JVI Accepted Manuscript Posted Online 15 July 2015 J. Virol. doi:10.1128/JVI.00993-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Expansion of SIV-specific CD8 T cell lines from SIV-naïve Mauritian cynomolgus

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macaques for adoptive transfer

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Running Title: Naïve expansion of SIV-specific CD8 T cells

5 6

Mariel S Mohns a, Justin M Greene a, Brian T Cain a, Ngoc H Pham a, Emma Gostick b,

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David A Price b, and David H O’Connor a,c#

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Department of Pathology, University of Wisconsin - Madison, Madison, Wisconsin,

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USAa; Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff,

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Wales, UKb; Wisconsin National Primate Research Center, University of Wisconsin -

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Madison, Madison, Wisconsin, USAc.

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#Corresponding Author: Dave O’Connor, [email protected]

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Word Count - Abstract: 313

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Word Count - Text: 4,454

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ABSTRACT

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CD8 T cells play a crucial role in the control of human immunodeficiency virus (HIV) and

29

simian immunodeficiency virus (SIV). However, the specific qualities and characteristics

30

of an effective CD8 T cell response remain unclear. Although targeting breadth, cross-

31

reactivity, polyfunctionality, avidity, and specificity are correlated with HIV control, further

32

investigation is needed to determine the precise contributions of these various attributes

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to CD8 T cell efficacy. We developed protocols for isolating and expanding SIV-specific

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CD8 T cells from SIV-naïve Mauritian cynomolgus macaques (MCM). These cells

35

exhibited an effector memory phenotype, produced cytokines in response to cognate

36

antigen, and suppressed viral replication in vitro. We further cultured cell lines specific

37

for four SIV-derived epitopes: Nef

38

254-262

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After autologous transfer into two MCM recipients, expanded CD8 T cells persisted in

40

peripheral blood and lung tissue for at least 24 weeks, and trafficked to multiple extra-

41

lymphoid tissues. However, these cells did not impact acute phase SIV load after

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challenge compared to historic controls. The expansion and autologous transfer of SIV-

43

specific T cells into naïve animals provides a unique model for exploring cellular

44

immunity and the control of SIV infection, and facilitates a systematic evaluation of

45

therapeutic adoptive transfer strategies for eradication of the latent reservoir.

103-111

RM9, Gag

389-394

GW9, Env

338-346

RF9, and Nef

LT9. These lines were up to 94.4% pure as determined by MHC-tetramer analysis.

46 47

IMPORTANCE

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CD8 T cells play a crucial role in the control of human immunodeficiency virus (HIV) and

49

simian immunodeficiency virus (SIV). Autologous adoptive transfer studies followed by

50

SIV challenge may help define the critical elements of an effective T cell response to HIV

51

and SIV infection. We developed protocols for isolating and expanding SIV-specific CD8

52

T cells from SIV-naïve Mauritian cynomolgus macaques. This is an important first step

53

toward the development of autologous transfer strategies to explore cellular immunity

54

and potential therapeutic applications in the SIV model.

55 56

INTRODUCTION

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CD8 T cells are essential for the control of human immunodeficiency virus (HIV)

58

and simian immunodeficiency virus (SIV) replication in the infected host. Experimental

59

depletion of CD8 T cells with monoclonal antibodies leads to an increase in plasma

60

viremia, and immune control is subsequently reestablished when these cells recrudesce

61

(1-3). Additionally, pressure from CD8 T cells selects for escape variants in the acute

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and chronic phases of HIV/SIV infection (4-7). However, qualitative differences clearly

63

exist between CD8 T cell populations. Both human and macaque cohorts that control

64

viral replication to low or undetectable levels are enriched for the expression of specific

65

major histocompatibility complex (MHC) class I alleles (8-11). These observations

66

suggest that CD8 T cell specificity for certain viral peptides presented on the target cell

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surface by particular MHC class I molecules is a key determinant of efficacy. Moreover,

68

epitope targeting breadth and polyfunctionality have been linked with CD8 T cell-

69

mediated control of HIV/SIV (12). Although a composite response directed against a

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broad array of epitopes may reduce the chances of viral escape at any particular site,

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reported associations with immune control are conflicting (13-16). Similarly, the ability of

72

CD8 T cells to deploy multiple effector functions in response to cognate antigen

73

encounter is likely beneficial, but it remains unclear to what extent the observed

74

correlations reflect causality (17, 18). Antigen avidity, variant epitope cross-recognition

75

and clonotype recruitment also play a key role in this complex scenario (19-23). Further

76

investigation of these various factors is therefore needed to distinguish effective from

77

ineffective CD8 T cell responses in HIV/SIV infection.

78

Although the development of antiretroviral therapy (ART) has greatly improved

79

the prognosis for HIV-infected individuals, complete eradication of the virus remains an

80

elusive goal. In addition to CD8 T cell-mediated pressure during the acute phase, rapid

81

seeding of the viral reservoir occurs within the first few days of infection (24).

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Establishment of a latent reservoir requires long-term ART to maintain control of the

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virus. However, recent studies have demonstrated the potential utility of a “shock and

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kill” strategy designed to force latent virus out of the reservoir and enable CD8 T cell-

85

mediated eradication (25, 26). Further studies of CD8 T cell efficacy may therefore

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contribute to the “kill” component of this novel therapeutic approach.

87

Non-human primate models provide a unique opportunity to explore immune

88

responses in HIV/SIV infection. We identified a geographically isolated population of

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Mauritian cynomolgus macaques (MCM) in which seven common haplotypes account for

90

all MHC diversity (27). These simple haplotypes facilitate studies of cellular immunity.

91

MCM are particularly useful for the investigation of therapeutic strategies because

92

confounding genetic variances that may influence the immune response are eliminated.

93

Experimental studies with MHC-matched MCM are therefore ideally suited to the

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development of adoptive transfer protocols as part of an eradication strategy.

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Adoptive transfer studies provide a powerful method for studying the cellular

96

immune response (28-32). Previous experiments in which bulk lymphocytes were

97

allogeneically transferred between MHC-matched MCM revealed limited donor cell

98

persistence beyond 14 days (31, 32), although increased donor cell persistence was

99

observed in MHC-identical siblings (30). Autologous adoptive transfer provides distinct

100

advantages in this context (30, 31). Furthermore, this approach prevents complications

101

related to donor matching and graft-versus-host effects. However, previous autologous

102

transfer experiments relied on the use of SIV-infected animals. In this study, we

103

developed a novel protocol to isolate and rapidly expand SIV-specific CD8 T cell lines

104

from SIV-naïve MCM. The expansion of bulk lymphocytes required for these

105

experiments represents a significant achievement considering the low precursor

106

frequencies present in uninfected animals. Autologous adoptive transfer of defined CD8

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T cell populations followed by infectious challenge with SIV may help define the critical

108

elements of an effective T cell response to HIV and SIV infection.

109 110

METHODS

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Animal care and ethics statement. Members of the Wisconsin National Primate

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Research Center (WNPRC) cared for all animals according to the regulations and

113

standards set by the International Animal Care and Use Committee (IACUC). Two

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animals with the M1/M3 MHC haplotype were selected for autologous transfer.

115

Genotyping was based on microsatellite analysis and the MHC class I alleles on these

116

haplotypes were characterized previously (27).

117 118

Autologous transfer and SIV challenge. Expanded cell lines were processed by Ficoll-

119

Paque PLUS (GE Health Sciences, Piscataway, NJ) density centrifugation and

120

resuspended in RPMI 1640 (HyClone, Logan, UT) supplemented with 10% fetal bovine

121

serum (HyClone), 1% antibiotic-antimitotic (HyClone), and 1% L-glutamine (HyClone)

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(R10 medium). Individual cell lines were counted and then combined into a single

123

homogenate for transfer. Cells were labeled with 80μM carboxyfluorescein succinimidyl

124

ester (CFSE) prior to infusion for tracking by flow cytometry as described previously (32).

125

After resuspension in 8-9ml of 1X phosphate-buffered saline (PBS) supplemented with

126

15U/ml heparin, cells were transfused into the saphenous vein by WNPRC staff. One

127

day post-transfer, animals were challenged intrarectally with 7,000 TCID50 of SIVmac239

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stock virus.

129

130

Peptides. Most of the peptides used in this study were synthesized by Genscript

131

(Piscataway, NJ), ProImmune (Sarasota, FL), or the Biotechnology Center at the

132

University of Wisconsin (Madison, WI) using standard tertiary butyloxycarbonyl or

133

fluorenyl-methoxycarbonyl solid-phase methods. The 15-mer peptides used to stimulate

134

CD8 T cell cultures and determine their restriction were provided by the NIH AIDS

135

Research and Reference Reagent Program (Germantown, MD). All peptide sequences

136

were derived from the SIVmac239 sequence.

137 138

Generation of MHC class I transferents. Transferents expressing single MCM MHC

139

class I alleles were generated using either K562 cells with plasmids synthesized by

140

GenScript or the 721.221 HLA-deficient human B-lymphoblastoid cell line (BLCL) with

141

full-length MHC-class I amplicons ligated into pcDNA3.1(+) (Invitrogen, Carlsbad, CA) as

142

described previously (33). Constructs were transfected using a Nucleofector Kit C

143

(Amaxa, Gaithersburg, MD). After 3 weeks in R10 culture, K562 cells or 721.221 cells

144

were stained with an anti-MHC class I antibody (W6/32) conjugated to phycoeythrin

145

(PE), provided courtesy of David Watkins. Cells expressing MHC class I were then

146

isolated by magnetic-activated cell sorting (MACS) using anti-PE microbeads with LS

147

columns (Miltenyi Biotec, Auburn, CA). Transferents were maintained under drug

148

selection conditions in R10 medium with G418 (Mediatech, Manassas, VA).

149 150

IFNγ ELISPOT assays. Peripheral blood mononuclear cells (PBMCs) were isolated

151

from EDTA-treated whole blood using Ficoll-Paque PLUS (GE Health Sciences) density

152

centrifugation. ELISPOT assays were conducted according to the manufacturer’s

153

protocol. Briefly, 105 cells in 100μl of R10 medium were added to precoated monkey

154

gamma interferon (IFNγ) ELISpotPLUS plates (Mabtech Inc., Mariemont, OH) with 10μM

155

peptide. All samples were repeated in duplicate or triplicate. Full-proteome peptide sets

156

comprised pools of 10 peptides, each at a concentration of 1μM. Concanavalin A (10μM)

157

was used as a positive control. Cells alone in the absence of stimulant were used as a

158

negative control. Wells were imaged using an AID ELISPOT reader, and spots were

159

counted using an automated program with parameters including size, intensity, and

160

gradient. Experimental responses exceeding the arithmetic mean of the negative control

161

wells plus two standard deviations were considered positive. The limit of detection was

162

set at 50 spot-forming cells per million PBMCs.

163 164

Expansion of SIV-specific CD8 T cell lines. PBMCs were isolated from EDTA-treated

165

whole blood by Ficoll-Paque PLUS (GE Health Sciences) density centrifugation. Cells

166

were split into Corning T75 cell culture flasks (Fisher Scientific, Pittsburgh, PA) with 20ml

167

of RPMI 1640 (HyClone) supplemented with 15% fetal bovine serum (HyClone) and

168

100IU/ml interleukin-2 (NIH AIDS Reagent Program, Germantown, MD) (R15-100

169

medium). Each T75 flask was stimulated with 10μl of 1mM peptide for each of the

170

following: Nef

171

(ProImmune). Every week thereafter, cell lines were restimulated with irradiated peptide-

172

pulsed BLCLs as described previously (34). After a total of 4 weeks, cells were tetramer-

173

sorted by MACS (Miltenyi Biotec) and transferred into G-Rex10 flasks (Wilson Wolf,

174

Saint Paul, MN) for rapid expansion. Resorting occurred every 2 weeks in addition to

175

restimulation until at least one cell line achieved greater than 50% specificity. Two weeks

176

prior to infusion, cell lines were transferred into G-Rex100 flasks (Wilson Wolf) for further

177

rapid expansion.

103-111

RM9, Gag

389-394

GW9, Env

338-346

RF9, and Nef

254-262

LT9

178 179

Tetramers. Biotinylated peptide-MHC class I (pMHCI) monomers were synthesized as

180

described previously (35). Tetramers were produced by mixing pMHCl monomers at a

181

4:1 molar ratio with purified streptavidin conjugated to either PE or allophycocyanin

182

(APC). The tetramer staining protocol was modified slightly from a previous description

183

(36). Briefly, approximately 1x106 cells were resuspended in 100μl of R10 medium with

184

1μg/ml of the appropriate tetramer for 30 min at 37°C. Cells were then surface stained

185

with 1μl of anti-CD3-AlexaFluor700, 2.5μl of anti-CD8-Pacific Blue, 5μl of anti-CD14-

186

ECD, and 5μl of anti-CD19-ECD (BD Biosciences, San Jose, CA) for 30 min at room

187

temperature, washed with 1X PBS supplemented with 10% fetal bovine serum

188

(fluorescence-activated cell sorting buffer; FACS buffer), and fixed with 2%

189

paraformaldehyde (PFA) (Fisher Scientific). Samples were acquired using an LSRII flow

190

cytometer (BD Biosciences), and data were analyzed with FlowJo software (TreeStar

191

Inc., Ashland, OR).

192 193

Phenotypic

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homogenates were thawed and resuspended in 100μl of R10 medium for each test

195

condition. Cells were then stained for 30 min at room temperature with the following

196

conjugates: (i) 1μl of anti-CD3-AlexaFluor700, 5μl of anti-CD4-APCH7, and 5μl of anti-

197

CD8-AmCyan (BD Biosciences); (ii) 5μl of anti-CD28-Pacific Blue, 5μl of anti-CCR7-

198

FITC, and 5μl of anti-CCR9-PerCPCy5.5 (BioLegend, San Diego, CA); (iii) 5μl of anti-

199

CD95-PECy7 (Fisher Scientific); (iv) 5μl of anti-α4β7-APC (NIH Nonhuman Primate

200

Reagent Resource); and (v) 5μl of anti-CXCR3-PE (eBioscience, San Diego, CA), After

201

washing with FACS buffer, cells were fixed with 2% PFA (Fisher Scientific). Samples

202

were acquired using an LSRII flow cytometer (BD Biosciences), and data were analyzed

203

with FlowJo software (TreeStar Inc.).

analysis.

Approximately 1x106 CD8 T cells from cryopreserved

204 205

Intracellular cytokine staining. Activation of CD8 T cell lines was measured via

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intracellular staining for IFNγ and tumor necrosis factor alpha (TNFα) as described

207

previously (34). Briefly, 5x104 peptide-pulsed BLCLs were cocultured with 1x105 CD8 T

208

cells from each line. The following peptides were tested in each case: Nef

209

Gag 389-394 GW9, Env 338-346 RF9, and Nef 254-262 LT9. Brefeldin A (20μl of a 1:100 dilution)

210

was added and the cells were incubated for 4 hr at 37°C on an angle. Cells were then

211

surface stained with 1μl of anti-CD3-AlexaFluor700, 2.5μl of anti-CD4-APC, and 2.5μl of

212

anti-CD8-Pacific Blue (BD Biosciences), washed once in FACS buffer, fixed with 2%

213

PFA (Fisher Scientific), and left overnight at 4°C. The following morning, cells were

214

permeabilized in FACS buffer containing 0.1% saponin (saponin buffer), stained with

215

pretitrated concentrations of anti-IFNγ-FITC and anti-TNFα-PE (BD Biosciences),

216

washed once in saponin buffer, and fixed with 2% PFA (Fischer Scientific). Samples

217

were acquired using an LSRII flow cytometer (BD Biosciences), and data were analyzed

218

with FlowJo software (TreeStar Inc.).

103-111

RM9,

219 220

Viral suppression assay. Effector cells and infected targets were prepared as

221

described previously (37). Briefly, targets were CD8-depleted by MACS (Miltenyi Biotec),

222

incubated with concavalin A (5μg/ml) overnight, and washed. After 4 days, targets were

223

washed again and plated at 2x106 cells/ml in 48-well plates. Virus was prepared by

224

layering SIVmac239 over sucrose and spinning at 14,000rpm for 60 min. Targets were

225

exposed to virus via magnetofection. Effectors were then plated with infected targets at

226

the appropriate ratio. After 4 days in culture, cells were surface stained with anti-CD3-

227

AlexaFluor700, anti-CD4-APC and anti-CD8-Pacific Blue as described above, then

228

stained intracellularly with a pretitrated concentration of anti-p27-FITC (NIH AIDS

229

Research and Reference Reagent Program) combined with bulk permeabilization

230

reagent (Invitrogen). After washing, cells were fixed with 2% PFA (Fischer Scientific) and

231

analyzed by flow cytometry as described above. Values were normalized by dividing the

232

average percentage of p27+ cells in the experimental wells by the average percentage

233

of p27+ cells in the infected control wells.

234 235

Isolation of tissues. Fresh PBMCs were isolated from EDTA-treated whole blood by

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Ficoll-Paque PLUS (GE Health Sciences) density centrifugation. Bronchoalveolar lavage

237

(BAL) fluid was passed through a 70μm filter (BD Biosciences) and centrifuged. Tissues

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were diced with a scalpel, pressed over a 100μm filter (BD Biosciences), and washed

239

through with R10 medium.

240 241

Plasma viral load. SIVmac239 plasma viral loads were measured as described

242

previously (38, 39). Briefly, viral RNA was reverse transcribed and then quantified using

243

a LightCycler 2.0 or LightCycler 480 instrument (Roche, Indianapolis, IN). Serial dilutions

244

of SIV Gag in vitro transcript were used as an internal standard curve for each run. The

245

limit of detection was 100 viral RNA copies/ml plasma.

246 247

RESULTS

248

Characterization of nine novel SIV-specific CD8 T cell responses in MCM.

249

Previously, we identified optimal SIV-derived epitope-specific CD8 T cell responses

250

restricted by MHC class I molecules encoded universally across all MCM haplotypes and

251

those confined to M1/M3 (27, 34, 40, 41). Although the M1 and M3 haplotypes are

252

extremely common, animals are often heterozygous for one of the other five major MHC

253

haplotypes. We therefore mapped additional responses for the M2, M4, M5, M6, and M7

254

haplotypes to account for the entire MHC diversity of MCM. Using a full-proteome

255

ELISPOT assay, we identified nine novel responses and established their MHC

256

restriction by testing SIV-specific CD8 T cell lines against BLCLs transfected with a

257

single MHC class I allele and pulsed with the appropriate 15-mer peptides (Figure 1A).

258

Furthermore, we determined the minimal optimal epitope sequence in each case by

259

pulsing BLCLs with serial dilutions of progressively truncated peptides (Figure 1B). In

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this way, we identified responses specific for Nef

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LP8, Env

262

Gag

263

Discovery of the dominant MHC-restricted SIV-specific CD8 T cell responses in MCM

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facilitated our selection of specific epitopes for the naïve expansion experiments.

661-669

255-263

QL9, Gag

34-41

VL8, VpX

19-27

254-262

GR9, Pol

LT9, Pol 639-648

1030-1038

DT10, Nef

RY8, Gag 216-223

437-444

DY8, and

NY9, and generated MHC-tetramers to confirm epitope specificity (Table 1).

265 266

Rapid expansion can generate highly specific CD8 T cell lines that express

267

common phenotypic homing markers. Early CD8 T cell responses typically recognize

268

epitopes present in the wild-type virus, but do not recognize epitope escape variants in

269

the later stages of infection. We suspected that these initially mobilized T cell

270

populations might have higher precursor frequencies, which would facilitate their

271

expansion from naïve animals. Accordingly, we hypothesized that epitopes targeted by

272

acute immunodominant CD8 T cell responses could be used to expand specific cell lines

273

from SIV-naïve animals for subsequent in vivo efficacy testing. Using PBMCs isolated

274

from two SIV-naïve MCM, we cultured cell lines specific for four SIV-derived CD8 T cell

275

epitopes: Nef

276

measured the specificity of these expanded lines by MHC-tetramer analysis prior to

277

transfer (Figure 2). Lines specific for Gag

278

animals and reached up to 94.4% purity. In contrast, the lines for Env 338-346 RF9 and Nef

279

254-262

280

cultures from cy0574 expanded to specificities of greater than 60%.

103-111

RM9, Gag

389-394

GW9, Env

389-394

338-346

RF9, and Nef

254-262

LT9. We

GW9 expanded vigorously in both

LT9 from cy0573 did not expand above 1% purity, whereas the corresponding

281

To determine whether these rapidly expanded CD8 T cell lines displayed homing

282

markers typically associated with an effector memory phenotype, we conducted a flow

283

cytometric analysis to evaluate surface expression of α4β7, CXCR3, CCR7, and CCR9

284

(Table 2). The combined pre-transfer homogenates from both cy0573 and cy0574 were

285

tested and compared to a PBMC control (cy0391). In both cy0573 and cy0574, the

286

expanded CD8 T cell populations expressed CXCR3 at frequencies >97%. Lower levels

287

of α4β7 expression were observed relative to the control, and neither CCR7 nor CCR9

288

were detected in significant amounts. These phenotypic characteristics are consistent

289

with effector memory differentiation.

290 291

Cytokine production in RM9-specific CD8 T cell lines. To evaluate functional

292

activation, each SIV-specific CD8 T cell line was cocultured with cognate peptide-pulsed

293

BLCLs, irrelevant peptide-pulsed BLCLs or unpulsed BLCLs. Intracellular production of

294

IFNγ and TNFα was measured in each case by flow cytometry (Figure 3). The Nef

295

RM9-specific CD8 T cell lines responded to cognate peptide stimulation with the highest

296

frequencies of cytokine-producing cells (cy0573: 12.4% IFNγ+TNFα+; cy0574: 47.7%

297

IFNγ+TNFα+). These lines were also tested against BLCLs pulsed with Nef

298

The frequencies of detected IFNγ+TNFα+ cells were 7.3% for cy0573 and 1.2% for

299

cy0574, compared to 7.1% and 1.4% respectively in the absence of peptide. Thus, there

300

was no cytokine production above baseline in the presence of an irrelevant peptide.

301

Strong responses were not observed in the CD8 T cell lines specific for Gag 389-394 GW9,

302

Env

303

111

304

SIV-derived epitope specificities.

338-346

RF9, and Nef

254-262

254-262

103-111

LT9.

LT9 (90%), while the remaining epitope-specific lines

313

were markedly less effective. Suppression by the combined homogenate was

314

comparable to that mediated by the CD8 T cell lines specific for Gag 389-394 GW9, Env 338-

315

346

316

corresponding cell lines from cy0573. In this case, CD8 T cells specific for Nef

317

RM9 suppressed viral replication by >30%, while the other epitope-specific lines and

318

combined homogenate were substantially less potent. Together with the intracellular

319

cytokine staining data, these results suggest that Nef

320

best option for therapeutic transfer in MCM.

RF9, and Nef

254-262

LT9 in isolation. Similar results were observed with the

103-111

103-111

RM9-specific cells are the

321 322

Autologous transfer of CD8 T cells into naïve animals. To prepare for autologous

323

transfer into naïve animals, the rapidly expanded cells were first counted to determine

324

the frequency of each SIV-specific line in the combined transfusate (Figure 5). A

325

combined total of 2.82E+09 cells was transferred into cy0573, while a combined total of

326

2.69E+09 cells was transferred into cy0574 (Figure 5A). We discovered contamination in

327

two of the G-Rex100 flasks on the day of transfer, so Nef

328

backup G-Rex10 flasks were used as substitutes, which resulted in lower overall

329

quantities of CD8 T cells with this specificity because G-Rex10 flasks have 1/10 of the

330

surface area of G-Rex100 flasks (Figure 5B). However, we estimated that the purity of

331

these Nef

332

in the transfusate.

103-111

103-111

RM9-specific cells from

RM9-specific lines was sufficient to include the smaller cell populations

333 334

Prolonged persistence of transfused CD8 T cells in multiple tissues. Ensuring cell

335

persistence in recipients is a major challenge for adoptive transfer. In the autologous

336

setting, however, this is less problematic because there is no genetic variance. We

337

monitored the persistence of autologously transferred CFSE+ donor cells in cy0573 and

338

cy0574 for a period of 24 weeks post-transfusion (Figure 6). Animal cy0573 was

339

necropsied at 37 days post-infection (38 days post-transfusion) due to complications

340

unrelated to the experiment. After transfer, there was a rapid decrease in CFSE+ CD8+

341

T cells over the first 2 weeks. Nevertheless, transferred cells remained detectable in

342

peripheral blood for at least 24 weeks post-transfer in cy0574 (Figure 6A). Substantially

343

higher frequencies of transferred cells were observed in BAL fluid. Moreover, these cells

344

showed evidence of ongoing proliferation (Figure 6B). In cy0573, we detected donor

345

cells at frequencies up to 45% in BAL fluid at 14 days post-infection; at the time of

346

necropsy, these cells were still detectable at a frequency of 4.9%. In cy0574, donor cells

347

persisted in BAL fluid until at least 8 weeks post-infection (0.45% CFSE+ CD8+).

348

Notably, these frequencies are higher than those observed in peripheral blood even at 1

349

week post-infection, suggesting that donor cells may become trapped or traffic

350

predominantly to the lung. In cy0573, other tissue sites were examined at necropsy

351

(Figure 6C). Donor cells were detected in spleen, bone marrow, and internal iliac lymph

352

nodes, with trace amounts present in other lymph nodes. These data provide evidence

353

that autologously transferred SIV-specific CD8 T cells traffic to multiple tissues and

354

persist for several weeks, and may have implications for control of the viral reservoir.

355 356

No observed impact of CD8 T cells on acute phase viral loads following

357

autologous transfer. Given that SIV-specific CD8 T cells control viral replication in vitro,

358

we hypothesized that we would detect a change in the acute phase viral load profile after

359

autologous transfer and subsequent infection. One day post-transfer, animals were

360

challenged intrarectally with 7,000 TCID50 of SIVmac239 stock virus. Historically, peak

361

viral load is typically 107 viral RNA copies/ml during acute phase SIV infection (38). In

362

both cy0573 and cy0574, plasma viral loads were consistent with historic controls,

363

peaking at 107 viral RNA copies/ml (Figure 7). Plasma viral loads were monitored

364

through 24 weeks post-infection with no detectable impact during the acute or chronic

365

phase.

366 367

DISCUSSION

368

MCM are an ideal experimental model for immunological studies because they

369

display limited genetic diversity. Here, we identified and characterized MHC class I-

370

restricted SIV-specific CD8 T cell responses accounting for all of the seven major

371

haplotypes. These newly defined immunodominant specificities expand the potential for

372

adoptive transfer studies in SIV-infected MCM (31, 32).

373

One of the major challenges with adoptive transfers is to ensure cell persistence

374

in recipient animals while maintaining a functional effector phenotype (28-32). Typically,

375

CD8 T cells expanded in vitro have a limited ability to proliferate, making it difficult to

376

achieve significant numbers required for adoptive transfer (28). In this study, we

377

developed a novel protocol to isolate and expand SIV-specific CD8 T cell lines from

378

naïve MCM. This rapid expansion method provides bulk quantities of CD8 T cells

379

suitable for transfer within a relatively short period of time. Although CD8 T cells

380

amplified in vitro typically display a fully differentiated phenotype, we found that rapid

381

expansion induced an effector memory profile with characteristic patterns of homing

382

marker expression. Additionally, we demonstrated that these expanded cells persisted

383

longer in the recipient animals compared to previous adoptive transfer studies (28-32).

384

More detailed phenotypic studies in vivo were precluded by CFSE-labeling prior to

385

transfer. We also observed important differences between expanded cells with distinct

386

specificities. CD8 T cells specific for Nef 103-111 RM9 were readily amplified and achieved

387

very high levels of purity. Moreover, the resulting Nef

388

lines were most effectively able to produce cytokines and suppress viral replication in

389

vitro. Accordingly, we predict that in vivo viral suppression would have been pronounced

103-111

RM9-specific CD8 T cell

390

had we been able transfer greater quantities of these cells. In contrast, CD8 T cells

391

specific for Gag

392

and therefore unlikely to suppress viral replication in vivo after transfer, although our

393

results do no preclude in vivo efficacy in future experiments. It is notable that we

394

observed an increase in viral replication post-transfer in our suppression assays. This

395

likely reflects the use of fresh PBMCs, which provide supplemental targets for SIV

396

infection. Moving forward, we will continue to optimize these protocols to culture CD8 T

397

cell lines specific for Nef

398

transfers to assess in vivo efficacy both during acute SIV infection and in the context of a

399

“shock and kill” strategy to purge viral reservoirs in previously infected animals.

389-394

GW9, Env

103-111

338-346

RF9, and Nef

254-262

LT9 were poorly functional

RM9 with the intention of performing further autologous

400

Previously, we showed that adoptively transferred cells persisted for up to 14

401

days in MHC-matched MCM (31, 32). In this autologous transfer study, CFSE+ donor

402

cells persisted through 24 weeks post-infection, significantly longer than previous

403

transfer studies conducted in our laboratory. Although donor cells were detected in

404

multiple tissue sites, the greatest frequencies were observed in the lung. This “trapping”

405

of these relatively large cells is a previously described phenomenon in transfer studies

406

both in the MCM model and in mice (28, 29, 42). Such anatomical preferences may

407

provide another explanation for the lack of impact on SIV load. It is therefore possible

408

that greater efficacy would be observed in vivo if more cells were able to traffic to other

409

tissues. Necropsy of animal cy0573 at 37 days post-infection did reveal trafficking to

410

spleen and bone marrow, with a minimal presence in lymph nodes. These trafficking

411

sites may provide additional insights into our understanding of how these potential

412

effector cells interact with the latently infected reservoir.

413

It may be possible to utilize a conditioning regimen to eliminate recipient immune

414

cells prior to transfer and increase cell trafficking. However, this approach would result in

415

a depletion of lymphocyte targets for viral infection. Alternatively, a “prime and pull”

416

vaccine strategy may be effective in establishing memory T cells at peripheral tissue

417

sites affected in the early phases of SIV infection (43). In this scheme, active T cells are

418

“primed” through conventional vaccination strategies and then “pulled” to a localized site

419

via topical chemokine application, resulting in protective immunity via the establishment

420

of a stable memory T cell population. This method prevented the development of

421

disease in HSV-2-infected mice, but demonstrated only a modest effect on antibody

422

responses to HIVgp140. We plan to utilize this strategy to target active T cells to the

423

localized sites of initial viral replication and bypass the “trapping” phenomenon in the

424

lung, potentially improving the impact on SIV.

425

In the present study, autologous transfer of SIV-specific CD8 T cells ultimately

426

failed to control SIV replication. Peak viral loads were comparable to historic controls,

427

and no significant changes were observed during the chronic phase. The lack of

428

cytokine production by CD8 T cells specific for Gag

429

254-262

430

approach to focus on Nef

431

cytokine production and suppression of viral replication. Trafficking cells out of the lung

432

using a “prime and pull” strategy may help direct T cells to the sites of viral replication,

433

while an activating agent in the context of ART may coax latent virus out of reservoir

434

sites. These supplemental strategies could further serve to enhance viral control. Thus,

435

the protocol developed here represents an important first step toward the optimization of

436

autologous transfer strategies with potential therapeutic utility.

389-394

GW9, Env 338-346 RF9, and Nef

LT9 may explain this lack of efficacy. In future studies, we plan to optimize our 103-111

RM9-specific CD8 T cells, which exhibited potent

437 438

ACKNOWLEDGEMENTS

439

This work was funded by the National Institutes of Health through grants R01 AI084787,

440

R01 AI077376-04A1, and R33 AI082880-03, and via the National Center for Research

441

Resources (grant P51 RR000167) and the Office of Research Infrastructure Programs

442

(grant P51 OD011106). The research was conducted in part at a facility constructed with

443

support from the Research Facilities Improvement Program (grants RR15459-01 and

444

RR020141-01). Reagents used in these studies were provided by the NIH Nonhuman

445

Primate Reagent Resource. DAP is a Wellcome Trust Senior Investigator. We thank all

446

staff at the Wisconsin National Primate Research Center for their contributions to this

447

work, and members of the AIDS Vaccine Research Laboratory Virology Services team

448

under the supervision of Thomas Friedrich for help with viral load measurements.

449 450

REFERENCES

451

1.

Jin X, Bauer DE, Tuttleton SE, Lewin S, Gettie A, Blanchard J, Irwin CE, Safrit

452

JT, Mittler J, Weinberger L, Kostrikis LG, Zhang L, Perelson AS, Ho DD. 1999.

453

Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian

454

immunodeficiency virus-infected macaques. J Exp Med 189:991–998.

455

2.

Schmitz JE, Kuroda MJ, Santra S, Sasseville VG, Simon MA, Lifton MA, Racz

456

P, Tenner-Racz K, Dalesandro M, Scallon BJ, Ghrayeb J, Forman MA,

457

Montefiori DC, Rieber EP, Letvin NL, Reimann KA. 1999. Control of viremia in

458

simian immunodeficiency virus infection by CD8+ lymphocytes. Science 283:857–

459

860.

460

3.

Friedrich TC, Valentine LE, Yant LJ, Rakasz EG, Piaskowski SM, Furlott JR,

461

Weisgrau KL, Burwitz B, May GE, Leon EJ, Soma T, Napoe G, Capuano SV,

462

Wilson NA, Watkins DI. 2007. Subdominant CD8+ T-cell responses are involved

463

in durable control of AIDS virus replication. J Virol 81:3465–3476.

464

4.

Allen TM, O’Connor DH, Jing P, Dzuris JL, Mothe BR, Vogel TU, Dunphy E,

465

Liebl ME, Emerson C, Wilson N, Kunstman KJ, Wang X, Allison DB, Hughes

466

AL, Desrosiers RC, Altman JD, Wolinsky SM, Sette A, Watkins DI. 2000. Tat-

467

specific cytotoxic T lymphocytes select for SIV escape variants during resolution of

468 469

primary viraemia. Nature 407:386–390. 5.

O’Connor DH, Allen TM, Vogel TU, Jing P, DeSouza IP, Dodds E, Dunphy EJ,

470

Melsaether C, Mothe B, Yamamoto H, Horton H, Wilson N, Hughes AL,

471

Watkins DI. 2002. Acute phase cytotoxic T lymphocyte escape is a hallmark of

472

simian immunodeficiency virus infection. Nat Med 8:493–499.

473

6.

Barouch DH, Kunstman J, Kuroda MJ, Schmitz JE, Santra S, Peyerl FW,

474

Krivulka GR, Beaudry K, Lifton MA, Gorgone DA, Montefiori DC, Lewis MG,

475

Wolinsky SM, Letvin NL. 2002. Eventual AIDS vaccine failure in a rhesus monkey

476

by viral escape from cytotoxic T lymphocytes. Nature 415:335–339.

477

7.

Price DA, Goulder PJ, Klenerman P, Sewell AK, Easterbrook PJ, Troop M,

478

Bangham CR, Phillips RE. 1997. Positive selection of HIV-1 cytotoxic T

479

lymphocyte escape variants during primary infection. Proc Natl Acad Sci U S A

480

94:1890–1895.

481

8.

Hendel H, Caillat-Zucman S, Lebuanec H, Carrington M, O’Brien S, Andrieu

482

JM, Schachter F, Zagury D, Rappaport J, Winkler C, Nelson GW, Zagury JF.

483

1999. New class I and II HLA alleles strongly associated with opposite patterns of

484

progression to AIDS. J Immunol 162:6942–6946.

485

9.

Migueles SA, Sabbaghian MS, Shupert WL, Bettinotti MP, Marincola FM,

486

Martino L, Hallahan CW, Selig SM, Schwartz D, Sullivan J, Connors M. 2000.

487

HLA B*5701 is highly associated with restriction of virus replication in a subgroup

488

of HIV-infected long term nonprogressors. Proc Natl Acad Sci U S A 97:2709–

489

2714.

490

10.

Loffredo JT, Maxwell J, Qi Y, Glidden CE, Borchardt GJ, Soma T, Bean AT,

491

Beal DR, Wilson NA, Rehrauer WM, Lifson JD, Carrington M, Watkins DI.

492

2007. Mamu-B*08-positive macaques control simian immunodeficiency virus

493

replication. J Virol 81:8827–8832.

494

11.

Yant LJ, Friedrich TC, Johnson RC, May GE, Maness NJ, Enz AM, Lifson JD,

495

O’Connor DH, Carrington M, Watkins DI. 2006. The high-frequency major

496

histocompatibility complex class I allele Mamu-B*17 is associated with control of

497

simian immunodeficiency virus SIVmac239 replication. J Virol 80:5074–5077.

498

12.

499 500

Freel SA, Saunders KO, Tomaras GD. 2011. CD8(+)T-cell-mediated control of HIV-1 and SIV infection. Immunol Res 49:135–146.

13.

Chouquet C, Autran B, Gomard E, Bouley JM, Calvez V, Katlama C,

501

Costagliola D, Riviere Y. 2002. Correlation between breadth of memory HIV-

502

specific cytotoxic T cells, viral load and disease progression in HIV infection. AIDS

503

16:2399–2407.

504

14.

Addo MM, Yu XG, Rathod A, Cohen D, Eldridge RL, Strick D, Johnston MN,

505

Corcoran C, Wurcel AG, Fitzpatrick CA, Feeney ME, Rodriguez WR, Basgoz

506

N, Draenert R, Stone DR, Brander C, Goulder PJ, Rosenberg ES, Altfeld M,

507

Walker BD. 2003. Comprehensive epitope analysis of human immunodeficiency

508

virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed

509

HIV-1 genome demonstrate broadly directed responses, but no correlation to viral

510

load. J Virol 77:2081–2092.

511

15.

Kiepiela P, Ngumbela K, Thobakgale C, Ramduth D, Honeyborne I, Moodley

512

E, Reddy S, de Pierres C, Mncube Z, Mkhwanazi N, Bishop K, van der Stok M,

513

Nair K, Khan N, Crawford H, Payne R, Leslie A, Prado J, Prendergast A,

514

Frater J, McCarthy N, Brander C, Learn GH, Nickle D, Rousseau C, Coovadia

515

H, Mullins JI, Heckerman D, Walker BD, Goulder P. 2007. CD8+ T-cell

516

responses to different HIV proteins have discordant associations with viral load.

517

Nat Med 13:46–53.

518 519

16.

Betts MR, Ambrozak DR, Douek DC, Bonhoeffer S, Brenchley JM, Casazza JP, Koup RA, Picker LJ. 2001. Analysis of total human immunodeficiency virus

520

(HIV)-specific CD4(+) and CD8(+) T-cell responses: relationship to viral load in

521

untreated HIV infection. J Virol 75:11983–11991.

522

17.

Betts MR, Nason MC, West SM, De Rosa SC, Migueles SA, Abraham J,

523

Lederman MM, Benito JM, Goepfert PA, Connors M, Roederer M, Koup RA.

524

2006. HIV nonprogressors preferentially maintain highly functional HIV-specific

525

CD8+ T cells. Blood 107:4781–4789.

526

18.

Kannanganat S, Kapogiannis BG, Ibegbu C, Chennareddi L, Goepfert P,

527

Robinson HL, Lennox J, Amara RR. 2007. Human immunodeficiency virus type 1

528

controllers but not noncontrollers maintain CD4 T cells coexpressing three

529

cytokines. J Virol 81:12071–12076.

530

19.

531 532

Appay V, Douek DC, Price DA. 2008. CD8+ T cell efficacy in vaccination and disease. Nat Med 14:623–628.

20.

Berger CT, Frahm N, Price DA, Mothe B, Ghebremichael M, Hartman KL,

533

Henry LM, Brenchley JM, Ruff LE, Venturi V, Pereyra F, Sidney J, Sette A,

534

Douek DC, Walker BD, Kaufmann DE, Brander C. 2011. High-functional-avidity

535

cytotoxic T lymphocyte responses to HLA-B-restricted Gag-derived epitopes

536

associated with relative HIV control. J Virol 85:9334–9345.

537

21.

Ladell K, Hashimoto M, Iglesias MC, Wilmann PG, McLaren JE, Gras S,

538

Chikata T, Kuse N, Fastenackels S, Gostick E, Bridgeman JS, Venturi V,

539

Arkoub ZA, Agut H, van Bockel DJ, Almeida JR, Douek DC, Meyer L, Venet A,

540

Takiguchi M, Rossjohn J, Price DA, Appay V. 2013. A molecular basis for the

541

control of preimmune escape variants by HIV-specific CD8+ T cells. Immunity

542

38:425–436.

543

22.

Price DA, Asher TE, Wilson NA, Nason MC, Brenchley JM, Metzler IS, Venturi

544

V, Gostick E, Chattopadhyay PK, Roederer M, Davenport MP, Watkins DI,

545

Douek DC. 2009. Public clonotype usage identifies protective Gag-specific CD8+

546 547

T cell responses in SIV infection. J Exp Med 206:923–936. 23.

Chen H, Ndhlovu ZM, Liu D, Porter LC, Fang JW, Darko S, Brockman MA,

548

Miura T, Brumme ZL, Schneidewind A, Piechocka-Trocha A, Cesa KT, Sela J,

549

Cung TD, Toth I, Pereyra F, Yu XG, Douek DC, Kaufmann DE, Allen TM,

550

Walker BD. 2012. TCR clonotypes modulate the protective effect of HLA class I

551

molecules in HIV-1 infection. Nat Immunol 13:691–700.

552

24.

Whitney JB, Hill AL, Sanisetty S, Penaloza-MacMaster P, Liu J, Shetty M,

553

Parenteau L, Cabral C, Shields J, Blackmore S, Smith JY, Brinkman AL, Peter

554

LE, Mathew SI, Smith KM, Borducchi EN, Rosenbloom DI, Lewis MG,

555

Hattersley J, Li B, Hesselgesser J, Geleziunas R, Robb ML, Kim JH, Michael

556

NL, Barouch DH. 2014. Rapid seeding of the viral reservoir prior to SIV viraemia

557

in rhesus monkeys. Nature 512:74–77.

558

25.

Halper-Stromberg A, Lu CL, Klein F, Horwitz JA, Bournazos S, Nogueira L,

559

Eisenreich TR, Liu C, Gazumyan A, Schaefer U, Furze RC, Seaman MS,

560

Prinjha R, Tarakhovsky A, Ravetch JV, Nussenzweig MC. 2014. Broadly

561

neutralizing antibodies and viral inducers decrease rebound from HIV-1 latent

562

reservoirs in humanized mice. Cell 158:989–999.

563

26.

Archin NM, Liberty AL, Kashuba AD, Choudhary SK, Kuruc JD, Crooks AM,

564

Parker DC, Anderson EM, Kearney MF, Strain MC, Richman DD, Hudgens MG,

565

Bosch RJ, Coffin JM, Eron JJ, Hazuda DJ, Margolis DM. 2012. Administration

566

of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. Nature

567

487:482–485.

568

27.

Wiseman RW, Wojcechowskyj JA, Greene JM, Blasky AJ, Gopon T, Soma T,

569

Friedrich TC, O’Connor SL, O’Connor DH. 2007. Simian immunodeficiency virus

570

SIVmac239 infection of major histocompatibility complex-identical cynomolgus

571

macaques from Mauritius. J Virol 81:349–361.

572

28.

Minang JT, Trivett MT, Bolton DL, Trubey CM, Estes JD, Li Y, Smedley J,

573

Pung R, Rosati M, Jalah R, Pavlakis GN, Felber BK, Piatak MJ, Roederer M,

574

Lifson JD, Ott DE, Ohlen C. 2010. Distribution, persistence, and efficacy of

575

adoptively transferred central and effector memory-derived autologous simian

576

immunodeficiency virus-specific CD8+ T cell clones in rhesus macaques during

577

acute infection. J Immunol 184:315–326.

578

29.

Bolton DL, Minang JT, Trivett MT, Song K, Tuscher JJ, Li Y, Piatak MJ,

579

O’Connor D, Lifson JD, Roederer M, Ohlen C. 2010. Trafficking, persistence,

580

and activation state of adoptively transferred allogeneic and autologous Simian

581

Immunodeficiency Virus-specific CD8(+) T cell clones during acute and chronic

582

infection of rhesus macaques. J Immunol 184:303–314.

583

30.

Mee ET, Stebbings R, Hall J, Giles E, Almond N, Rose NJ. 2014. Allogeneic

584

lymphocyte transfer in MHC-identical siblings and MHC-identical unrelated

585

Mauritian cynomolgus macaques. PLoS One 9:e88670.

586

31.

Greene JM, Burwitz BJ, Blasky AJ, Mattila TL, Hong JJ, Rakasz EG, Wiseman

587

RW, Hasenkrug KJ, Skinner PJ, O’Connor SL, O’Connor DH. 2008. Allogeneic

588

lymphocytes persist and traffic in feral MHC-matched mauritian cynomolgus

589

macaques. PLoS One 3:e2384.

590

32.

Greene JM, Lhost JJ, Hines PJ, Scarlotta M, Harris M, Burwitz BJ, Budde ML,

591

Dudley DM, Pham N, Cain B, Mac Nair CE, Weiker MK, O’Connor SL, Friedrich

592

TC, O’Connor DH. 2013. Adoptive transfer of lymphocytes isolated from simian

593

immunodeficiency virus SIVmac239Deltanef-vaccinated macaques does not affect

594

acute-phase viral loads but may reduce chronic-phase viral loads in major

595

histocompatibility complex-matched recipients. J Virol 87:7382–7392.

596 597

33.

Shimizu Y, DeMars R. 1989. Production of human cells expressing individual transferred HLA-A,-B,-C genes using an HLA-A,-B,-C null human cell line. J

598 599

Immunol 142:3320–3328. 34.

Burwitz BJ, Pendley CJ, Greene JM, Detmer AM, Lhost JJ, Karl JA,

600

Piaskowski SM, Rudersdorf RA, Wallace LT, Bimber BN, Loffredo JT, Cox

601

DG, Bardet W, Hildebrand W, Wiseman RW, O’Connor SL, O’Connor DH.

602

2009. Mauritian cynomolgus macaques share two exceptionally common major

603

histocompatibility complex class I alleles that restrict simian immunodeficiency

604

virus-specific CD8+ T cells. J Virol 83:6011–6019.

605

35.

Price DA, Brenchley JM, Ruff LE, Betts MR, Hill BJ, Roederer M, Koup RA,

606

Migueles SA, Gostick E, Wooldridge L, Sewell AK, Connors M, Douek DC.

607

2005. Avidity for antigen shapes clonal dominance in CD8+ T cell populations

608

specific for persistent DNA viruses. J Exp Med 202:1349–1361.

609

36.

Wooldridge L, van den Berg HA, Glick M, Gostick E, Laugel B, Hutchinson

610

SL, Milicic A, Brenchley JM, Douek DC, Price DA, Sewell AK. 2005. Interaction

611

between the CD8 coreceptor and major histocompatibility complex class I

612

stabilizes T cell receptor-antigen complexes at the cell surface. J Biol Chem

613

280:27491–27501.

614

37.

Greene JM, Lhost JJ, Burwitz BJ, Budde ML, Macnair CE, Weiker MK, Gostick

615

E, Friedrich TC, Broman KW, Price DA, O’Connor SL, O’Connor DH. 2010.

616

Extralymphoid CD8+ T cells resident in tissue from simian immunodeficiency virus

617

SIVmac239{Delta}nef-vaccinated macaques suppress SIVmac239 replication ex

618

vivo. J Virol 84:3362–3372.

619

38.

Cline AN, Bess JW, Piatak MJ, Lifson JD. 2005. Highly sensitive SIV plasma

620

viral load assay: practical considerations, realistic performance expectations, and

621

application to reverse engineering of vaccines for AIDS. J Med Primatol 34:303–

622

312.

623

39.

O’Connor SL, Lhost JJ, Becker EA, Detmer AM, Johnson RC, Macnair CE,

624

Wiseman RW, Karl JA, Greene JM, Burwitz BJ, Bimber BN, Lank SM, Tuscher

625

JJ, Mee ET, Rose NJ, Desrosiers RC, Hughes AL, Friedrich TC, Carrington M,

626

O’Connor DH. 2010. MHC heterozygote advantage in simian immunodeficiency

627

virus-infected Mauritian cynomolgus macaques. Sci Transl Med 2:22ra18.

628

40.

Budde ML, Wiseman RW, Karl JA, Hanczaruk B, Simen BB, O’Connor DH.

629

2010. Characterization of Mauritian cynomolgus macaque major histocompatibility

630

complex class I haplotypes by high-resolution pyrosequencing. Immunogenetics

631

62:773–780.

632

41.

Budde ML, Lhost JJ, Burwitz BJ, Becker EA, Burns CM, O’Connor SL, Karl

633

JA, Wiseman RW, Bimber BN, Zhang GL, Hildebrand W, Brusic V, O’Connor

634

DH. 2011. Transcriptionally abundant major histocompatibility complex class I

635

alleles are fundamental to nonhuman primate simian immunodeficiency virus-

636

specific CD8+ T cell responses. J Virol 85:3250–3261.

637

42.

Schrepfer S, Deuse T, Reichenspurner H, Fischbein MP, Robbins RC,

638

Pelletier MP. 2007. Stem cell transplantation: the lung barrier. Transplant Proc

639

39:573–576.

640 641 642 643 644 645

43.

Shin H, Iwasaki A. 2012. A vaccine strategy that protects against genital herpes by establishing local memory T cells. Nature 491:463–467.

646

FIGURE LEGENDS

647

Figure 1 Identification of SIV-specific CD8 T cell responses. Peptide-pulsed BLCLs

648

were cultured with SIV-specific CD8 T cell lines. Activation was measured by

649

intracellular cytokine staining with anti-IFNγ-FITC and anti-TNFα-PE. A) Representative

650

activation of CD8 T cell line Env

651

matched BLCLs or transferents expressing specific alleles. B) Representative

652

determination of minimal optimal epitope sequence for Gag 255-263 NY9. MHC-matched

653

BLCLs were pulsed with serial dilutions of prospective optimal peptides. Responses

654

were normalized to the percentage of the maximum response.

661-669

QL9. CD8 T cell lines were tested against MHC-

655 656

Figure 2 Specificity of CD8 T cell lines determined by MHC-tetramer analysis. The

657

specificity of each SIV-specific CD8 T cell line was assessed prior to CFSE-labeling and

658

transfer. Plots are pre-gated on CD3+ lymphocytes. Frequencies of tetramer+ CD8+

659

cells are shown in the depicted gates.

660 661

Figure 3 Cytokine production by SIV-specific CD8 T cell lines. CD8 T cell lines

662

specific for Nef

663

cocultured with peptide-pulsed BLCLs as indicated. The frequency of dual IFNγ and

664

TNFα cytokine-producing cells measured by flow cytometry under each condition is

665

displayed on the y-axis for each SIV-specific CD8 T cell line (x-axis).

103-111

RM9, Gag

389-394

GW9, Env

338-346

RF9, and Nef

254-262

LT9 were

666 667

Figure 4 Suppression of viral replication by CD8 T cell lines. Viral suppression

668

assays were conducted with each SIV-specific CD8 T cell line and combined

669

homogenate as indicated. Nef

670

suppressed viral replication most effectively compared to the other specificities. PBMCs

671

from recipient blood taken immediately pre-transfer, immediately post-transfer, and 1

103-111

RM9-specific CD8 T cell lines from both animals

672

day post-transfer appeared to increase viral replication. Values were normalized by

673

dividing the average percentage of p27+ cells in the experimental wells by the average

674

percentage of p27+ cells in the control wells (no effectors).

675 676

Figure 5 Cell counts and composition of transfusate. A) Cell counts for SIV-specific

677

CD8 T cell lines. Due to contamination in two of the G-Rex100 flasks on the day of

678

transfer, Nef

679

in lower absolute numbers for this specificity. B) Percentage of cells from each SIV-

680

specific CD8 T cell line that contributed to the total transfusate.

103-111

RM9-specific cells from backup G-Rex10 flasks were used, resulting

681 682

Figure 6 Cell trafficking and persistence in multiple tissues. Animal cy0573 was

683

necropsied at 37 days post-infection. A) CFSE+ donor cells persisted in peripheral blood

684

for at least 24 weeks post-infection. B) CFSE+ donor cells trafficked at high frequencies

685

to the lung, as measured in BAL fluid, and persisted until at least 8 weeks post-transfer.

686

C) CFSE+ donor cells trafficked to spleen and bone marrow, as detected post-necropsy.

687

Trafficking to lymph nodes, especially the internal iliac lymph nodes, was also observed.

688 689

Figure 7 SIVmac239 challenge after autologous CD8 T cell transfer. Animals were

690

challenged intrarectally with a standard high dose of SIVmac239 (7,000 TCID50) one day

691

after autologous transfer of SIV-specific CD8 T cells. Quantitative PCR was used to

692

detect viral RNA copies in plasma. Animal cy0573 was necropsied at 37 days post-

693

infection. Historic control represents median viremia values from a previous study where

694

animals were challenged with 1,000 TCID50 (red).

695

Table 1 Characterization of MHC-restricted SIV-specific CD8 T cell responses

Protein and amino acids positive for restriction

Sequence positive for restriction as determined by indicated method Full-proteome ELISPOT assay

Tat 59-67 (39)

Peptide scanning

SIV-specific CD8 T cell line established

Allele specificity

Haplotype

Optimal epitope sequence

Optimal epitope name

Tetramer response observed

CCYHCQFCFC

Yes

A1*063:02

Universal

CCYHCQFCF

Tat CF9

RPKQAWCWF

Yes

A1*063:02

Universal

RPKQAWCWF

Env RF9

Yes

Yes

A1*063:02

Universal

RPKVPLRTM

Nef RM9

Yes

AAQQRGPRKPIKCWN

Yes

A1*063:02

Universal

GPRKPIKCW

Gag GW9

Yes

IWGQVPKFHLPVEKD

Yes

A4*01:01

Universal

QVPKFHLP

Pol QP8

Yes

TARGLLNMADKKETR

Yes

B*104:01

M1

LNMADKKET

Nef LT9

Yes

Yes

B*150:01:01

M2

RKAKIIKDY

Pol RY8

Yes

Yes

B*075:01

M3

SFPDPPTDTP

Rev SP10

Yes

Gag TV9

Env 338-346 (39)

QPINDRPKQAWCWFG

Nef 103-111 (32)

VRPKVPLRTMSYKLA

Gag 386-394 (32) Pol 591-598 (32) Nef 254-262 Pol 1030-1038

KVVPRRKAKIIKDYG

Rev 59-68 (39)

RIYSFPDPPTDTPLD

SFPDPPTDT

Gag 459-467 (39)

TAPPEDPAV

Yes

B*075:01

M3

TAPPEDPAV

Env 620-628 (39)

TVPWPNASL

Yes

B*075:01

M3

TVPWPNASL

Env TL9

Gag 221-229 (39)

PAPQQGQLR

Yes

B*075:01

M3

PAPQQGQLR

Gag PR9

Gag 146-154 (39)

VHLPLSPRTLNAWVK

Yes

B*075:01

M3

HLPLSPRTL

Gag HL9

Yes

Tat 42-29 (39)

SQLYRPLEACYNTCY

HLPLSPRTL

Yes

B*075:01

M3

QLYRPLEA

Tat QA8

Yes

Gag 28-37 (39)

GKKKYMLKHVVWAAN

Yes

B*011:01

M3

KYMLKHVVWA

Gag KA10

Gag 437-444

LGPWGKKPRNFPMAQ

Yes

B*147:01

M4

LGPWGKKP

Gag LP8

Env 661-669

EAQIQQEKNMYELQK

Yes

A1*031:01

M4

QQEKNMYEL

Env QL9

Gag 34-41

YMLKHVVWAANELDR

Yes

B*050:04

M5

VVWAANEL

Gag VL8

Yes

VpX 19-27

TIGEAFEWLNRTVEE

Yes

B*095:01

M6

GEAFEWLNR

VpX GR9

Yes

Pol 639-648

VKDPIEGEETYYTDG

Yes

A1*060:05

M7

DPIEGEETYT

Pol DT10

Nef 216-223

EVLAWKFDPTLAYTY

Yes

A1*060:05

M7

DPTLAYTY

Nef DY8

Gag 255-263

WMYRQQNPIPVGNIY

Yes

A1*060:05

M7

NPIPVGNIY

Gag NY9

Yes

Yes

Table 2 Phenotypic homing markers expressed by CD8 T cell lines Cell line α4β7 CCR7 CXCR3 CCR9 cy0391*

73.3

26.8

50.4

2.65

cy0573

36.6

0.17

97.2

2.46

cy0574

17.4

0.59

97.2

2.63

*PBMC control sample from a previous adoptive transfer experiment at 1 year post infection

A) T Cell Line: cy0389 Env661-669 QL9 MHC-Matched BLCLs

Mafa A1*031

Mafa B*147:01

10 5

TNFα-PE

10

4

10 3

10

2

0

0

10

2

10

3

10

4

10

5

0

10

2

10

3

10

4

10

5

0

10

2

10

3

10

4

10

5

IFNγ-FITC

% Maximum Response

B) T Cell Line: cy0390 Gag 255-263 NY9 100

404 (QQNPIPVGNIY) 406 (QNPIPVGNIY) 409 (NPIPVGNIY) 412 (NPIPVGNI) 562 (WMYRQQNPIPVGNIY)

50

0 1

.1

.01

.001

.0001

0

Peptide Concentration (mM)

Figure 1 Identification of SIV-specific CD8 T cell responses. Peptide-pulsed BLCLs were cultured with SIV-specific CD8 T cell lines. Activation was measured by intracellular cytokine staining with anti-IFNγ-FITC and anti-TNFα-PE. A) Representative activation of CD8 T cell line Env 661-669 QL9. CD8 T cell lines were tested against MHC-matched BLCLs or transferents expressing specific alleles. B) Representative determination of minimal optimal epitope sequence for Gag 255-263 NY9. MHC-matched BLCLs were pulsed with serial dilutions of prospective optimal peptides. Responses were normalized to the percentage of the maximum response.

cy0573 RM9

Tetramer

10 5

10

4

10

3

10 5

49.6%

10

0

10

3

10

2

10

3

10

4

10

3

5

0

RF9

10 5

10

94.4%

0

0

4

GW9

10 4

10 4

10

2

10

3

10

4

10

5

LT9

10 5

0.93%

10

0.72%

3

0

0

0

10

2

10

3

10

4

10

5

0

10

2

10

3

10

4

10

5

CD8 cy0574 10 5

10

4

RM9

10 5

81.7%

10 4

Tetramer

10 3

10

0

10

4

10

3

86.6%

3

0

0

10 5

GW9

10 2

10 3

10 4

10 5

RF9

0

10 5

76.7%

10 4

10

0

10

2

10

3

10

4

10

5

LT9

68.2%

3

0

0

10

2

10

3

10

4

10

5

0

10

2

10

3

10

4

10

5

CD8 Figure 2 Specificity of CD8 T cell lines determined by MHC-tetramer analysis. The specificity of each SIV-specific CD8 T cell line was assessed prior to CFSE-labeling and transfer. Plots are pre-gated on CD3+ lymphocytes. Frequencies of tetramer+ CD8+ cells are shown in the depicted gates.

50 Peptide Stim.

40 30

RM9

15

GW9

% IFNγ+ TNFα+

RF9 LT9 No Peptide 10

5

cy0573

LT 9

RF 9

G

W

9

9 RM

9 LT

RF 9

9 W G

RM

9

0

cy0574 SIV-specific CD8 T cell lines

Figure 3 Cytokine production by SIV-specific CD8 T cell lines. CD8 T cell lines specific for Nef 103-111 RM9, Gag 389-394 GW9, Env 338-346 RF9, and Nef 254-262 LT9 were cocultured with peptide-pulsed BLCLs as indicated. The frequency of dual IFN γ and TNFα cytokine producing cells measured by flow cytometry under each condition is displayed on the y-axis for each SIV-specific CD8 T cell line (x-axis).

cy0573 cy0574

Normalized % p27+

1.5

1.0

0.5

5m

in I n f e 5m p r e c t e -tr d in po a n s st -tr fer an sf 1 e da r y po st R M 9 G W 9 R F9 H om L og T9 en U n i ate nf ec te d

0

Figure 4 Suppression of viral replication by CD8 T cell lines. Viral suppression assays were conducted with each SIV-specific CD8 T cell line and combined homogenate as indicated. Nef 103-111 RM9-specific CD8 T cell lines from both animals suppressed viral replication most effectively compared to the other specificities. PBMCs from recipient blood taken immediately pre-transfer, immediately post-transfer, and 1 day post-transfer appeared to increase viral replication. Values were normalized by dividing the average percentage of p27+ cells in the experimental wells by the average percentage of p27+ cells in the control wells (no effectors).

A) Cell counts for SIV-specific CD8 T cell lines

SIV-specific CD8 Tcell line

cy0573

cy0574

RM9

4.92E+07

6.98E+07

GW9

1.01E+09

1.41E+09

RF9

8.78E+08

2.60E+08

LT9

8.87E+08

9.51E+08

2.82E+09

2.69E+09

Combined Total

B) Frequency of transferred cells 2%

3% RM9 GW9

31%

RF9 36%

35% 52%

31%

cy0573

LT9

10%

cy0574

Figure 5 Cell counts and composition of transfusate. A) Cell counts for SIV-specific CD8 T cell lines. Due to contamination in two of the G-Rex100 flasks on the day of transfer, Nef 103-111 RM9-specific cells from backup G-Rex10 flasks were used, resulting in lower absolute numbers for this specificity. B) Percentage of cells from each SIV-specific CD8 T cell line that contributed to the total transfusate.

A) Persistence in PBMC

% CFSE+ CD8+ T Cells

0.40 0.20

cy0573

0.15 0.05

cy0574

0.03

0.02

0.01

0

0

18

0

16

0

14

0

12

10

90

80

70

60

50

40

30

20

0

10

0.00

Days Post-Transfer

C) Persistence in tissues post-necropsy

B) Persistence in bronchoalveolar lavage cells

5.0 37 dpi

4

10

22.4%

5

0

10

2

10

3

10

4

10

4.91%

5

0

10

2

10

3

10

4

10

5

cy0574 56 dpi

10 4

10

4

10 3

10

3

3

0

10

10

2

10

31.4% 0 10 2

10 3

10 4

2

4.19%

0

10 5

0

10

2

84 dpi

10

3

10

4

10

5

0

10 5

10

4

10

4

10 4

10

3

10

3

10 3

10

2

10

2

10 2

0.22% 10 3

10 4

10 5

0.06%

0

0

10

2

10

3

10

10 3

10 4

10 5

168 dpi

10 5

10 2

10 2

140 dpi

5

0

0.447%

0

10

0

ne

10 4

0.00

Bo

5

PB M C

10

4

5

0.18%

0

0

10 2

10 3

10 4

10 5

0.0008

0.0006

0.0004

0.0002

0.0000

LN

CFSE

10

0.05

Ax illa ry

CD3

28 dpi 10 5

% CFSE+ CD8+ T Cells

14 dpi 5

10

0.10

en

10

0.15

le

3

0

Sp

10

0

r

2

2

ve

45% 0 10

10

Li

2

3

on

3

0

10

ol

10

10

10 3

3.0 0.20

C

4

w

10

ro

10 4

ar

4

10

4.0

M

10 5

BA L

28 dpi 10 5

In gu in al LN In te rn al Ilia c LN M es en te ric M LN es oc ol on ic LN

14 dpi 10 5

% CFSE+ CD8+ T Cells

cy0573

Figure 6 Cell trafficking and persistence in multiple tissues. Animal cy0573 was necropsied at 37 days post-infection. A) CFSE+ donor cells persisted in peripheral blood for at least 24 weeks post-infection. B) CFSE+ donor cells trafficked at high frequencies to the lung, as measured in BAL fluid, and persisted until at least 8 weeks post-transfer. C) CFSE+ donor cells trafficked to spleen and bone marrow, as detected post-necropsy. Trafficking to lymph nodes, especially the internal iliac lymph nodes, was also observed.

Plasma Viral RNA Copies/ml

10 8

cy0573 cy0574

10 6

Historic Control

10 4

10 2

10 0 0

50

100

150

200

Days post infection

Figure 7 SIVmac239 challenge after autologous CD8 T cell transfer. Animals were challenged intrarectally with a standard high dose of SIVmac239 (7,000 TCID50) one day after autologous transfer of SIV-specific CD8 T cells. Quantitative PCR was used to detect vRNA copies in plasma. Animal cy0573 was necropsied at 37 days post-infection. A control animal from a previous study was challenged with 1,000 TCID50 (red).