1 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 …cancerimmunolres.aacrjournals.org/content/canimm/early/2015/02/20/... · 4 Satoshi Ueha1, *, Shoji Yokochi 1, 2,

Anti-CD4 and immune checkpoint antibody synergy

1

Robust antitumor effects of combined anti-CD4-depleting antibody and anti-PD-1/PD-L1 1

immune checkpoint antibody treatment in mice 2

3

Satoshi Ueha1, *, Shoji Yokochi1, 2, *, Yoshiro Ishiwata1, 2, *, Haru Ogiwara1, Krishant Chand1, 4

Takuya Nakajima1, Kosuke Hachiga1, 2, Shigeyuki Shichino1, Yuya Terashima1, Etsuko Toda1, 5

Francis HW Shand3, Kazuhiro Kakimi4, Satoru Ito1, 2, and Kouji Matsushima1 6

7 1Department of Molecular Preventive Medicine, Graduate School of Medicine, The University 8

of Tokyo; 2IDAC Theranostics, Inc.; 3Department of Pharmacology and Therapeutics, The 9

University of Melbourne; 4Department of Immunotherapeutics, The University of Tokyo 10

Hospital. 11 * S. Ueha, S. Yokochi and Y. Ishiwata contributed equally to this paper. 12

13

Running title: Anti-CD4 and immune checkpoint antibody synergy 14

15

Keywords: cancer treatment, CD4 depletion, immune checkpoint antibodies, CTL, Treg 16

17

Correspondence: Kouji Matsushima 18

Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of 19

Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 Japan 20

Telephone: 81-3-5841-3431; E-mail: [email protected] 21

22

Conflicts of interest: SY, SU, SI and KM own stock in IDAC Theranostics, Inc. We declare no 23

other conflicts of interest. 24

Financial support: This work was supported by the Japan Science and Technology Agency 25

CREST program; Grants-in-Aid for Scientific Research (C) 25460491 (to S.U.) and (B) 26

25293113 (to K.M.) from the Japanese Ministry of Education, Culture, Sports, Science and 27

Technology; and Health and Labor Science Research Grants for Research for Promotion of 28

Cancer Control (Applied Research for Innovative Treatment of Cancer). 29

30

Word count: 5,967/ 6,000 31

Figures: 6 main, 9 supplementary 32

Tables: 2 supplementary 33

on February 28, 2019. © 2015 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 20, 2015; DOI: 10.1158/2326-6066.CIR-14-0190

http://cancerimmunolres.aacrjournals.org/


2

ABSTRACT 34

Depletion of CD4+ cells in tumor-bearing mice has strong antitumor effects. However, the 35

mechanisms underlying these effects and the therapeutic benefits of CD4+ cell depletion relative 36

to other immunotherapies have not been fully evaluated. Here, we investigated the antitumor 37

effects of an anti-CD4-depleting monoclonal antibody (mAb) as a monotherapy or in 38

combination with immune checkpoint mAbs. In B16F10, Colon 26 or LLC subcutaneous tumor 39

models, administration of the anti-CD4 mAb alone had strong antitumor effects that were 40

superior to those elicited by CD25+ Treg depletion or other immune checkpoint mAbs, and 41

which were completely reversed by CD8+ cell depletion. CD4+ cell depletion led to the 42

proliferation of tumor-specific CD8+ T cells in the draining lymph node and increased 43

infiltration of PD-1+CD8+ T cells into the tumor, with a shift towards type I immunity within the 44

tumor. Combination treatment with the anti-CD4 mAb and immune checkpoint mAbs, 45

particularly anti-PD-1 or anti-PD-L1 mAbs, synergistically suppressed tumor growth and 46

greatly prolonged survival. To our knowledge, this work represents the first report of robust 47

synergy between anti-CD4 and anti-PD-1 or anti-PD-L1 mAb therapies. 48





3

INTRODUCTION 49

Immune checkpoint modulators such as those targeting cytotoxic 50

T-lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) have 51

attracted attention due to their extraordinary antitumor effects in patients with advanced 52

melanoma, lung cancer, and renal cancer (1, 2). A monoclonal antibody (mAb) against CTLA-4 53

(ipilimumab) that enhances both early T-cell activation and CTL function was approved for 54

treatment of patients with advanced melanoma in the USA in 2011. An anti-PD-1 mAb 55

(nivolumab) that protects activated T cells from exhaustion in peripheral tissues was approved 56

for treatment of patients with melanoma in Japan and in the US in 2014. In addition, other 57

mAbs against CTLA-4 (tremelimumab), PD-1 (pembrolizumab) and programmed death-ligand 58

1 (PD-L1, a ligand for PD-1) are currently undergoing clinical trials to evaluate their antitumor 59

efficacy. However, despite clear survival benefits in a subset of tumor patients, other groups of 60

patients are refractory to these single agent therapies. 61

62

Combination therapies comprising immune checkpoint modulators that have different 63

points of action, targeting for example the activation and expansion of T cells in lymphoid 64

tissues and the exhaustion and deletion of T cells in the effector site, represent promising 65

strategies for tumor immunotherapy (1). Synergistic anti-tumor effects in advanced melanoma 66

have been reported with a combination of anti-CTLA-4 and anti-PD-1 mAbs (3). The antitumor 67

efficacy of other combinations of regulators of lymphocyte activation and expansion (e.g. 68

Lymphocyte activation gene-3/LAG-3, OX40/CD134) and of lymphocyte exhaustion and 69

deletion (e.g. T-cell immunoglobulin mucin-3/TIM-3, 4-1BB/CD137, B- and T-lymphocyte 70

attenuator/BTLA, glucocorticoid-induced TNF-receptor/GITR) are currently under investigation. 71

Because immune checkpoint modulators play both positive and negative roles in the immune 72

inhibitory pathway with some redundancy, identification of optimal therapeutic combinations 73

remains a considerable challenge. 74

75

Another approach to immune checkpoint modulation involves depleting 76

immunosuppressive leukocyte populations such as forkhead box P3 (Foxp3)+CD25+ regulatory 77

T cells (Treg), Th2 cells, T regulatory (Tr) 1/3 cells (4), myeloid-derived suppressor cells 78

(MDSC) and indoleamine-2,3-dioxygenase (IDO)+ plasmacytoid DCs (pDC) (5-7). Several 79

groups have suggested that depletion of CD4+ cells, including Tregs, Th2 cells, Tr1/3 cells, and 80

a subpopulation of MDSCs and pDCs, results in strong antitumor effects in mouse models due 81

to the enhancement of CTL responses (8-12). These antitumor effects may be associated with 82

the modulation of multiple immune checkpoints caused by CD4+ cell depletion. However, the 83

relative advantage of CD4+ cell depletion over other immune checkpoint mAb-based treatments 84





4

remains unclear. Encouraged by the positive reports surrounding the benefits of anti-CD4 mAb 85

treatment in mice, and by the recent clinical data supporting anti-CTLA-4 and anti-PD-1 mAb 86

therapies, here we examine whether treatments that combine an anti-CD4 mAb and immune 87

checkpoint modulators produce synergistic antitumor activity. 88

89

Thus, in the present study we used comprehensive immunologic analyses to compare the 90

antitumor effects of an anti-CD4-depleting mAb with those of a variety of mAbs against 91

immune checkpoint molecules, including PD-1, PD-L1, PD-L2, CTLA-4, OX40, LAG-3, 92

TIM-3, BTLA and GITR, in mouse subcutaneous tumor models. We also investigated the 93

antitumor effects of treatments that combined an anti-CD4 mAb and antibodies against these 94

immune checkpoint molecules. We report that treatment with an anti-CD4 mAb alone induces 95

strong antitumor effects and expansion of tumor-specific CD8+ T cells, and that combination of 96

an anti-CD4 mAb with anti-PD-1 or anti-PD-L1 mAbs results in striking synergy in the 97

suppression of tumor growth.98





5

MATERIALS AND METHODS 99

Mouse. Seven-week-old female C57BL/6 and male BALB/c mice were purchased from Japan 100

SLC. Fluorescent ubiquitination-based cell cycle indicator (Fucci) double transgenic mice were 101

generated by crossbreeding FucciG1-#639 and FucciS/G2/M-#474 animals (obtained from Dr. A. 102

Miyawaki through the RIKEN BRC) as described previously (13). Mice transgenic for the 103

gp100 melanoma antigen-specific Pmel-1-TCR or the ovalbumin specific OT-I TCR were 104

purchased from The Jackson Laboratory. Each experimental group contained 8 mice except 105

where otherwise specified. All animal experiments were conducted in accordance with 106

institutional guidelines with the approval of the Animal Care and Use Committee of the 107

University of Tokyo. 108

109

Cell lines and tumor models. B16F10 and LLC were obtained from the American Type Culture 110

Collection. Colon 26 was obtained from the Cell Resource Center for Biomedical Research, 111

Institute of Development, Aging, and Cancer, Tohoku University. B16F10 cells expressing the 112

truncated form of human low-affinity nerve growth factor receptor (∆hLNGFR/hCD271) were 113

generated by retroviral transduction and 2 subsequent rounds of in vivo passaging 114

(Supplementary Fig. S1). B16F10 cells (5 × 105 per mouse), LLC cells (5 × 105 per mouse) and 115

Colon 26 cells (2 × 105 per mouse) were inoculated s.c. into the right flanks of C57BL/6 or 116

BALB/c mice. Tumor diameter was measured twice weekly and used to calculate tumor volume 117

(mm3) [(major axis; mm) × (minor axis; mm) 2 × 0.5236]. 118

119

In vivo antibody treatment. Anti-CD4 (clone GK1.5), anti-CD8 (clone YTS169.4), anti-PD-1 120

(clone J43), anti-PD-L1 (clone 10F.9G2), anti-PD-L2 (clone TY25), anti-OX40 (clone OX-86), 121

anti-CTLA-4 (clone 9D9), anti-LAG-3 (clone C9B7W), anti-BTLA (clone 6A6), anti-TIM-3 122

(clone RMT3-23), anti-GITR (clone DTA-1) and anti-CD25 (clone PC-61.5.3) mAbs were 123

purchased from BioXcell. Antibodies were injected intraperitoneally at a dose of 200 μg per 124

mouse. Anti-CD4 mAb (200 μg/mouse) was administered in a single dose or in successive doses 125

on days 5 and 9 after tumor inoculation. Immune checkpoint antibodies (200 μg/mouse) were 126

administered on days 4, 8, 14 and 18 after tumor-inoculation. Combination treatments with the 127

anti-CD4 mAb and anti-immune checkpoint antibodies were administered under the same 128

conditions as respective single agent protocols. 129

130

Immunohistological analysis. Immunofluorescent staining was performed as described 131

previously (14-16) using primary antibodies and the appropriate fluorophore-conjugated 132

secondary Abs as listed in Table S1, then photographed using a SP5 confocal microscope (Leica 133

Microsystems). 134





6

135

Flow cytometry. Intravascular leukocytes were stained by intravenous injection of 136

fluorophore-conjugated mAb (3 μg/mouse) against CD45 or CD45.2 3 min prior to collecting 137

tissues. Single-cell suspensions were prepared by enzymatic or mechanical dissociation of 138

tissues with or without subsequent density separation, as described previously (17, 18). 139

Flow-Count fluorospheres (Beckman Coulter) were used to determine cell numbers and 140

normalize cell concentrations prior to antibody staining. Cells were pretreated with Fc Block 141

(anti-mouse CD16/CD32 mAb; clone 2.4G2, BioXcell), then stained with mix of 142

fluorophore-conjugated anti-mouse mAbs as indicated in Table S1. Data were acquired on a 143

Gallios flow cytometer (Beckman Coulter) and analyzed using FlowJo software (version 9.7.5; 144

FlowJo, LLC). Non-viable cells were excluded from the analysis based on forward and side 145

scatter profiles and propidium iodide staining. 146

147

Quantitative reverse transcription real-time polymerase chain reaction. Total RNA was 148

extracted using a RNeasy Mini kit (Qiagen) and converted to cDNA using ReverTra Ace qPCR 149

RT Master Mix with gDNA Remover (Toyobo) according to the manufacturer's instructions. 150

Real-time quantitative PCR analysis was performed using THUNDERBIRD Probe qPCR Mix 151

or THUNDERBIRD SYBR qPCR Mix (Toyobo) and an ABI 7500 sequence detector system 152

(Life Technologies). The primers used for the PCR reaction are listed in Table S2. The 153

expression levels of each gene were normalized to Rps3 expression level for each sample. 154

155

Statistics. Unless otherwise stated, data are presented as mean ± SE. Statistical analyses were 156

performed using GraphPad Prism software (version 6.0e, GraphPad Software). For comparisons 157

between groups in the in vivo study we used one-way ANOVA with Dunnett’s post-hoc test. For 158

comparisons between the means of two variables we used paired Student’s t-tests. Comparisons 159

of survival data between groups were made using the log-rank test after Kaplan-Meier analysis. 160

A P-value < 0.05 was considered to be statistically significant. 161

162





7

RESULTS 163

An optimized anti-CD4 mAb treatment protocol exerts robust antitumor effects 164

We began by optimizing the protocol for anti-CD4 mAb administration in B16F10, 165

Lewis lung carcinoma (LLC) and Colon 26 tumor models. Mice bearing subcutaneous tumors 166

received a single intraperitoneal injection of 200 μg anti-CD4 mAb 2 days before (day –2) or 0, 167

3, 5 or 9 days after tumor inoculation. In all three models, administration of anti-CD4 mAb on 168

days 3 and 5 significantly suppressed tumor growth (Supplementary Fig. S2A–C). B16F10 169

tumor growth, but not LLC and Colon 26 tumor growth, was also inhibited by mAb 170

administration on days –2 and 0 (Supplementary Fig. S2A). However, the growth of LLC and 171

Colon 26 tumors was not significantly affected by mAb administration at days –2 and 0 172

(Supplementary Fig. S2B and C). Successive administration of the anti-CD4 mAb on days 5 and 173

9 resulted in the greatest inhibition of tumor growth in all three models (data not shown). Doses 174

of anti-CD4 mAb (3.1 or 12.5 μg/mouse) that were insufficient to cause CD4 lymphocyte 175

depletion had no inhibitory effect on tumor growth in the melanoma model (Supplementary Fig. 176

S2D and E). Based on these results, for subsequent studies we adopted a protocol of 177

administering the anti-CD4 mAb at a dose of 200 μg/mouse successively on days 5 and 9 after 178

tumor inoculation. 179

We next compared the antitumor effects of the anti-CD4 mAb against those of a 180

variety of immune checkpoint mAbs (PD-1, PD-L1, PD-L2, CTLA-4, OX40, LAG-3, TIM-3, 181

BTLA and GITR) in the B16F10 model, because melanoma is a major target of anti-immune 182

checkpoint antibody therapy. We found that twice-weekly injections of immune checkpoint 183

antibodies were sufficient to produce the same level of anti-tumor effect as achieved with daily 184

injections (data not shown). Among the mAbs tested, the anti-CD4 mAb was the most effective 185

single-agent treatment in terms of tumor growth inhibition and survival (Fig. 1A–C). 186

Collectively, these results confirm the potent antitumor effects of anti-CD4 mAb treatment in 187

mice and reveal a surprising advantage of anti-CD4 mAb treatment over immune checkpoint 188

mAb treatment. 189

190

Anti-CD4 mAb treatment depletes CD4+ T cells and pDCs 191

To determine which cells are depleted by anti-CD4 mAb therapy, we next examined 192

changes in cell populations with immunosuppressive potential following anti-CD4 mAb 193

administration at day 5 in mice bearing B16F10 tumors. Flow cytometric analysis revealed that 194

numbers of CD4+ T cells including Foxp3+CD25+ Tregs decreased 50- to 100-fold over days 2 195

to 9 following anti-CD4 mAb administration (7 to 14 days after tumor inoculation), as 196

compared to cell numbers in phase-matched untreated tumor-bearing mice (Supplementary Fig. 197

S3A–C). When LLC tumor-bearing mice were administered anti-CD4 mAb on days 5 and 9, 198





8

CD4+ T cells disappeared from the blood until at least day 15 after the first mAb administration 199

(Supplementary Fig. S3D). pDCs, a subset of which are positive for CD4 and have been 200

implicated in the suppression of antitumor immune responses (7), also decreased 3- to 10-fold 201

over days 2 to 9 following mAb treatment (Supplementary Fig. S3A–C). MDSC subpopulations, 202

including neutrophils and Ly-6Chi or Ly-6Clo monocytes, were not significantly affected by mAb 203

treatment (data not shown). These results indicate that CD4+ T cells (including Tregs) and pDCs 204

are the targets of anti-CD4 mAb therapy. 205

206

Anti-CD4 mAb treatment increases the number of tumor-infiltrating CD8+ T cells 207

We next investigated the effects of anti-CD4 mAb therapy on tumor-infiltrating CD8+ 208

T-cell populations. Intravascular staining (IVS) is a technique that allows circulating leukocytes 209

present in tissue blood vessels (which represent a proportion of total leukocytes recovered) to be 210

distinguished from cells actually infiltrating the parenchyma of tissues, including tumors (19). 211

In untreated B16F10 tumors, about 15% of CD8+ T cells were positive for IVS, and the 212

frequency of PD-1+CD137+ tumor-reactive cells (20) was about 10-fold lower in this population 213

than in the IVS-negative parenchymal cell population (Supplementary Fig. S4A and B). 214

Anti-CD4 mAb treatment significantly increased the frequency and number of IVS-CD45- CD8+ 215

T cells in the tumor (Fig. 2A and B). The increased number of CD8+ T cells in the tumors of 216

anti-CD4 mAb-treated mice was also evident in histological sections (Fig. 2C). Furthermore, the 217

IVS–CD8+ T cells induced by anti-CD4 mAb treatment contained a higher proportion of 218

PD-1+CD137+ tumor-reactive cells (Fig. 2D and E), had greater potential to produce IFNγ in 219

response to ex vivo PMA/ionomycin stimulation (Fig. 2F and G), and showed higher specific 220

killing activity against B16F10 tumor cells (Supplementary Fig. S5A–C), compared to T cells 221

from the untreated group. In the LLC and Colon 26 tumor models, anti-CD4 mAb-treated mice 222

displayed decreased tumor growth, systemically increased CD8+CD44hiPD-1+ T cells, and 223

upregulation of LAG-3, TIM-3, and CTLA-4 in tumor-infiltrating CD8+ T cells (Supplementary 224

Fig. S6A–D). Collectively, these results suggest that anti-CD4 mAb treatment enhances 225

antitumor CD8+ T-cell responses and induces a shift towards type I immunity within the tumor. 226

227

Anti-CD4 mAb treatment promotes expansion of tumor-specific CD8+ T cells in the 228

draining lymph node 229

To further investigate the effects of anti-CD4 mAb treatment on tumor-specific CD8+ 230

T-cell responses, we adoptively transferred melanoma antigen-specific Pmel-1 TCR transgenic 231

CD8+ T cells (21) into mice 1 day before inoculation with B16F10 tumors (day –1) 232

(Supplementary Fig. S7A and B). On day 14 after tumor inoculation, numbers of Pmel-1 CD8+ 233

T cells in the blood, draining lymph node (dLN), non-dLN (ndLN), spleen and tumor were 10- 234





9

to 100-fold higher in anti-CD4 mAb-treated mice compared to that in untreated mice 235

(Supplementary Fig. S7C and D). As tumors grew, Pmel-1 CD8+ T-cell numbers were 236

unchanged or decreased in untreated group mice, whereas Pmel-1 CD8+ T-cell numbers 237

increased in anti-CD4 mAb-treated mice (Supplementary Fig. S7E). To determine the site of 238

Pmel-1 CD8+ T-cell expansion, we administered BrdU one hour prior to collecting tissues. The 239

number of BrdU+ proliferating Pmel-1 CD8+ T cells in the dLN far outnumbered those in the 240

tumor, irrespective of anti-CD4 mAb treatment (Supplementary Fig. S7F and G). Importantly, 241

proliferating cell numbers decreased between days 9 and 14 in untreated mice, but increased in 242

anti-CD4 mAb-treated mice (Supplementary Fig. S7H). Similar CD4-depletion-induced 243

proliferation was also observed in endogenous polyclonal CD8+ T cells (data not shown). These 244

data suggest that anti-CD4 mAb treatment protects tumor-reactive CD8+ T cells from deletion, a 245

mechanism of peripheral tolerance in which the continuous and excessive exposure of 246

antigen-specific T cells to cognate antigens eventually results in the loss of the antigen-specific 247

T-cell clones. 248

To confirm the effects of anti-CD4 mAb treatment on the proliferation of CD8+ T cells, 249

we used fluorescent ubiquitination-based cell-cycle indicator (Fucci) double transgenic mice. In 250

Fucci mice, Fucci-orange (mKO2) and Fucci-green (mAG) are expressed reciprocally in the 251

G0/G1 and S/G2/M phases of the cell cycle, respectively (13, 18). In the B16F10 tumor model, 252

anti-CD4 mAb treatment significantly increased the proportion of mAG+ proliferating cells 253

among CD8+CD44hi T cells in both the dLN and non-dLN, compared to the proportion of these 254

cells in untreated control mice (Supplementary Fig. S7I and J). 255

To determine whether this CD4 depletion-induced proliferation was specific for 256

tumor-specific CD8+ T cells or was a tumor antigen-independent response such as homeostatic 257

proliferation (22), we adoptively transferred a CFSE-labeled mixture of Pmel-1, 258

ovalbumin-specific OT-I and polyclonal CD8+ T cells into B16 tumor-bearing or tumor-free 259

mice with or without anti-CD4 mAb treatment (Supplementary Fig. S8A). Pmel-1 but not OT-I 260

or polyclonal CD8+ T cells selectively proliferated in the dLN of B16 tumor-bearing mice 261

(Supplementary Fig. S8B–E). These results indicate that CD4 depletion-induced T-cell 262

expansion is specific for tumor-specific CD8+ T cells. Collectively, these results suggest that 263

anti-CD4 mAb treatment systemically increases the availability of tumor-specific CD8+ T cells 264

by enhancing their proliferation in the dLN in a tumor-associated antigen-dependent manner. 265

266

Enhanced CD8+ T-cell responses underlie the antitumor effects of anti-CD4 mAb 267

treatment 268

To determine whether enhanced CTL responses are responsible for the antitumor 269

effects of anti-CD4 mAb treatment, we administered the anti-CD4 mAb together with an 270





10

anti-CD8-depleting mAb. When the anti-CD8-depleting mAb was administered together with 271

the anti-CD4 mAb, the inhibitory effect of anti-CD4 mAb treatment on tumor growth was 272

completely reversed (Fig. 3A and B). We also investigated whether treatment with an 273

anti-CD25-depleting mAb, which is widely used to deplete Foxp3+CD25+ Tregs in mice (23), 274

could produce the same effect as anti-CD4 mAb treatment. Under our administration protocol, 275

tumor growth in the anti-CD25 mAb-treated group was almost equivalent to that observed in 276

untreated mice (Fig. 3A and B). These results suggest that the tumor-specific CD8+ T cells that 277

are induced by CD4 mAb treatment are responsible for the antitumor effects of the treatment, 278

and that anti-CD4 mAb treatment might deplete immunosuppressive populations more 279

efficiently than anti-CD25 mAb treatment. 280

281

Combination treatment with anti-CD4 and anti-PD-1 or anti-PD-L1 mAbs synergistically 282

enhances antitumor effects 283

Next, we examined whether synergistic antitumor effects could be achieved by 284

supplementing anti-CD4 mAb treatment with various immune checkpoint mAbs, particularly 285

those targeting the exhaustion and deletion phase of the immune response. We devised a 286

combination treatment protocol of anti-CD4 mAb with immune checkpoint antibodies as 287

depicted in Fig. 4A. Strikingly, combination treatment with anti-CD4 and anti-PD-L1 mAbs, 288

and to a lesser extent anti-CD4 and anti-PD-1 mAbs, resulted in dramatic synergistic inhibition 289

of tumor growth in the B16F10 melanoma model (Fig. 4B and C). Combination treatment with 290

anti-CD4 and anti-CTLA-4, anti-TIM-3, anti-BTLA and anti-GITR mAbs also had additive or 291

synergistic effects (Fig. 4B and C), but anti-PD-L2, anti-OX40 and anti-LAG-3 mAbs produced 292

no synergistic antitumor effect when combined with the anti-CD4 mAb (Fig. 4B and C). 293

Survival was also prolonged by combination treatment with anti-CD4 and anti-PD-L1 mAbs 294

compared to anti-CD4 mAb monotherapy, but not by other combinations of anti-CD4 and 295

immune checkpoint mAbs (Fig. 4D). Importantly, depletion of CD8+ T cells completely 296

abrogated the tumor growth inhibition induced by the combination of anti-CD4 and anti-PD-1 297

or PD-L1 mAbs, indicating that CD8+ T cells play a critical role in the antitumor effects of the 298

combination treatment (Fig. 4E). 299

To determine whether the synergistic antitumor effects of anti-CD4 and anti-PD-1 or 300

anti-PD-L1 mAb treatment are common to other tumor types and mouse strains, we examined 301

the effect of combination treatment in the Colon 26 subcutaneous tumor model in BALB/c mice. 302

The anti-PD-1 or anti-PD-L1 mAb treatment alone did not inhibit tumor growth, whereas 303

combination treatment with anti-CD4 and anti-PD-1 or anti-PD-L1 mAbs resulted in strong 304

synergistic inhibition of tumor growth (Fig. 5A and B). These effects were completely reversed 305

by treatment with an anti-CD8 depleting mAb (Fig. 5B). Notably, we observed complete 306





11

remission in three of ten mice treated with the anti-CD4/anti-PD-1 mAb combination, and in six 307

of ten mice treated with the anti-CD4/anti-PD-L1 mAb combination. In addition, the six mice 308

that rejected the tumor in the anti-CD4/anti-PD-L1 mAb-treated group were resistant to 309

re-challenge with Colon 26 tumor cells at a dose 5 times higher than that used in the initial 310

inoculation (Fig. 5C). Collectively, these results indicate that combination treatment with 311

anti-CD4 and anti-PD-1 or anti-PD-L1 mAbs has a dramatic and robust antitumor effect that is 312

mediated by antitumor CD8+ T cells. 313

314

Blockade of the PD-1/PD-L1 signaling axis increases the number of PD-1+ tumor-reactive 315

CD8+ T cells in the circulation 316

Finally, we investigated the cellular and molecular mechanisms underlying the synergy 317

between anti-CD4 and anti-PD-1 or anti-PD-L1 mAbs in the B16F10 melanoma model. 318

Quantitative RT-PCR analysis of whole tumor tissue demonstrated that anti-CD4 mAb treatment 319

alone augmented expression of the antitumor cytokine genes Ifng and Tnf, the IFNγ-inducible 320

genes Cxcl10 and Cd274/PD-L1 (24, 25), and genes encoding the pro-apoptotic molecules Fasl, 321

Prf1/perforin, and Gzmb/Granzyme B, compared with the expression levels of these genes in 322

untreated tumors (Supplementary Fig. S9A and B). The upregulation of PD-L1 by anti-CD4 323

mAb treatment was also observed at the protein level (Supplementary Fig. S9C). However, no 324

additive or synergistic effects on gene expression were observed in groups receiving 325

combination treatment with anti-CD4 and anti-PD-1 or PD-L1 mAbs. Consistent with these 326

results, the proportion of IFNγ-producing and TNFα-producing cells within the 327

tumor-infiltrating CD8+ T-cell population was equivalent between mice receiving anti-CD4 328

mAb alone and mice receiving the combination of anti-CD4 and anti-PD-1 or anti-PD-L1 mAbs 329

(data not shown). 330

We next analyzed the effects of anti-PD-1 and anti-PD-L1 mAbs on the PD-1+CD8+ T 331

cells that increase in number in the systemic circulation in response to anti-CD4 mAb treatment. 332

We examined cell populations expressing the effector/memory T-cell marker CD44 and the 333

activation marker CD137. Combination treatment with anti-CD4 and anti-PD-L1 mAbs 334

increased the frequency of CD44hiPD-1+ cells amongst CD8+ T cells in the blood, dLN and 335

non-dLN, compared to that in mice receiving the anti-CD4 mAb alone (blood data shown in Fig. 336

6A and B). In blood CD8+ T cells, expression levels of PD-1 on cells within the CD44hiPD-1+ 337

population and the frequency of PD-1+CD137+ cells were significantly higher in mice that 338

received the combination of anti-CD4 and anti-PD-L1 mAbs compared to the corresponding 339

expression levels and frequency in mice that received the anti-CD4 mAb alone (Fig. 6A–C). In 340

contrast, combination treatment with anti-CD4 and anti-PD-1 mAbs decreased the frequency of 341

the CD44hiPD-1+ population among blood CD8+ T cells, and decreased the expression levels of 342





12

PD-1 on cells within the CD44hi PD-1+ population (Fig. 6A, E and F). However, the frequency 343

of the CD44hiCD137+ tumor-reactive cell population was higher in mice receiving the 344

combination of anti-CD4 and anti-PD-1 mAbs compared to mice receiving the anti-CD4 mAb 345

alone (Fig. 6A, E and F), suggesting that anti-PD-1 mAb treatment does not actually decrease 346

the number of tumor-reactive CD8+ T cells in the blood, but rather decreases the level of PD-1 347

expression on these cells. On the other hand, the frequency of PD-1+ cells among 348

tumor-infiltrating CD8+ T cells in anti-CD4 mAb-treated mice was not affected by treatment 349

with anti-PD-1 or anti-PD-L1 mAbs (Fig. 6D and G). 350





13

DISCUSSION 351

The recent success of anti-CTLA-4 and anti-PD-1 mAb therapies in the clinic has 352

highlighted the potential of immunotherapy for the treatment of cancer (2, 3, 26-29). However, 353

the development of immunotherapy for widespread clinical use remains in its early stages. 354

Extensive efforts have been directed toward enhancing endogenous antitumor immunity by 355

dampening the influence of immunosuppressive mechanisms. Treatment strategies have 356

included combinations of antibodies with other antibodies and with other immunotherapies or 357

anti-cancer therapeutics. In the present study, we demonstrate that antibody-mediated depletion 358

of CD4+ cells from tumor-bearing mice results in enhanced polyclonal PD-1+CD137+ 359

tumor-reactive and monoclonal tumor-specific Pmel-1 CD8+ T-cell responses, and strong 360

inhibition of tumor growth. Combination treatment with the anti-CD4 mAb and various immune 361

checkpoint mAbs, particularly anti-PD-1 and anti-PD-L1 mAbs, revealed striking synergy in 362

suppressing tumor growth and prolonging survival. 363

Several previous reports have described antitumor activity of anti-CD4 mAb treatment 364

in solid tumor models in C57BL/6 mice, including subcutaneous tumors induced by inoculation 365

with B16 melanoma cells (9, 11, 12), recurrent TC1 lung cancer cells (30), or embryo cells 366

expressing the adenovirus-derived E1A protein (10). Although the efficacy of immunotherapy in 367

mouse tumor models often depends on tumor type, taken together, these reports from 368

independent groups and our results from the present study suggest that anti-CD4 mAb treatment 369

is likely to have broad spectrum antitumor activity against solid tumors. Optimization of the 370

anti-CD4 mAb administration protocol revealed robust antitumor effects when mice received 371

the mAb on days 3 or 5, rather than when mice receive the mAb prior to tumor inoculation (day 372

–2). These results suggest that pretreatment is not necessary. However, priming and/or the 373

pre-existence of activated CD8+ T cells are important for effective anti-CD4 mAb therapy. 374

Although the mechanistic link between the timing of anti-CD4 antibody administration and the 375

efficacy of treatment remains to be elucidated, administration of the antibody to patients with 376

early-stage cancer or whose tumor burden has been reduced by surgical resection, irradiation or 377

chemotherapeutics is likely to be most beneficial. 378

A dose of anti-CD4 mAb sufficient to deplete most CD4+ cells was required in order 379

for antitumor effects to be observed. The CD4+ cell population includes Foxp3+ CD25+ Tregs, 380

Th2 cells, Tr1/3 cells (4) and IDO+ immunosuppressive pDCs (7). Considering that markedly 381

increased proliferation of tumor-specific CD8+ T cells was observed in the dLN, anti-CD4 mAb 382

treatment is likely to augment proliferation of tumor-reactive CD8+ T cells through the removal 383

of these CD4+ immunosuppressive cells from the dLN. In addition, anti-CD4 mAb treatment 384

increased the proportion of PD-1+CD137+ tumor-reactive cells and IFNγ-producing cells among 385

tumor-infiltrating CD8+ T cells in the B16F10 model, suggesting that anti-CD4 mAb treatment 386





14

augmented both the quantity and quality of tumor-specific CD8+ T-cell responses. We recently 387

demonstrated that IFNγ- and TNFα-induced cell-cycle arrest is an important mechanism 388

underlying the antitumor effects induced by tumor-specific CD8+ T-cell transfer (31). The shift 389

towards IFNγ-dominant type I immunity, which was evident in the strong induction of IFNγ and 390

TNFα in tumor-infiltrating CD8+ T cells after anti-CD4 mAb treatment, is likely to play a 391

central role in the antitumor effects that we observed (32). Notably, depletion of CD25+ Tregs 392

by administration of an anti-CD25 mAb on days 5 and 9 post tumor inoculation did not 393

reproduce the antitumor effect of anti-CD4 mAb treatment. Because some Foxp3+ Tregs have 394

low-to-negative CD25 expression, residual Foxp3+ CD25–/lo Tregs may have contributed to this 395

discrepancy. Moreover, the antitumor effects of anti-CD25 mAb treatment have been reported to 396

be optimal when the mAb is administered prior to tumor inoculation (33, 34), because when 397

administered after tumor inoculation the anti-CD25 mAb depletes not only Tregs but also other 398

activated lymphocytes expressing CD25. The involvement of Treg and other CD4+ 399

immunosuppressive populations in the suppression of CD8+ T cell-mediated antitumor 400

responses remains to be elucidated. 401

The synergy that occurs in combination treatment with anti-CD4 and anti-PD-1 or 402

anti-PD-L1 mAbs is likely due to the blockade of PD-1/PD-L1 signaling in PD-1+ activated 403

CD8+ T cells that are induced by anti-CD4 mAb treatment. We did not detect any synergistic 404

effect in terms of the quantity and quality of the tumor-infiltrating CD8+ T-cell response 405

promoted by anti-CD4 and anti-PD-1 or anti-PD-L1 mAb treatment. However, the frequency of 406

the PD-1+CD137+ and CD44hiCD137+ tumor-reactive populations increased among CD8+ T 407

cells in the blood upon blockade of the PD-1/PD-L1 signaling axis. Considering that T cells 408

continuously traffic between peripheral and secondary lymphoid tissues via the lymph-blood 409

circulation, the blockade of PD-1/PD-L1 signaling may prevent exhaustion or deletion of 410

tumor-reactive PD-1+CD8+ T cells in the tumor and allow them to migrate into the dLN, thus 411

sustaining antitumor CD8+ T-cell responses. In addition, anti-CD4 mAb treatment increased the 412

number of IFNγ-producing PD-1+ CD8+ T cells in the tumor, resulting in the upregulation of 413

IFNγ-inducible genes including PD-L1. Although the shift towards IFNγ-dominant type-1 414

immunity within the tumor contributes to the inhibition of tumor growth, it also promotes the 415

exhaustion or deletion of tumor-infiltrating PD-1+CD8+ T cells by enhancing PD-1/PD-L1 416

signaling. It is therefore likely that the synergy of the anti-CD4 and anti-PD-1 or anti-PD-L1 417

mAb combination treatment arises due to the blockade of this adverse negative feedback 418

mechanism. 419

We are in the process of developing a humanized anti-CD4 mAb with potent 420

antibody-dependent cell-mediated cytotoxicity (ADCC) as an anti-cancer therapeutic. Because 421

CD4+ T cells play important roles in both humoral and cellular immunity, the heightened risk of 422





15

infectious diseases that may be associated with transient CD4+ T-cell depletion should be 423

carefully evaluated in clinical trials. In addition, trials should seek to maximize clinical efficacy 424

and safety through rigorous optimization of the antibody administration protocol. In pre-clinical 425

studies in nonhuman primates, no serious adverse effects were detected after several weeks of 426

treatment with our humanized anti-human CD4 mAb that resulted in CD4+ T-cell depletion. In 427

addition, no severe adverse effects have been observed during phase-II clinical trials for T-cell 428

malignancy with long-term administration of other humanized anti-CD4 mAbs (35, 36). 429

Preexisting humoral immune mediators, such as immunoglobulin, plasma cells and memory B 430

cells, CD8+ T-cell responses, and unimpaired natural immunity are likely to provide basal 431

protection against infectious diseases during CD4+ T cell-depleting therapies. On the other hand, 432

consideration should also be given to the potential for the acute exacerbation of chronic diseases 433

associated with viral infection (e.g. hepatitis C and B) due to excessive activation of effector 434

and memory CD8+ T cells after CD4+ cell depletion. 435

In conclusion, our study represents the first report of robust antitumor effects of 436

combination treatment with an anti-CD4-depleting antibody and anti-PD-1 or anti-PD-L1 437

immune checkpoint antibodies in mice. We have also characterized the immunologic bases for 438

the synergy between these agents. Recent clinical trials suggest that anti-PD-1, anti-PD-L1, or 439

anti-CTLA-4 mAbs, or combinations of these agents, are not effective against all types of solid 440

tumors. Our findings suggest that combination treatment with an anti-CD4 mAb and immune 441

checkpoint mAbs, particularly anti-PD-1 or anti-PD-L1 mAbs, is likely to result in greater 442

clinical efficacy against a broader ranges of cancers. 443





16

ACKNOWLEDGMENTS 444

We thank A. Miyawaki, A. Sakaue-Sawano and the RIKEN BioResource Center for providing 445

FucciG1 and FucciS/G2/M mice; A. Hosoi for assistance with Pmel-1-B16F10 experiments; H. 446

Yamazaki, K. Tsuji, and K. Yoshioka for animal care; A. Yamashita, S. Aoki and S. Fujita for 447

expert technical assistance; and M. Otsuji, K. Takeda and S. Shibayama for helpful discussions. 448

449

FOOTNOTES 450

Author contributions: S.Y., S.U., Y.I., S.I. and K.M. designed research; S.Y., S.U., Y.I., H.O., 451

K.C., T.N., K.H., Y.T., E.T., A.H. performed research; S.Y., S.U., Y.I., H.O., K.C., S.S., K.K., S.I. 452

and K.M. analyzed data; S.Y., S.U., Y.I., K.C., F.H.W.S., K.K., S.I. and K.M. wrote the paper. 453





17

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554

555





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FIGURE LEGENDS 556

Figure 1. Antitumor effects of anti-CD4 mAb treatment. 557

Mice bearing B16F10 melanoma tumors were injected intraperitoneally with anti-CD4 mAb 558

(200 μg/mouse) on days 5 and 9 or anti-immune checkpoint mAbs on days 4, 8, 14 and 18 after 559

tumor inoculation. (A) Tumor growth curves. (B) Tumor volume on day 16 (upper panel) or day 560

15 (lower panel). (C) Survival following tumor inoculation (8 mice per group). (A, B) Data 561

represent mean ± SE of 8 mice per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to 562

control). 563

564

Figure 2. Anti-CD4 mAb treatment increases the number of tumor-infiltrating CD8+ T 565

cells. 566

Mice bearing B16F10 (A, B, D–G) or B16F10-∆hLNGFR (C) tumors were injected 567

intraperitoneally with anti-CD4 mAb on days 5 and 9, and tumor-infiltrating CD8+ T cells were 568

analyzed on day 14 after tumor inoculation. Control mice received an injection of vehicle only. 569

For flow cytometric analyses, mice were given an intravenous injection of anti-CD45.2 Ab 3 570

min prior to the collection of tissues to enable identification of cells in the blood compartment 571

(intravascular staining, IVS). (A) Flow cytometry plots of parenchymal leukocyte compartments 572

(CD45+ IVS-CD45.2–). (B) The number of parenchymal CD8+ T cells in tumor. (C) Distribution 573

of CD8+ T cells in the tumor. Green, CD8; red, ∆hLNGFR; blue, propidium iodide (PI). 574

Enlargements in white boxes show non-necrotic areas, yellow box shows necrotic area. Scale 575

bar represents 200 μm. (D) Flow cytometry plots and (E) frequencies of PD-1+ CD137+ 576

tumor-reactive cells among the parenchymal CD8+ T-cell population. (F) Flow cytometry plots 577

and (G) frequencies of IFNγ- and TNFα-producing cells among the parenchymal CD8+ T-cell 578

population following ex vivo re-stimulation with PMA and ionomycin. Data represent mean ± 579

SE of 4 mice per group and are representative of at least four independent experiments. 580

Numbers in flow cytometry plots indicate mean frequencies within live cells (A) or parental 581

populations (D and F). ***, P < 0.001 (compared to control). 582

583

Figure 3. CD8+ T cells play a pivotal role in the antitumor effects of anti-CD4 mAb 584

treatment. 585

Mice bearing B16F10 tumors were injected intraperitoneally with anti-CD4, anti-CD8 and/or 586

anti-CD25 mAbs (200 μg/mouse) on days 5 and 9 after tumor inoculation. (A) Tumor growth 587

curves. (B) Tumor volume on day 15 after tumor inoculation. Data represent mean ± SE of 8 588

mice per group. **, P < 0.05 (compared to control); ††, P < 0.01 (comparison as indicated). 589

590

Figure 4. Combination treatment with anti-CD4 and anti-PD-1 or anti-PD-L1 mAbs has 591





21

synergistic antitumor effects. 592

Mice bearing B16F10 tumors received anti-CD4 mAb, anti-immune checkpoint mAb, or a 593

combination of these, according to the treatment schedule shown in (A). (B) Tumor volume on 594

day 16 (left) or 15 (right). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to control); #, 595

P=0.021 (compared to αCD4); ††, P < 0.01; †††, P < 0.001 (comparisons as indicated). (C) 596

Tumor growth curves. (D) Survival plots representative of two independent experiments. *, P < 597

0.05; **, P < 0.01 ***, P < 0.001 ****, P < 0.0001 (compared to control); †, P < 0.05; ††, P < 598

0.01; †††, P < 0.001 (compared to αCD4). (E) Anti-CD8 mAb was administered together with 599

anti-CD4 mAb and tumor volumes were measured on day 16. **, P < 0.01 (compared to 600

control). Data represent mean ± SE of 8 mice per group. In the text as (data not shown) but 601

should insert into Figure 4. 602

603

Figure 5. Combination treatment with anti-CD4 and anti-PD-1 or anti-PD-L1 mAbs 604

induces long-term antitumor CD8+ T-cell memory. 605

Mice bearing Colon 26 tumors received anti-CD4, anti-PD-L1, anti-PD-1 or anti-CD8 mAbs or 606

a combination of these according to the treatment schedule shown in Fig. 4A. (A) Tumor growth 607

curves. (B) Tumor volume on day 18. **, P < 0.01; ***, P < 0.001 (compared to control); #, 608

P=0.029; ###, P < 0.001 (compared to αCD4); †††, P < 0.001 (comparisons as indicated). (C) 609

The six mice that achieved complete remission of Colon 26 tumors after anti-CD4 and 610

anti-PD-L1 treatment were re-challenged on day 39 with Colon 26 tumor cells at five-times the 611

cell number of the initial challenge. Arrow indicates day of re-challenge. *, P < 0.05; **, P < 612

0.01 (compared to control). (A and B) Data represent mean ± SE of 10 mice per group. 613

614

Figure 6. Anti-PD-L1 and anti-PD-1 treatments target PD-1+ CD8+ T cells that are induced 615

by anti-CD4 treatment. 616

Mice bearing B16F10 tumors were treated with anti-CD4, anti-PD-L1 or anti-PD-1 mAbs, or a 617

combination of these according to the treatment schedule shown in Fig. 4A. (A) Flow cytometry 618

plots of blood CD8+ T cells. (B and E) Proportions of CD44hi PD-1+ cells, PD-1+ CD137+ cells 619

or CD44hi CD137+ cells among blood CD8+ T cells on day 14. (C and F) Mean fluorescent 620

intensity (MFI) of PD-1 expression on CD8+ CD44hi PD-1+ cells in the blood. (D and G) 621

Proportions of PD-1+ cells among tumor-infiltrating CD8+ T cells. (B–D) show anti-PD-L1 mAb 622

experiments; (E–G) show anti-PD-1 mAb experiments. Data represent mean ± SE of 4 mice per 623

group and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 624

0.001. 625






















Published OnlineFirst February 20, 2015.Cancer Immunol Res Satoshi Ueha, Shoji Yokochi, Yoshiro Ishiwata, et al. treatment in miceantibody and anti-PD-1/PD-L1 immune checkpoint antibody Robust anti-tumor effects of combined anti-CD4 depleting

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1 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 …cancerimmunolres.aacrjournals.org/content/canimm/early/2015/02/20/... · 4 Satoshi Ueha1, *, Shoji Yokochi 1, 2,