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Two Probiotic Bacillus Spores Enhance Cellular Immunity in

BALB /C Mice

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2017-0373.R2

Manuscript Type: Article

Date Submitted by the Author: 03-Oct-2017

Complete List of Authors: Gong, Li; Zhejiang University,, Huang, Qin; Zhejiang University, ; Hainan Tropical Ocean University, College of Life Sciences and Ecology Fu, Aikun; Zhejiang University, Wu, Yanping; Zhejiang University Li, Ya-Li; Zhejiang University; College of life sciences, Hunan Normal

University Xu, Xiaogang; Zhejiang University Huang, Yi; Zhejiang University; Guangxi University Yu, Dong-You; Zhejiang University Li, Wei-fen; Zhejiang University

Is the invited manuscript for consideration in a Special

Issue? : N/A

Keyword: Bacillus subtilis, Bacillus subtilis subsp. natto, cellular immune response, natural killer cell, splenic lymphocyte subset

https://mc06.manuscriptcentral.com/cjm-pubs

Canadian Journal of Microbiology

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Two Probiotic Bacillus Spores Enhance Cellular Immunity in BALB /C Mice 1

Li Gong1, Qin Huang

1,2, Aikun Fu

1, YanPing Wu

1, Yali Li

3, Xiaogang Xu

1, Yi Huang

1,4, Dongyou Yu

1, 2

Weifen Li1,* 3

*Corresponding Author: 4

Professor Weifen Li 5

Telephone: +86-13107728728 6

E-mail: [email protected] 7

Fax: 86-571-86091820 8

Address of authors: 9

1Key Laboratory of Molecular Animal Nutrition, Ministry of Education, Institute of Feed Science, College of 10

Animal Science, Zhejiang University, 310058 Hangzhou, China 11

2College of Life Sciences and Ecology, Hainan Tropical Ocean University, Hainan Province, 572022 Sanya, 12

China 13

3School of Life Sciences, Hunan Normal University, 410006 Changsha, China 14

4College of Animal Science and Technology, Guangxi University, 530005 Nanning, China 15

16

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Abstract 17

Previous studies found that Bacillus subtilis BS02 (B. subtilis BS02) and B. subtilis subsp. natto BS04 (B. 18

subtilis natto BS04) isolated in our laboratory could activate immune response of murine macrophages in vitro. 19

This study aims to investigate the effects of dietary supplementation with Bacillus spores on the systemic 20

cellular immune response in BALB/C mice. Results showed that both B. subtilis BS02 and B. subtilis natto 21

BS04 enhanced the phagocytic function of mononuclear phagocyte system (MPS) and the cytotoxicity of natural 22

killer (NK) cells. In addition, B. subtilis BS02 could increase the respiratory burst activity of blood phagocytes. 23

Furthermore, B. subtilis BS02 and B. subtilis natto BS04 increased the percentage of IFN-γ producing CD4+ 24

cells and CD8+ T cells, but only BS04 increased the percentage of CD3

+ cells and CD3

+CD4

+ cells in 25

splenocytes. However, there were no effects on other subsets of splenic lymphocytes and mitogen-induced 26

splenic lymphocyte proliferation. All data suggested that oral administration of B. subtilis BS02 or B. subtilis 27

natto BS04 could significantly enhance cellular immunity in BALB/C mice by increasing phagocytic activity of 28

MPS and cytotoxic activity of NK cells in strain-specific manner. 29

Keywords: Bacillus subtilis, Bacillus subtilis subsp. natto, cellular immune response, natural killer cell, splenic 30

lymphocyte subset 31

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Introduction 32

Probiotics are live microbes that, when administered in adequate amount, confer a health benefit to the host (Hill 33

et al. 2014; Sanders 2008). Probiotics have been reported to interact with intestinal resident microflora and 34

epithelial and immune cells in mucosa (Lu and Walker 2001). Thus, they can benefit the gut microbiota by 35

competitively excluding pathogenic microorganisms (Zareie et al. 2006) or producing antimicrobials to inhibit 36

the growth and colonization of pathogenic bacteria (Urdaci et al. 2004), and regulating host’s local and systemic 37

immune responses (Corthesy et al. 2007). Oral administration of probiotics mainly exerts direct effects on 38

antigen presenting cells (APC, such as dendritic cells and macrophages) firstly. Then, cytokines and maturation 39

markers expressed by probiotics-stimulated APC induce T-cell response (Cella et al. 1996; Mohamadzadeh et al. 40

2005) . T-cell blasts can enter the bloodstream through the efferent lymph after the activation of APC 41

(Macpherson and Harris 2004) , subsequently influencing the distant immune response in lymphatic organs (de 42

Moreno de LeBlanc et al. 2008; Marranzino et al. 2012). 43

The well investigated probiotic for their immunomodulation properties are lactic acid bacteria, such as 44

Lactobacillus rhamnosus GG, Lactobacillus casei Shirota, Bifidobacterium animalis Bb-12, Lactobacillus 45

johnsonii La1 and Bifidobacterium lactis DR10 (Ashraf and Shah 2014). Some spore-forming Bacillus species 46

have also been used as probiotics for the prevention or treatment of gastrointestinal disorders (Cutting 2011). 47

Extensive investigations confirmed that spores of Bacillus protected by multiple layers of proteins are robust and 48

able to resist extreme environments such as excessive temperature, desiccation, exposure to solvents, and other 49

noxious chemicals (Hinc et al. 2013; Knecht et al. 2011). The spore can effectively improve feed utilization, 50

modulate intestinal microflora, enhance the immunity, and even improve antagonism to pathogens (Amuguni 51

and Tzipori 2012; Barnes et al. 2007). 52

Our previous studies have demonstrated that B. subtilis BS02 and B. subtilis natto BS04 possess 53

immunomodulatory activities on murine macrophages in vitro (Xu et al. 2012). The main focus of the present 54

study was to evaluate effects of B. subtilis BS02 and B. subtilis natto BS04 on the systemic cellular immune 55

response in BALB /C mice. 56

57

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Materials and methods 58

Animals and diets 59

Sixty 4-week-old female BALB/C mice were randomly distributed into three groups. The control group (n =20) 60

was fed on the controlled diet, and two probiotic treatment groups (n =20) were fed on the spore of B. subtilis 61

BS02 and B. subtilis natto BS04 (1 × 108

CFU per kilogram of feed) in addition to the controlled diet for 8 62

weeks, respectively (Fig. 1). The experimental protocol, animal care, and treatment were approved by the 63

Committee on Animal Care and Use of Zhejiang University. Spores were prepared by the exhaustion method 64

using Difco Sporulation Medium (Feavers et al. 1990). 65

66

Carbon clearance assay 67

After the feeding trial, six mice were sampled from each group for carbon clearance assay (CCA). CCA was 68

performed to estimate the phagocytic function of the MPS according to the method previously described (Biozzi 69

et al. 1953). Briefly, India ink was injected into the tail vein of mice at a dose of 0.01 mL/g body weight. Blood 70

was collected from the retro-orbital venous plexus at 2 and 7 min time points after carbon injection. 0.02 mL 71

blood was hemolyzed with 2 mL 0.1% Na2CO3 solution, and OD680 was measured. The phagocytic index (K) 72

was calculated using the following formula: K = (log Cl – log C2)/ (t2 - tl), where C1 and C2 stand for blood 73

carbon concentration at time t1 and t2, respectively. 74

75

Respiratory burst activity 76

Another six mice from each group were sampled for blood and spleens. Respiratory burst activity of blood 77

phagocytes was determined by nitro blue tetrazolium (NBT) assay (Choi et al. 2006) and the absorbance was 78

recorded with an ELISA reader at 540 nm. 79

80

Sampling and preparation of splenic lymphocytes 81

Lymphocytes were obtained from spleens. Briefly, spleens were treated by repeating injection with PBS, and the 82

cell-rich fluid was collected. After lysis of red blood cells, the cell suspension was incubated for 2 h in Petri 83

dishes to remove adherent cells. Then, cells were washed, counted and adjusted to 2 × 106 cells/mL in complete 84

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RPMI-1640 medium for analyzing the ratios and functionalities of lymphocytes. 85

86

NK cell cytotoxicity assay 87

Splenic lymphocytes were incubated for 72 h with a mix of IL-12 (PeproTech) and IL-18 (MBL), and then 88

collected as effector cells. YAC-1 cell line was selected as target cells. 50 µL effector cells and 50 µL YAC-1 89

target cells (1×105 cells/well) were added into 96-well plates at the effector cell: target cell (E: T) ratio of 25:1. 90

The plates were centrifuged and incubated at 37 °C, 5% CO2. After 12 h of incubation, 10 µL WST-8 reagent 91

(Dojindo Laboratories) was added (Kamiyama et al. 2005), and then the plates were additionally incubated for 4 92

h. In control group, target cells and effector cells were incubated alone with culture medium. Cell culture 93

medium was used as the blank (B). Then, the OD value at 450 nm was determined in a microplate reader. NK 94

cell cytotoxicity was calculated by the following formula: cytotoxicity (%) = (ODT–(ODT+E–ODE)–ODB)/(ODT–95

ODB) ×100. 96

97

Lymphocyte proliferation 98

Proliferation responses in total splenic lymphocytes were performed as previously described with modifications 99

(Miyamoto et al. 2002). WST-8 assay was carried out to assess lymphocyte proliferation, because it is more 100

reproducible and sensitive than MTT assay. Briefly, in a final volume of 100 µL of RPMI-1640 medium 101

containing 5% fetal calf serum, whole splenic lymphocyte preparations (2 × 106 cells/well) were cultured in 102

quadruplets in flat-bottomed 96-well microtiter plates in the presence or absence of lipopolysaccharide (LPS), 103

phytohemagglutinin (PHA) and CONA with B. subtilis BS02 or B. subtilis natto BS04. Plates were incubated at 104

37 °C, in 5% CO2 for 48 h. After incubation, 10 uL WST-8 working solution (Dojindo Laboratories) was added 105

to each well, and the plates were incubated for another 4 h. The OD was measured at 450 nm using the ELISA 106

reader. Stimulation index (SI) was calculated by the following formula: SI = (mean OD of mitogen-stimulated 107

proliferation)/ (mean OD of non-stimulated proliferation). 108

109

Phenotype analysis of splenic lymphocytes by flow cytometry 110

Cell suspensions (100 µL/tube) were stained with antibodies (eBioscience) in the following combinations: Tube 111

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1, APC anti-CD3ε, PE anti-CD4 and PerCP-Cy5.5 anti-CD8, FITC anti-CD45R (B220); Tube 2, APC anti-CD3ε 112

and FITC anti-CD49b. Relevant isotype controls were used. Percentages of following lymphocyte subsets were 113

measured: CD3+

(T lymphocytes), CD3+CD4

+ (helper T lymphocytes), CD3

+CD8

+ (cytotoxic T lymphocytes), 114

CD45R (B220)+ (B lymphocytes), and CD3

－CD49b

+ (NK cells). The percentage of CD4

+ cells was divided by 115

the percentage of CD8+

cells to calculate the CD4: CD8 ratio. 116

117

Flow cytometric analysis of intracellular IFN-γ or IL-4 in CD4+ and CD8

+ T cells 118

For stimulated cells, 100 µL cell suspensions were incubated with phorbol 12-myristate 13-acetate (PMA, 7.5 119

µL, 1 µg/mL, Alexis) and ionomycin (6 µL, 50 µg/mL, Alexis) in the presence of Brefeldin A (BFA, 6 µL, 0.5 120

mg/mL, Alexis) for 4 h at 37 °C, 5% CO2, whilst only BFA was added to unstimulated cells. Stimulated and un-121

stimulated (BFA alone) samples were then labeled with CD3-APC and CD8-PerCP-Cy5.5 at room temperature 122

(RT) for 15 min. They were fixed and permeabilized using FIX & PERM Kit (MultiSciences) according to the 123

manufacturer's instructions. Then the cells were stained at RT for 15 min for intracellular cytokines using 124

antibodies (eBioscience): IFN-γ-FITC and IL-4-PE, or appropriate controls (FITC and PE Rat IgG1 isotype 125

controls). Cells were washed, and resuspended in 0.5 mL PBS. Samples were analyzed within 24 h using a 126

FACSCalibur flow cytometer (Becton Dickinson) equipped with a 488-nm argon laser and CELLQuest software 127

was used. Percentages of CD3+4

+IFN-γ

+ cells (Th1 cells), CD3

+4

+ IL-4

+ cells (Th2 cells), CD3

+8

+ IFN-γ

+ cells 128

(Tc1 cells), and CD3+8

+ IL-4

+ cells (Tc2 cells) were analyzed. 129

130

Statistical analysis 131

All values are expressed as means ± SD. Differences between the control group and the treatment groups was 132

examined by Student's t-test using SPSS 16.0 for Windows. P-values less than 0.01 or 0.05 were considered 133

statistically significant and are indicated by asterisks in the figures. 134

135

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Results 136

The phagocytic function of the MPS 137

The mononuclear phagocyte system (MPS) comprises monocytes, macrophages and dendritic cells (DCs), which 138

collectively play key roles in innate immune responses and trigger adaptive immunity (Hoebe et al. 2004). 139

Phagocytosis was the most sensitive parameter of all the cellular innate immune responses. As depicted in Fig. 2, 140

phagocytic index of the MPS in B. subtilis BS02 and B. subtilis natto BS04 group was significantly higher than 141

that of the control group (P<0.01), respectively. In addition, phagocytic index of the MPS in the BS04 was 142

stronger than BS02 group. 143

144

Respiratory burst activity of blood phagocytes 145

Respiratory burst is regarded as a potent response in phagocytic cells to degrade internalized particles and 146

bacteria (Babior 1984). Compared to the control group, respiratory burst activity of blood phagocytes was 147

significantly increased (P<0.01) in BS02 group, while no significant difference was observed in BS04 group 148

(Fig. 3). 149

150

The cytotoxic activity of NK cells against Yac-1 cells 151

NK cells display MHC-unrestricted cytotoxicity and contribute to modulate both innate and adapted immune 152

responses (Lauwerys et al. 2000). We found that the cytotoxicity of NK cell against Yac-1 cells significantly 153

increased in two probiotic treatment groups compared with the control (P<0.01) (Fig. 4). 154

155

Flow cytometric analysis of spleen lymphocyte subsets 156

Lymphocytes are essential players in the adaptive immune response. CD4+ T helper cells modulate the level and 157

the direction of immune response through the release of Th1- or Th2-type cytokines (Zhu and Paul 2008), while 158

CD8+

T cells expand both humoral and cellular responses in vivo (Shoukry et al. 2003). CD4/CD8 ratio is a 159

strong marker of immune activation and immune senescence (Bruno et al. 2017). Our flow cytometric 160

analysis showed that, compared with the control group, there were no statistically significant differences in 161

splenic lymphocyte subsets, including the percentage of CD3+, CD3

+CD4

+, CD3

+CD8

+, CD45R (B220)

+, CD3

-162

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CD49b +

cells (Fig. 5a) and the CD4: CD8 ratio (Fig. 5b), in two Bacillus-treated groups, except that the strain 163

BS04 could significantly increase the percentage of CD3+ cells and CD3

+CD4

+ cells (Fig. 5a). 164

165

The mitogen-induced splenic lymphocyte proliferation 166

The next is to study whether the higher percentage of CD3+ cells and CD3+CD4+ cells in BS04 group is the result 167

of proliferation of splenic lymphocyte. However, we found that there were no statistically significant differences 168

for lymphocyte proliferative response induced by T cell mitogen ConA or phytohemagglutinin (PHA), and by B 169

cell mitogen lipopolysaccharide (LPS) either for 48 h or for 72 h in both groups (Fig. 6), suggesting that BS04-170

mediated higher percentage of CD3+ cells and CD3+CD4+ cells were not associated with proliferation of splenic 171

lymphocyte. 172

173

Analysis of IFN-γ-producing or IL-4-producing CD4+ and CD8

+ T cells 174

Effector CD4+ T cells are of two general subtypes, T helper 1 (Th1) and T helper 2 (Th2), which have distinct 175

and opposing activities. The expression of Th1-associated cytokines, IFN-γ, an effector of cellular responses, 176

produced principally by CD4 Th1 cells, CD8 Tc1 cells and NK cells (Szabo et al. 2002), increased in the spleens 177

from mice orally administered with B. subtilis (Duc le et al. 2004; Huang et al. 2008). Thus, we evaluated 178

percentages of IFN-γ-producing or IL-4 producing CD4+ and CD8+ T cells. Phorbol 12-myristate 13-acetate 179

(PMA)-ionomycin stimulation had no effect on the CD8 expression, but CD4 expression diminished after PMA 180

stimulation (Rostaing et al. 1999). Since the multiparameter staining of the unstimulated T-cell lymphocytes 181

showed that the vast majority of the CD8- cells were CD4

+ cells (data not shown), those cells which did not stain 182

for CD8 (left-hand quadrants) were assumed to represent the CD4+ T cells (Fig. 7 a, b, c, d). Percentages of IFN-183

γ producing CD4+ cells and CD8

+ T cells (i.e., Th1 and Tc1 cells) in the Bacillus fed group were significantly 184

higher (P<0.01) than that in the control group (Fig. 7 a, b, e), whereas only relatively low percentages (less than 185

1%) of IL-4 producing CD4+ cells and CD8

+ T cells (i.e., Th2 and Tc2 cells) were observed in both groups (data 186

not shown, deducing it impossible to apply statistical data analysis) (Fig.7 c, d). 187

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Discussion 188

Supplementation with probiotics is increasingly considered as an eco-friendly prophylactic measure in health 189

management. Most available researches have demonstrated that probiotics prevent disease by boosting immune 190

function. Some probiotic strains can stimulate the host's mucosal immune system (Boirivant and Strober 2007). 191

Local immune responses in the gut can also alter immunity in organs distal from the intestine (Belkaid and Naik 192

2013). Among probiotics, Bacillus strains are gaining more and more attention. Considerable efforts have been 193

devoted to demonstrating the benefits of Bacillus on growth, physiological status, and immune responses 194

(Barnes et al. 2007; Duc le et al. 2004). The present study described the effects of B. subtilis BS02 and B. subtilis 195

natto BS04 on the systemic immunity. A probiotic bacterium should be non-pathogenic to the hosts and this need 196

to be ascertained prior to application (Sorokulova et al. 2008). The safety of Bacillus used in this study has been 197

measured by detecting the activity of lactate dehydrogenase (LDH) in Caco-2 cell culture supernatant and we 198

found that Bacillus had no effect on cell membrane integrity (Huang et al. 2013). The numbers and percentage of 199

active CD4 and CD8 cells are critical in maintaining cellular immune response. The impaired CD4/CD8 200

equilibrium is the characteristic feature of a variety of immune disorders (Ashraf and Shah 2014). These two 201

Bacillus strains showed little effect on the subsets of spleen lymphocytes (Fig. 5), which indicated that the two 202

Bacillus strains are nontoxic, and safe to use. 203

T lymphocyte cells are an important population in immune system. Flow cytometric analysis showed that 204

B. subtilis BS02 spores had no effect on the percentage of splenic lymphocyte subsets (Fig. 5). However, BS04 205

increased the levels of CD3+ cells and CD3+CD4+ cells without stimulation of proliferation of splenic 206

lymphocytes (Fig. 6). CD4+ T cells play a central role in immune protection. It is well known that CD4

+ T cells 207

recognize antigenic peptides in the context of MHC-class II molecules and provide enhanced protection against 208

pathogen challenge (Aagaard et al. 2009). CD4+T cells also play a crucial role in directing the adaptive immune 209

response. Therefore, it is conceivable that BS04 may enhance the response of T cells, reducing the ability of 210

pathogens to escape recognition by the immune system. 211

Accumulating evidence demonstrates that dietary supplementation with probiotics augments innate immune 212

function including phagocytic activity of MPS and cytotoxic activity of NK cells (Gill et al. 2001; Neumann et 213

al. 1998). Many studies focused on Lactobacillus showed that probiotics could enhance cellular immune 214

response characterized by activation of macrophages and NK cells (Ashraf and Shah 2014; Dong et al. 2012b). 215

In the current study, oral administration of B. subtilis BS02 and B. subtilis natto BS04 spores enhanced the 216

phagocytic function of the MPS and the cytotoxic activity of NK cells (Fig. 2 and Fig. 4). In addition, B. subtilis 217

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BS02 increased the respiratory burst activity (Fig. 3) which is one of the important innate immune mechanisms 218

available with phagocytes that are aimed at killing bacterial invaders (Babior 1984). Our results were in line 219

with the results reported by Shida et al (2006), who found that peripheral blood mononuclear cells 220

stimulated by Lactobacillus casei strain Shirota helped T cells to secrete IFN-γ and augmented the activity 221

of NK cell. Our previous study showed that orally administered B. subtilis natto B4 could remarkedly 222

increase the production of IFN-γ in murine macrophages (Xu et al. 2012). All these results indicated that 223

these two Bacillus strains could improve the systemic innate immunity of mice with species and strain 224

specificity. 225

In peripheral tissues, the NK cells have been proposed to play a relevant role in T cell priming (Mailliard et 226

al. 2003; Moretta 2002). Activated NK cells can also boost ongoing adaptive responses via producing IFN-γ, 227

which promotes Th1 cell polarization (Kikuchi et al. 2004; Martin-Fontecha et al. 2004). Dong et al (2012) 228

reported that NK cell activity was significantly increased by all six Lactobacillus strains, and that the 229

Lactobacillus strains tended to promote T helper 1 cytokines (Dong et al. 2012a) . Our data demonstrated that 230

pretreatment with B. subtilis BS02 and B. subtilis natto BS04 spore enhanced percentages of IFN-γ producing 231

cell (Fig. 7), suggesting that these two B. subtilis spores could directly induce Th1 activation. It was reasonable 232

to expect that administration of probiotic B. subtilis BS02 and B. subtilis natto BS04 increased NK cytotoxicity 233

and Th1 immune response in BALB/C mice. 234

In summary, oral administration of B. subtilis BS02 or B. subtilis subsp. natto BS04 spores could induce 235

systemic immune response and promote Th1 cellular immunity indicated by enhancing percentages of IFN-γ 236

producing cell, phagocytic activity of MPS and cytotoxic activity of NK cells. 237

238

Acknowledgments 239

This study was supported by the national 863 project of China (2013AA102803D), the National Natural Science 240

Foundation of China (No. 31472128), Natural Science Foundation of Zhejiang province (LY13C170003), and 241

the Major Science and Technology Project of Zhejiang Province (No. 2006C12086), China. We thank all authors 242

of the study for their participation. 243

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366 367

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Figure Legends 368

Fig 1. The experimental scheme 369

Fig 2. Phagocytic index of the MPS 370

Each bar represents the mean ± SD (n=6). * represents p<0.05, ** <0.01 compared with control mice, the same 371

as below. 372

Fig 3. Respiratory burst activity of blood phagocytes 373

Each bar represents the mean ± SD (n=6). 374

Fig 4. Cytotoxicity of NK cells 375

The cytotoxic activity of NK cells against Yac-1 cells. Each bar represents the mean ± SD (n=6). 376

Fig 5. Effects of dietary Bacillus on splenic lymphocytes 377

(a) Percentages of splenic lymphocyte subsets, (b) The CD4: CD8 ratio. 378


Fig 6. Mitogen-induced lymphocyte proliferation 380


Fig 7. Flow cytometric analysis of IFN-γ or IL-4 producingCD4+ and CD8

+ T cells 382

(a, b) representative plots of IFN-γ producing CD4+ and CD8

+ T cells, (c, d) representative plots of IL-4 383

producing CD4+ and CD8

+ T cells. Th cells (CD3

+ CD 4

+IFN-γ

+), Th2 cells (CD3

+ CD 4

+ IL-4

+ cells), Tc1 cells 384

(CD3+ CD 8

+ IFN-γ

+ cells), Tc2 cells (CD3

+ CD 8

+ IL-4

+ cells). 385

(e) Percentages of IFN-γ producing CD4+ and CD8

+ T cells. 386

Two-colors dot plots were generated after gating on CD3+ cells; and within this gate CD8

+ or CD8－ cells 387

expressing IFN-γ or IL-4 were then determined. The quadrants were placed on the basis of the staining of 388

negative controls, i.e., cells stained with irrelevant Isotype control monoclonal antibodies. PerCP-Cy5.5 anti-389

CD8 and FITC anti-IFN-γ are, respectively, on the X-axis and on the Y-axis. PerCP-Cy5.5 anti-CD8 is on the X-390

axis and PE anti-IL-4 on the Y-axis. Each bar represents the mean ± SD (n=6). 391

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