate. Proliferationwas measured by radioactive thymidine incorporationon day 5 of culture. PHA-treated and untreated healthy donor PBMCs wereused as positive and negative controls, respectively.

Alternatively, HAM/TSP PBMCs were labeled with carboxyfluoresceindiacetate succinimidyl ester (Life Technologies), according to the manufac-turer’s instructions, and cultured as described earlier without or withBNZ132-1-40 at 10 μM. At day 5, the cells were washed, stained for cell-surface markers [CD3-pacific blue (PB), CD4-APC, and CD8-PerCP-Cy5.5 (BDBiosciences)], and the data acquired on LSRII flow cytometer (BD Biosciences)and analyzed using FlowJo software (Tree star).

The effects of BNZ132-1–40 on the proliferation of different lymphocytesubsets were also examined by Ki-67 stain at day 5 of culture. For this, PBMCswere cultured without or with BNZ132-1-40 at 10 μM. At day 5, the cellswere stained for cell-surface markers [CD3-PB, CD4-Alexa Fluor700, CD8-PerCP-Cy5.5, and CD25-APC (BD Biosciences)] and then fixed and per-meabilized using the Foxp3 Fix/Perm kit (eBioscience), according to themanufacturer’s instructions, before staining with FITC-conjugated Ki-67 an-tibody (BD Biosciences).

Activation Markers Analysis. For phosphorylated STAT5 (pSTAT5) detection,HAM/TSP PBMCs were cultured as described earlier, with or without BNZ132-1-40, at 10 μM for 20 h or 5 d. The cells were then fixed in 1.5% (vol/vol)paraformaldehyde and permeabilized with 90% (vol/vol) methanol. Afterwashing, the cells were stained with antibodies against pSTAT5-PE, CD3-APC, CD4-FITC, and CD8-PE-Cy7 (BD Biosciences).

The effects of BNZ132-1-40 peptide on the expression of CD122, CD25, andCD132 were examined by flow cytometry. HAM/TSP PBMCs were stained with

A B

Fig. 5. BNZ132-1-40 is more effective than a combination of blocking anti-CD25 and anti-CD122 antibodies in reducing HAM/TSP PBMC SP. (A) In-hibition of pSTAT5 expression in PBMCs from three patients with HAM/TSPwith BNZ132-1-40 (10 μM) or with a combination of Anti-Tac and Mikβ1(each 5 μM). STAT5 phosphorylation in the lymphocytes was examined at20 h of culture. (B) Inhibition of PBMC SP in three patients with HAM/TSPwith BNZ132-1-40 (10 μM) or with a combination of Anti-Tac and Mikβ1(each 5 μM).

HAM 9

HAM 10

0 102 103 104 105 0 102 103 104 105

0 102 103 104 105 0 102 103 104 105

0 1

02

103

1

04

105

0 1

02

103

1

04

105

0 1

02

103

1

04

105

0 1

02

103

1

04

105

CD

8 C

D8

Tax tetramer Tax tetramer

Tax tetramer Tax tetramer

Fig. 6. BNZ132-1-40 (10 μm) reduces the SP of Tax-specific CD8+ T cells.PBMCs from two HLA-A 0201 HAM/TSP subjects (HAM9 and HAM10) werecultured for 6 d with and without BNZ132-1-40.

11034 | www.pnas.org/cgi/doi/10.1073/pnas.1412626112 Massoud et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

4, 2

020

Page 6: Common γ-chain blocking peptide reduces in vitro immune … · 2015-08-31 · Common γ-chain blocking peptide reduces in vitro immune activation markers in HTLV-1-associated myelopathy/tropical

CD3-PB, CD4-Alexa Fluor700, CD8-PerCP-Cy5.5, CD122-PE, CD25-FITC (BDBiosciences), and CD132-APC (Biolegend) at baseline and after culture at 24and 48 h.

Analysis of Tax-Specific Cells and CTL Spontaneous Degranulation. Analysis ofantigen-specific CD8+ T cells was performed using a PE-conjugated HTLV-1Tax11–19 peptide (LLFGYPVYV)-loaded HLA-A*0201 tetramer (provided byNational Institutes of Health AIDS Research and Reference Reagent Pro-gram). PBMCs from patients with HAM/TSP known to have the allele HLAA*0201 were cultured without or with BNZ132-1-40 at 10 μM. At day 6 ofculture, the cells were stained with Tax-tetramer, CD3-PB, CD4-APC, andCD8-PerCP-Cy5.5 (BD Biosciences).

For spontaneous CTL degranulation, HAM/TSP PBMCs were cultured in a96-well U-bottom plate at 2 × 105 cells per well, with or without BNZ132-1-4010 μM, and analysis was performed as previously described (19).

Statistical Analysis. Paired t test was used to compare the differential effectsof BNZ132-1-40 on the proliferation (Ki-67) and activation (pSTAT5 andCD25 expression) in the different lymphocyte subsets (CD4+ vs. CD8+ T cells)and to compare the effects of BNZ132-1-40 (on SP and pSTAT5) with those ofa combination of monoclonal antibodies (anti-CD25 and anti-CD122). Linearregression analysis was used to examine the correlation between the effectson pSTAT5 and CD132 and the inhibition of SP. All statistical analysis wasdone using Prism software version 6.0 (Graphpad).

1. Proietti FA, Carneiro-Proietti AB, Catalan-Soares BC, Murphy EL (2005) Global epi-demiology of HTLV-I infection and associated diseases. Oncogene 24(39):6058–6068.

2. Araujo AQ, Silva MT (2006) The HTLV-1 neurological complex. Lancet Neurol 5(12):1068–1076.

3. Lima MA, Harab RC, Schor D, Andrada-Serpa MJ, Araújo AQ (2007) Subacute pro-gression of human T-lymphotropic virus type I-associated myelopathy/tropical spasticparaparesis. J Neurovirol 13(5):468–473.

4. Umehara F, et al. (1993) Immunocytochemical analysis of the cellular infiltrate in thespinal cord lesions in HTLV-I-associated myelopathy. J Neuropathol Exp Neurol 52(4):424–430.

5. Aye MM, et al. (2000) Histopathological analysis of four autopsy cases of HTLV-I-associated myelopathy/tropical spastic paraparesis: Inflammatory changes occur si-multaneously in the entire central nervous system. Acta Neuropathol 100(3):245–252.

6. Sakai JA, Nagai M, Brennan MB, Mora CA, Jacobson S (2001) In vitro spontaneouslymphoproliferation in patients with human T-cell lymphotropic virus type I-associ-ated neurologic disease: Predominant expansion of CD8+ T cells. Blood 98(5):1506–1511.

7. Yamano Y, et al. (2002) Correlation of human T-cell lymphotropic virus type 1 (HTLV-1)mRNA with proviral DNA load, virus-specific CD8(+) T cells, and disease severity in HTLV-1-associated myelopathy (HAM/TSP). Blood 99(1):88–94.

8. Jacobson S, Shida H, McFarlin DE, Fauci AS, Koenig S (1990) Circulating CD8+ cytotoxicT lymphocytes specific for HTLV-I pX in patients with HTLV-I associated neurologicaldisease. Nature 348(6298):245–248.

9. Kubota R, Kawanishi T, Matsubara H, Manns A, Jacobson S (1998) Demonstration ofhuman T lymphotropic virus type I (HTLV-I) tax-specific CD8+ lymphocytes directly inperipheral blood of HTLV-I-associated myelopathy/tropical spastic paraparesis pa-tients by intracellular cytokine detection. J Immunol 161(1):482–488.

10. Johnson-Nauroth JM, Graber J, Yao K, Jacobson S, Calabresi PA (2006) Memorylineage relationships in HTLV-1-specific CD8+ cytotoxic T cells. J Neuroimmunol176(1-2):115–124.

11. Yamane H, Paul WE (2012) Cytokines of the γ(c) family control CD4+ T cell differ-entiation and function. Nat Immunol 13(11):1037–1044.

12. Liao W, Lin JX, Leonard WJ (2013) Interleukin-2 at the crossroads of effector re-sponses, tolerance, and immunotherapy. Immunity 38(1):13–25.

13. Verbist KC, Klonowski KD (2012) Functions of IL-15 in anti-viral immunity: Multiplicityand variety. Cytokine 59(3):467–478.

14. Azimi N, Jacobson S, Leist T, Waldmann TA (1999) Involvement of IL-15 in the path-ogenesis of human T lymphotropic virus type I-associated myelopathy/tropical spasticparaparesis: Implications for therapy with a monoclonal antibody directed to the IL-2/15R beta receptor. J Immunol 163(7):4064–4072.

15. Lehky TJ, et al. (1998) Reduction in HTLV-I proviral load and spontaneous lympho-proliferation in HTLV-I-associated myelopathy/tropical spastic paraparesis patientstreated with humanized anti-Tac. Ann Neurol 44(6):942–947.

16. Ju W, et al. (2011) CP-690,550, a therapeutic agent, inhibits cytokine-mediated Jak3activation and proliferation of T cells from patients with ATL and HAM/TSP. Blood117(6):1938–1946.

17. Salgado E, Maneiro JR, Carmona L, Gomez-Reino JJ (2014) Safety profile of proteinkinase inhibitors in rheumatoid arthritis: Systematic review and meta-analysis. AnnRheum Dis 73(5):871–882.

18. Azimi N, Nagai M, Jacobson S, Waldmann TA (2001) IL-15 plays a major role in thepersistence of Tax-specific CD8 cells in HAM/TSP patients. Proc Natl Acad Sci USA98(25):14559–14564.

19. Enose-Akahata Y, Oh U, Grant C, Jacobson S (2008) Retrovirally induced CTL de-granulation mediated by IL-15 expression and infection of mononuclear phagocytesin patients with HTLV-I-associated neurologic disease. Blood 112(6):2400–2410.

20. Peloponese JM, Yeung ML, Jeang KT (2006) Modulation of nuclear factor-kappaB byhuman T cell leukemia virus type 1 Tax protein: Implications for oncogenesis andinflammation. Immunol Res 34(1):1–12.

21. Chen J, et al. (2008) Induction of the IL-9 gene by HTLV-I Tax stimulates the sponta-neous proliferation of primary adult T-cell leukemia cells by a paracrine mechanism.Blood 111(10):5163–5172.

22. Abadie V, Discepolo V, Jabri B (2012) Intraepithelial lymphocytes in celiac diseaseimmunopathology. Semin Immunopathol 34(4):551–566.

23. Dunphy S, Gardiner CM (2011) NK cells and psoriasis. J Biomed Biotechnol 2011:248317.

24. Tefferi A, et al. (2011) Circulating interleukin (IL)-8, IL-2R, IL-12, and IL-15 levels areindependently prognostic in primary myelofibrosis: A comprehensive cytokine pro-filing study. J Clin Oncol 29(10):1356–1363.

A

B C

Fig. 7. BNZ132-1-40 (10 μm) inhibits the sponta-neous degranulation and IFN-γ secretion by CD8+

cytotoxic T cells. (A) Representative dot plots fromone patient with HAM/TSP illustrating spontaneousdegranulation and IFN-γ secretion by CD8+ CTLs.(B) Spontaneous degranulation and IFN-γ secretion byCD8+ CTLs from four patients with HAM/TSP. (C) Per-centage inhibition, by BNZ132-1-40, of spontaneousdegranulation and IFN-γ secretion by CD8+ CTLs fromthe four tested patients with HAM/TSP.

Massoud et al. PNAS | September 1, 2015 | vol. 112 | no. 35 | 11035

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Oct

ober

4, 2

020