Clinical and Experimental Immunology O R I G I N AL ART I CLE doi:10.1111/j.1365-2249.2010.04173.

Selective effects of Lactobacillus casei Shirota on T cell activation,

natural killer cell activity and cytokine production cei_4173 378..388

H. Dong,* I. Rowland,* K. M. Tuohy,* Summary

L. V. Thomas† and P. Yaqoob*
*Department of Food and Nutritional Sciences,
Modulation of host immunity is an important potential mechanism by which
University of Reading, Reading, and †Yakult UK probiotics confer health benefits. This study was designed to investigate the
Ltd, Artemis, Ruislip, UK effects of a probiotic strain, Lactobacillus casei Shirota (LcS), on immune
function using human peripheral blood mononuclear cells (PBMC) in vitro.
In addition, the role of monocytes in LcS-induced immunity was also
explored. LcS promoted natural killer (NK) cell activity and preferentially
induced expression of CD69 and CD25 on CD8+ and CD56+ subsets in the
absence of any other stimulus. LcS also induced production of interleukin
(IL)-1b, IL-6, tumour necrosis factor (TNF)-a, IL-12 and IL-10 in the absence
of lipopolysaccharide (LPS). In the presence of LPS, LcS enhanced IL-1b
production but inhibited LPS-induced IL-10 and IL-6 production, and had no
further effect on TNF-a and IL-12 production. Monocyte depletion reduced
significantly the impact of LcS on lymphocyte activation, cytokine production
and natural killer (NK) cell activity. In conclusion, LcS activated cytotoxic
lymphocytes preferentially in both the innate and specific immune systems,
which suggests that LcS could potentiate the destruction of infected cells in
the body. LcS also induced both proinflammatory and anti-inflammatory
Accepted for publication 16 March 2010 cytokine production in the absence of LPS, but in some cases inhibited LPS-
Correspondence: Dr P. Yaqoob, Department of induced cytokine production. Monocytes play an important role in LcS-
Food and Nutritional Sciences, University of induced immunological responses.
Reading, Whiteknights, PO Box 226, Reading
RG6 6AP, UK. Keywords: cytokines, immunomodulation, L. casei Shirota, NK cell activity,
E-mail: [email protected] T lymphocytes

‘live microorganisms, that when included in foods can influ-

Introduction
ence the composition and activity of the gut microbiota,
One of the most promising areas of development in the modulate the inflammatory response, improve the nonspe-
human nutritional field over the last two decades has been cific intestinal barrier, and reinforce or modulate the
the use of probiotics and recognition of their role in human mucosal and the systemic immune responses’ [1]. It has also
health and disease. Lactic acid-producing bacteria are the been suggested that the selection of an appropriate probiotic
most commonly used probiotics in foods and supplements. strain for its inclusion in a probiotic preparation should be
The means by which probiotic bacteria elicit their health made on the basis of its capacity to induce an improved gut
effects are not understood fully, but may include competitive immune response [2].
exclusion of enteric pathogens, neutralization of dietary car- Probiotics have been shown to reduce the duration and
cinogens, production of anti-microbial metabolites and total symptom score of the common cold in healthy volun-
modulation of mucosal and systemic immune function [1]. teers [3], to decrease the duration of infection in older
According to the currently adopted definition by the Food in-patients [4] and to reduce the incidence of influenza-like
and Agriculture Organization/World Health Organization illness in healthy subjects [5]. Human intervention studies
(FAO/WHO), probiotics are defined as ‘live microorganisms have shown that probiotics enhance innate immunity,
which when administered in adequate amounts confer a including natural killer (NK) cell activity, phagocytic activity
health benefit on the host’. However, in recognition of the and respiratory burst [5–11]. However, the reported effects
increasing evidence for effects of probiotics on immune of probiotics on cytokine production are complex and
function, this definition has sometimes been extended to inconsistent. Some probiotic strains, such as Lactobacillus

378 © 2010 British Society for Immunology, Clinical and Experimental Immunology, 161: 378–388
 Probiotics and immune function

rhamnosus GG, and a mixture of probiotic strains of L. harvested in the exponential phase, resuspended in
gasseri CECT 5714 and L. coryniformis CECT 5711, appear to phosphate-buffered saline (PBS; Oxoid), centrifuged twice at
enhance production of the anti-inflammatory cytokine, 1960 g (Sanyo/MSE Micro Centaur, Haverhill, USA) for
interleukin (IL)-10, while having no effect or slightly 5 min and resuspended at the required concentration in
decreasing production of the proinflammatory cytokines, RPMI-1640 (Autogen Bioclear, Wiltshire, UK) containing
IL-6, interferon (IFN)-g and tumour necrosis factor 0·75 mM glutamine (Autogen Bioclear).
(TNF)-a. However, a number of human studies report no
effect of probiotics on the ex vivo production of cytokines
Preparation of PBMC and monocyte-depleted PBMC
[5,7,12,13]. Furthermore, there are few data on the effects of
(MD-PBMC)
probiotics on cellular immunity, either from human studies
or from in vitro experiments. Fasted blood samples were taken from 19 healthy adult
It is becoming clear that the immunomodulatory effects of donors aged 28–44 years in sodium heparin vacutainer tubes
probiotics are strain-dependent. L. casei Shirota (LcS), a (Greiner Bio-One Ltd, Gloucestershire, UK). Blood was
commercial probiotic strain, increases the number of ben- layered over an equal volume of Lympholyte (Cedarlane
eficial intestinal bacteria and improves the balance between Laboratories Ltd, Tyne & Wear, UK) and centrifuged at 930 g
beneficial and potentially harmful intestinal bacteria [14], for 15 min at room temperature. The plasma was removed
prevents the recurrence of superficial bladder cancer and of into a sterile 15 ml tube for later use. Cells were harvested
colorectal polyps with moderate atypia [15,16], improves from the interface, washed once, resuspended in RPMI-1640
chronic constipation [17], enhances NK cell activity [18] and medium (containing glutamine) and the above steps were
modifies allergen-induced immune responses in allergic then repeated to achieve a lower degree of erythrocyte
rhinitis [19]. However, the effects of LcS on immune func- contamination. The pellet was finally resuspended in RPMI-
tion are still poorly understood, particularly with respect to 1640 and the cell number was adjusted to 2 ¥ 106 cells/ml.
acquired immunity. In an in vitro study using human periph- For monocyte depletion experiments, monocytes were
eral blood mononuclear cells (PBMCs), LcS was reported to removed from PBMC using anti-CD14 magnetic beads (BD
enhance NK cell activity and induce IL-12 production [20]. Biosciences, Oxford, UK) and the efficiency of depletion
In another in vitro study, heat-killed LcS stimulated IL-10, (<1·5% monocytes) was verified by flow cytometry. The
IL-12, TNF-a and IFN-g production, promoted NK cell MD-PBMCs were washed twice with PBS before being
activity and activated CD69 expression on NK cells. The adjusted to 2 ¥ 106 cells/ml.
study also indicated that monocytes are important for these
effects [21].
In vitro culture conditions
The aim of the current study was to characterize the
immunomodulatory properties of LcS in an in vitro system PBMC were incubated in 24-well plates in the presence of
using PBMCs, particularly with respect to T lymphocyte 105, 106 or 107 colony-forming units (CFU)/ml LcS and 2·5%
activation, and to determine the role of monocytes in these autologous plasma for 24h at 37°C in an air/CO2 (19 : 1)
effects. atmosphere. The ratios of PBMC and LcS were 10 : 1, 1 : 1
and 1 : 10, respectively. For lymphocyte activation experi-
ments, PBMC were incubated in the presence or absence of
Materials and methods
2·5 mg/ml concanavalin A (ConA; Sigma) and for cytokine
production they were incubated in the presence or absence
LcS preparation
of 1 mg/ml lipopolysaccharide (LPS; Sigma). At the end of
Stock cultures of LcS, isolated from the fermented milk the incubation, cells were stained for activation marker mea-
product Yakult (Yakult Europe B.V., Almere, the Nether- surement, and supernatants were collected and stored at
lands), were grown on de Man, Rogosa and Sharpe (MRS) -20°C for later analysis of cytokine production. Non-
agar (Oxoid, Hampshire, UK) for 48 h at 37°C in an anaerobic stimulated cultures were used as negative controls. For
cabinet (MACS MG 1000; Don Whitley Scientific, West York- monocyte depletion experiments, PBMCs or MD-PBMCs
shire, UK) with a gas mixture of 10% H2, 10% CO2 and 80% were incubated with LcS at 106 CFU/ml for 24 h in the pres-
N2 by volume, subsequently preserved in Microbank® mixed ence or absence of ConA or LPS.
vials (ProLab Diagnostics, Neston, UK) at -80°C and kept as
a stock for future use. For liquid culture, one pure colony was
Lymphocyte activation analysis
taken from an MRS nutrient agar plate and grown overnight
in 10 ml of pre-reduced MRS broth (Oxoid) with 0·05% Cells were removed gently from wells using cell scrapers and
L-cysteine hydrochloride (Sigma, Dorset, UK) in a shaking plastic pipettes, stained with appropriate combinations of
incubator (cooled orbital incubator; Gallenkamp, Loughbor- fluorescently labelled monoclonal antibodies, including
ough, UK) at 37°C, and 0·5 ml of the overnight culture was fluorescein isothiocyanate (FITC)-labelled anti-CD69 or
inoculated into another 10 ml MRS broth. The bacteria were anti-CD25 and phycoerythrin (PE)-labelled anti-CD3, anti-

© 2010 British Society for Immunology, Clinical and Experimental Immunology, 161: 378–388 379
H. Dong et al.

CD4, anti-CD8 or anti-CD56, and fixed with Cell Fix (all 106 CFU/ml, representing a ratio of PBMC and LcS 1 : 1
from BD Biosciences). The fixed cells were analysed on a (P < 0·001) compared with unstimulated cultures (Fig. 1a).
Becton Dickinson FACSCalibur flow cytometer (BD Bio- LcS-induced CD69 expression by CD8+ lymphocytes was
sciences) within 24 h. The lymphocytes were gated and fluo- particularly marked, although CD4+ cells were also stimu-
rescence data for 10 000 events were collected and analysed lated by LcS (Fig. 1b). LcS alone also stimulated expression
using Becton Dickinson CellQuest software. of CD25 (Fig. 1c). In this case, however, LcS increased
expression of CD25 by CD8+ cells significantly, but not by
CD4+ cells (Fig. 1d), suggesting preferential activation of the
Cytokine analysis
CD8+ subset.
The concentrations of IL-1b, IL-6, IL-10, IL-12 (p70) and In the presence of ConA, LcS had no further effect on
TNF-a in the supernatants of PBMC or MD-PBMC cultures expression of CD69 or CD25 compared with those cultures
were measured by commercially available enzyme-linked stimulated by ConA alone (Fig. 1). At suboptimal ConA con-
immunosorbent assay (ELISA) kits (BD Biosciences), centrations (1·25 and 0·625 mg/ml), there was a tendency for
according to the manufacturer’s instructions. LcS to enhance mitogen-stimulated CD69 and CD25 expres-
sion compared with ConA alone, but the effects did not
reach statistical significance (data not shown).
NK cell activity
Freshly prepared PBMC and MD-PBMC or 24 h-incubated
LcS induces cytokine production by PBMC and whole
PBMC or MD-PBMC with medium or LcS at 106 CFU/ml
blood cultures
were adjusted to a concentration of 5 ¥ 106 cells/ml. Viable
target cells (K562) were enumerated by microscopy of PBMC were exposed to LcS at 105, 106 or 107 CFU/ml in the
Trypan blue-stained cell preparations and 5 ¥ 106 cells were presence or absence of LPS, and the results showed that LcS
collected and washed twice with PBS before incubation with at 106 CFU/ml had the maximum effect on induction of the
carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) cytokine production (data not shown). Figure 2 shows
(1 mg/ml; Sigma, Dorset, UK) for 45 min at 37°C in an air/ cytokine production induced by LcS at 106 CFU/ml, in the
CO2 (19 : 1) atmosphere. After incubation, the target cells presence or absence of LPS. In the absence of LPS, LcS
were washed twice and resuspended in 1 ml of complete strongly induced production of IL-1b by PBMC (P < 0·001)
medium composed of RPMI-1640 medium, 0·75 mM compared with non-stimulated cultures. Stimulation with
glutamine and 10% newborn calf serum (Sigma, Dorset, LPS also induced IL-1b production, but to a significantly
UK). PBMC or MD-PBMC were incubated with CFDA-SE- lesser degree than LcS (P < 0·05). When PBMC were exposed
labelled target cells for 2 h at 37°C in an air/CO2 (19 : 1) to both LPS and LcS the IL-1b-inducing effects were addi-
atmosphere at effector to target cell ratios of 100 : 1, 50 : 1, tive, in that LcS enhanced IL-1b production by LPS-
25 : 1 and 12·5 : 1. Twenty microlitres of propidium iodide stimulated cells significantly (P < 0·05).
(PI) (Sigma) at 1 mg/ml were added to the samples prior to In the absence of LPS, LcS induced production of IL-6
analysis on the flow cytometer. The results were expressed as by PBMC significantly (P < 0·001) compared with non-
the percentage lysis of the target cells. stimulated cultures. However, induction of IL-6 by LPS was
greater than that by LcS (P = 0·09; Fig. 2). When PBMC were
exposed to both LcS and LPS, IL-6 production was reduced
Statistics
significantly compared with LPS alone, suggesting that
All the values are presented as mean and standard deviation. although LcS itself induced IL-6 production, it inhibited
All data were analysed using spss version 15·0. Significant LPS-induced IL-6 production.
differences among treatments were evaluated by two-way The effects of LcS on TNF-a production were similar to
analysis of variance (anova) or Student’s t-test when those on production of IL-1b. LcS alone strongly induced
applicable. The criterion for statistical significance was production of TNF-a by PBMC, and also induced greater
defined as P < 0·05. TNF-a production by LPS-stimulated PBMC cultures than
LPS alone (P < 0·05). LPS alone had no effect on induction
of TNF-a.
Results
LcS induced IL-12 production by PBMC (P < 0·01) com-
pared with unstimulated cultures, but had no further effect
LcS induces CD69 expression on CD4+ and CD8+
on LPS-treated cultures. The results also suggested that LPS
lymphocytes and CD25 expression on CD8+
was unable to induce IL-12.
lymphocytes
LcS alone induced IL-10 production significantly in
Approximately 2·5% of unstimulated lymphocytes expressed PBMC cultures (P < 0·001) compared with non-stimulated
CD69 (Fig. 1a). In the absence of ConA, LcS increased CD69 cultures, but to a lesser extent than LPS (P < 0·05). Similar to
expression by lymphocytes, with a maximal effect at IL-6 production, LcS modulated the effect of LPS-induced

380 © 2010 British Society for Immunology, Clinical and Experimental Immunology, 161: 378–388
 Probiotics and immune function

(a) Fig. 1. Effect of Lactobacillus casei Shirota (LcS) on expression of

70 CD69 and CD25 by lymphocytes. Peripheral blood mononuclear cells
(PBMCs) were cultured with live L. casei Shirota at 105, 106 or 107
CD69 expression (%)

60
colony-forming units (CFU)/ml in the presence or absence of
50
concanavalin A (ConA) (2·5 mg/ml). Non-stimulated cultures were
40
shown as negative control. Each bar represents the mean of 6 to 19
30 *** *** blood donors and the error bars represent standard deviation [49].
20 *P < 0·05, **P < 0·01, ***P < 0·001 compared with non-stimulated
***
10 cultures.
0 䉳
−LcS LcS LcS LcS −LcS LcS LcS LcS
105 106 107 105 106 107
IL-10 production. Although not significant at 106 CFU/ml,
LcS at 107 CFU/ml reduced IL-10 production significantly by
−ConA ConA
LPS-stimulated PMBC compared with LPS alone (data not
(b)
shown).
45 In summary, production of IL-1b, IL-6, IL-10, IL-12 and
CD4+ TNF-a by unstimulated PBMC was very low. When stimu-
CD69 expression (%)

40
CD8+
35 lated by LcS alone, production of all cytokines analysed was
30 induced, but to different extents. Conversely, LPS induced
25
production of IL-1b, IL-6 and IL-10, but not TNF-a or
20 ***
*** IL-12. When PBMC were exposed to both LcS and LPS,
15
10 *** production of IL-1b and TNF-a was enhanced, but that of
5 *** *** IL-6 and IL-10 was inhibited, and there was no effect on
0 IL-12 production compared with cytokines induced by LPS
−LcS LcS LcS LcS −LcS LcS LcS LcS
alone.
105 106 107 105 106 107
In addition to assessing the effects of LcS on cytokine
−ConA ConA
production, a secondary aim of the study was to verify
whether PBMC samples can be replaced by whole blood
(c)
samples for cytokine measurement. Table 1 shows the effects
70
of LcS on cytokine production measured in the supernatants
CD25 expression (%)

60 of both PBMC and whole blood cultures. Whole blood cul-

50 tures produced very similar results, and levels of cytokines
40 produced by PBMC and whole blood were correlated highly
30 (P < 0·01, r = 0·69–0·83, data not shown), suggesting that
20 *** *** *** whole blood cultures could be employed for these types of
10 experiments.
0
−LcS LcS LcS LcS −LcS LcS LcS LcS
105 106 107 105 106 107 Role of monocytes in LcS-induced immunomodulation
−ConA ConA To study the role of monocytes in LcS-induced immuno-
modulation, the effects of LcS at 106 CFU/ml on expres-
(d) sion of activation markers and cytokine production of
45 CD4 PBMC and monocyte-depleted PBMC (MD-PBMC) were
CD25 expression (%)

40 CD8 compared. In this experiment, activation markers were

35
30
examined on CD3+, CD4+, CD8+ and CD56+ subsets of lym-
25 phocytes in PBMC and MD-PBMC. LcS stimulated a signifi-
20 cant increase in CD69 expression on all the above subsets in
15 PBMC (P < 0·05). Following depletion of monocytes, LcS
10 *** ***
5 ***
still increased the expression of CD69 on CD3+, CD8+ and
0 CD56+ subsets relative to no stimulus, but did not increase
−LcS LcS LcS LcS −LcS LcS LcS LcS expression on CD4+ T cells (Fig. 3a). However, the degree of
105 106 107 105 106 107 induction of CD69 by LcS on MD-PBMC was significantly
−ConA ConA lower than that on PBMC (Fig. 3a). LcS induced CD25
expression only on CD8+ and CD56+ subsets in PBMC, and
also MD-PBMC, but expression of this activation marker
was reduced significantly by monocyte depletion (Fig. 3b).

© 2010 British Society for Immunology, Clinical and Experimental Immunology, 161: 378–388 381
 Cytokine production (pg/ml) Cytokine production (pg/ml)

382
0
250
500
750
1000
1250
1500
1750
2000
2250
2500
0
5 000
10 000
15 000
20 000
25 000
30 000
35 000
40 000
45 000
H. Dong et al.

LcS
LPS
LcS
LPS

***

Medium

**

non-stimulated cultures.
LcS+LPS
Medium

IL-1β
***
LcS+LPS

IL-12
P<0·05 ***

duction induced by LPS.

P<0·05

Effect of LcS on NK cell activity

***

IL-6
***

***
P<0·05

[49]. *P < 0·05, **P < 0·01,***P < 0·001 compared with
IL-10
P<0·05
***
***

presence or absence of lipopolysaccharide (LPS). PBMCs were

***
TNF-α
P<0·001

cytokines by peripheral blood mononuclear cells (PBMCs) in the

Fig. 2. Effect of Lactobacillus casei Shirota (LcS) on production of

were shown as negative control. Each bar represents the mean of 6

in the presence or absence LPS (1 mg/ml). Non-stimulated cultures

to 12 blood donors and the error bars represent standard deviation

***
P<0·05

cultured with live LcS at 106 colony-forming units (CFU)/ml for 24 h

before (fresh) and after 24 h incubation with medium or LcS

(Fig. 4). For cells stimulated by LPS, monocyte depletion
by monocyte depletion, whereas IL-10 production was

bated in medium for 24h than in fresh PBMC (Fig. 5a,b).

effect of LcS, monocyte depletion totally inhibited IL-10 pro-
LcS-induced cytokine production. LcS-induced production
(Fig. 4), indicating that monocytes play an important role in
towards a decrease in IL-12 production induced by LcS

and IL-10 compared with PBMC (Fig. 4). Similar to the

resulted in a significant decrease in production of IL-1b, IL-6
of IL-1b, IL-6, TNF-a and IL-12 was not inhibited entirely
Monocyte depletion resulted in a significant decrease in

NK cell activity was assessed in PBMC and MD-PBMC

the production of IL-1b, IL-6, TNF-a and IL-10, and a trend

inhibited totally compared with the unstimulated cultures

at 106 CFU/ml. NK cell activity was higher in PBMC incu-

Table 1. Cytokine production by peripheral blood mononuclear cells (PBMC) and whole blood cultures.
Cytokine production (pg/ml)

-LPS LPS

Cytokines Cultures -LcS LcS105 LcS106 LcS107 -LcS LcS105 LcS106 LcS107

TNF-a PBMC 336·94 ⫾ 639·32 3 229·18 ⫾ 1 748·03 7 624·68 ⫾ 6 445·59 5 052·09 ⫾ 4 320·58 830·88 ⫾ 633·70 2 412·14 ⫾ 933·49 2 529·82 ⫾ 1 423·40 2 295·21 ⫾ 1 602·16
Whole blood 0 ⫾ 7·69 581·68 ⫾ 369·57 1 520·11 ⫾ 890·06 912·41 ⫾ 328·26 430·04 ⫾ 126·26 554·85 ⫾ 175·66 745·85 ⫾ 349·46 439·31 ⫾ 139·83
IL-1b PBMC 265·92 ⫾ 245·54 5 426·13 ⫾ 3 294·24 15 768·87 ⫾ 7 837·44 13 574·36 ⫾ 7 420·27 3 630·13 ⫾ 1 518·16 8 456·79 ⫾ 3 618·13 15 450·98 ⫾ 6 756·7 13 837·80 ⫾ 6 452·38
Whole blood 0 ⫾ 30·88 279·16 ⫾ 184·39 1 491·46 ⫾ 459·71 2 240·31 ⫾ 825·11 1 028·43 ⫾ 424·33 1 424·44 ⫾ 387·77 2 500·89 ⫾ 782·03 2 666·67 ⫾ 799·76
IL-6 PBMC 1027·36 ⫾ 1260·59 17 130·94 ⫾ 7 704·93 13 246·97 ⫾ 7 608·38 7 895·36 ⫾ 3 128·75 20 523·39 ⫾ 7 664·50 25 572·54 ⫾ 11 198·91 13 771·86 ⫾ 7 608·80 7 213·60 ⫾ 2 417·80
Whole blood 53·05 ⫾ 68·82 818·04 ⫾ 517·75 1 860·72 ⫾ 965·38 728·20 ⫾ 229·01 4 433·98 ⫾ 1 418·17 5 155·58 ⫾ 2 088·81 4 304·88 ⫾ 1 885·20 1 490·41 ⫾ 664·85
IL-10 PBMC 46·12 ⫾ 23·77 594·06 ⫾ 491·96 525·54 ⫾ 568·90 131·17 ⫾ 87·13 964·33 ⫾ 630·68 1 461·81 ⫾ 1 218·84 883·24 ⫾ 855·63 278·86 ⫾ 220·46
Whole blood 12·53 ⫾ 32·40 45·96 ⫾ 54·01 93·47 ⫾ 92·99 56·65 ⫾ 36·69 442·97 ⫾ 240·21 416·38 ⫾ 242·87 305·58 ⫾ 199·57 115·15 ⫾ 66·81
IL-12 PBMC 62·80 ⫾ 85·14 84·15 ⫾ 56·01 210·40 ⫾ 192·57 180·44 ⫾ 160·12 69·00 ⫾ 91·19 61·44 ⫾ 100·44 92·49 ⫾ 80·70 106·56 ⫾ 116·22
Whole blood 14·64 ⫾ 10·99 29·08 ⫾ 23·24 85·24 ⫾ 99·67 11·61 ⫾ 7·70 15·65 ⫾ 10·62 18·84 ⫾ 13·48 30·20 ⫾ 33·35 7·72 ⫾ 7·21

Cytokine production was measured in the supernatants of PBMC and whole blood cultures incubated with three different L. casei Shirota (LcS) concentrations, 105, 106 and 107 colony-forming units
(CFU)/ml, in the presence or absence of lipopolysaccharide (LPS) (1 mg/ml). The values represent mean ⫾ standard deviation (s.d.) of six volunteers. IL, interleukin; TNF, tumour necrosis factor.

© 2010 British Society for Immunology, Clinical and Experimental Immunology, 161: 378–388
 Probiotics and immune function

(a) present study demonstrates that the probiotic strain, LcS,

25 PBMC promotes T cell activation, as evidenced by induction of
MD-PBMC
CD69 and CD25 expression by T lymphocytes. Importantly,
20 there was evidence of preferential activation of CD8+ T cells.
CD69 expression (%)

LcS also enhanced CD69 and CD25 expression on the CD56+

Increase of

15 subset, increased NK cell activity and induced cytokine pro-

duction by human PBMC in vitro.
10 ***

***
** Effects of LcS on lymphocyte activation
5
* After T cell activation via CD3/T cell receptor (TCR) or via
**
0 CD2 (the alternate T cell activation pathway), the first mea-
Lymphocytes CD3+ CD4+ CD8+ CD56+ surable surface marker that is induced is CD69 [22]. It has
(b) been reported that quantitative flow cytometric determina-
25 tion of CD69 expression on T lymphocytes has several
PBMC advantages over traditional lymphocyte proliferation assays
MD-PBMC and is a useful diagnostic tool for detailed assessment of T
20
CD25 expression (%)

lymphocyte and subset activation [23].

CD25, the a chain of the IL-2 receptor, is a late activation
Increase of

15
marker induced during lymphoid cell activation, reaching a
peak at 96 h after stimulation by phytohaemagglutinin
10
(PHA) [24]. CD25 is expressed on activated T cells, B cells
P =0·07 and monocytes. Formation of the high-affinity IL-2 receptor
5
*** allows T cell proliferation and differentiation to be driven by
*** IL-2 [25]. Previous studies have shown overall agreement
0 with CD25 or CD69 and proliferation assays when a recall
Lymphocytes CD3+ CD4+ CD8+ CD56+
specific antigen was used [24].
Fig. 3. Effect of monocyte depletion on Lactobacillus casei Shirota CD25 and CD69 have been regarded generally as activa-
(LcS)-induced CD69 and CD25 expression by peripheral blood tion markers without discrimination of their significance for
mononuclear cells (PBMCs) and monocyte-depleted (MD)-PBMCs. proliferation. However, Clausen [26] reported that there was
(a) CD69 expression; (b) CD25 expression. PBMCs or MD-PBMCs
discrimination between CD25 and CD69 as activation
were incubated with medium or live LcS at 106 colony-forming units
markers on cytotoxic CD56+ lymphocytes and that CD25
(CFU)/ml for 24 h. Data are shown as mean ⫾ standard deviation of
should be viewed as an indicator for proliferative potential,
6 to 19 subjects. *P < 0·05, **P < 0·01, ***P < 0·001 compared between
PBMC and MD-PBMC cultures. whereas CD69 is a marker for cytotoxic activity. However,
further evidence is required to justify this.
Studies on the effect of LcS on cellular immunity are very
However, when PBMC were exposed to LcS at 106 CFU/ml
limited. Our data demonstrate that LcS stimulated CD69
for 24 h, NK cell activity was increased greatly compared
expression by CD3+, CD4+, CD8+ and CD56+ lymphocytes in
with that in the medium-only control (Fig. 5b). NK cell
the absence of mitogen. It also stimulated expression of
activity in 24 h incubated MD-PBMC with medium was
CD25, but only by CD8+ and CD56+ lymphocytes. This is the
greater than that in fresh PBMC or PBMC incubated for 24 h
first time that such an effect of LcS on T lymphocytes, and in
with medium (P < 0·01). When MD-PBMC were stimulated
particular CD8+ subsets, has been shown. Similar effects of
with LcS for 24 h, NK cell activity was increased compared
other probiotic strains have been reported in a few cases. For
with MD-PBMC cultured with medium alone (P < 0·01,
example, Castellazzi et al. [27] demonstrated that L. para-
Fig. 5c). However, the magnitude of the increase of NK cell
casei I 1688, L. salivarius I 1794 and a mixture of the two
activity stimulated by LcS was reduced significantly by
strains increased the percentage of CD4+/CD25+ cells
monocyte depletion. For example, at a 50 : 1 ratio of effector
(T helper-activated regulatory T cells), CD8+/CD25+ (T
to target cells, the magnitude of the increase of NK cell
suppressor/cytotoxic-activated cells) and CD16+/CD56+ (NK
activity by LcS was 3·14-fold in PBMC and 1·65-fold in
cells) (P < 0·05). Another in vitro experiment showed that
MD-PBMC. Thus, monocyte depletion reduced NK cell
two Lactobacillus strains, L. johnsonii La 1 and L. sakei LTH
activity induced by LcS.
681, increased expression of CD69 by CD8+ cells [28]. In a
human trial by Meyer [29], the expression of CD69 by T
Discussion
lymphocytes was increased by consumption of both conven-
Evidence has accumulated to suggest that probiotics confer tional yogurt and a probiotic product, and this activation
health benefits via modulation of immune function. The was especially significant in the CD8+ subset, with a lesser

© 2010 British Society for Immunology, Clinical and Experimental Immunology, 161: 378–388 383
H. Dong et al.

20 000 Fig. 4. Effect of monocyte depletion on Lactobacillus casei Shirota

IL-1β production (pg/ml) PBMC
(LcS)- and lipopolysaccharide (LPS)-induced cytokine production.
MD-PBMC Peripheral blood mononuclear cells (PBMCs) or monocyte-depleted
15 000
(MD)-PBMCs were incubated with live LcS at 106 colony-forming
Increase of

units (CFU)/ml or LPS at 1 mg/ml for 24 h. Increase of cytokine

10 000
productions of interleukin (IL)-1b, IL-6, tumour necrosis factor
(TNF)-a, IL-12 and IL-10 in the supernatants of the cultures were
5 000 measured by enzyme-linked immunosorbent assay. Data are shown as
***
* mean ⫾ standard deviation of 6 to 14 subjects. *P < 0·05, **P < 0·01,
0 ***P < 0·001 compared between PBMC and MD-PBMC cultures.
LcS LPS
䉳
50 000 PBMC
effect on CD4+ lymphocytes. In a randomized placebo-
IL-6 production (pg/ml)

40 000 MD-PBMC
controlled human trial [12], the effect of Saccharomyces bou-
lardii administration was studied in healthy children aged
Increase of

30 000
between 6 months and 10 years who were admitted for acute
20 000 diarrhoea. The patients who were supplemented with S. bou-
lardii for 7 days showed a significant decrease in daily stool
*
10 000 frequency and a significant increase in the percentage of CD8
*
lymphocytes and serum immunoglobulin A (IgA) compared
0
LcS LPS with the placebo group. In a randomized, double-blind,
placebo-controlled intervention study by de Vrese [3], L.
500 PBMC gasseri PA 16/8, Bifidobacterium longum SP 07/3 and B.
IL-12 production (pg/ml)

400 MD-PBMD bifidum MF 20/5 were supplemented for 3 months in 479

healthy adults. Cellular immune parameters were evaluated
Increase of

300 in a randomly drawn subgroup of 122 volunteers before and

after 14 days of supplementation. The results showed that
200
the total symptom score, the duration of common cold epi-
100 sodes and days with fever during an episode were signifi-
* cantly lower in the probiotic group. There was also a
0 significant increase in the percentage of cytotoxic/T suppres-
LcS LPS
sor cells (CD8+) after probiotic supplement. Our data are
14 000 thus consistent with these studies, indicating that probiotics
TNF-α production (pg/ml)

12 000 enhance the numbers/activation of cytotoxic lymphocytes

10 000 and may enhance cytotoxic activity. It remains to be deter-
Increase of

8 000 mined whether these effects are strain-dependent, or a

6 000 general feature of lactobacillus and bifidobacterium species.
Several human studies have shown that probiotics, includ-
4 000
*
ing LcS, enhance NK cell activity [5,7,8,10,13,18,30–32]. The
2 000
results in the current study showed a direct enhancement of
0 NK cell activity in human PBMC stimulated by LcS in vitro,
LcS LPS
which is in agreement with another in vitro study [21]
3000 employing heat-killed LcS and showing that both NK cell
PBMC
IL-10 production (pg/ml)

2500 MD-PBMD activation and activity were enhanced by LcS stimulation.

These data suggest that LcS could potentiate the destruction
Increase of

2000
of infected cells in the body and enhance host defence.
1500 Further research to investigate mechanisms is required and
1000 human trials are needed to confirm these immunomodula-
tory effects of LcS in vivo.
500
** **
0
LcS LPS Effects of LcS on cytokine production by PBMC
Probiotics clearly modulate cytokine production and the
effects appear to be strain-specific [33]. Data on the effects of
LcS on cytokine production, however, are limited. The
current study demonstrates that live LcS greatly induces pro-

384 © 2010 British Society for Immunology, Clinical and Experimental Immunology, 161: 378–388
 Probiotics and immune function

(a) In the current study, LPS (the main component of the cell
90 Fresh wall of Gram-negative bacteria) induced greater production
PBMC
80 MD-PBMC of IL-10 and IL-6, lower production of IL-1b than LcS, but
Specific lysis (%)

70
had no significant effect on production of IL-12 or TNF-a.
60
50 According to other studies employing human PBMC, Escheri-
40 chia coli and LPS are weaker inducers of IL-12, TNF-a and
30 IFN-g, but far more potent inducers of the anti-inflammatory
20
10
cytokine, IL-10, than lactic acid bacteria [34–37]. In an in vitro
0 study, LPS was a more potent inducer of IL-10 than several
100/1 50/1 25/1 12·5/1 live B. longum strains, but a weak inducer of TNF-a produc-
(b) tion [38,39]. In another in vitro study, LPS induced produc-
24-h incubation of PBMC medium tion of IL-1b, IL-10, IL-12 and, in particular, a large amount of
90
LcS
80 IL-6, by human myeloid dendritic cells, but did not induce
*
70 production of TNF-a [38]. On the other hand, Lactobacillus
Specific lysis (%)

60 ***
strains have been reported to induce production of IL-12,
50
40 ***
TNF-a and IFN-g, as well as IL-10 [40,41]. In addition, our
30 results also confirmed that PBMC samples can be replaced by
*
20 whole blood samples for cytokine measurement [42].
10 The current study showed that LcS enhanced LPS-induced
0
100/1 50/1 25/1 12·5/1 IL-1b, but inhibited LPS-induced IL-10 and IL-6 production
and had no further effect on production of IL-12 or TNF-a.
(c)
24-h incubation of MD-PBMC medium Interestingly, Mohamadzadeh et al. [38] showed that pro-
90 * LcS duction of IL-12 by myeloid dendritic cells was sustained
80
in response to three Lactobacillus species in the presence of
Specific lysis (%)

70 *
60 LPS, whereas LPS-induced IL-10 production was greatly
50 * inhibited. These results indicate that the effects of probiotics
40 on inflammatory responses might be strain-dependent, and
30
20 also dependent upon host immune status. The interpreta-
10 tion of these effects of lactobacilli is clearly complex, but it is
0 suggested that the literature to date supports evidence for an
100/1 50/1 25/1 12·5/1
augmentation of innate immune defences by probiotics,
coupled with an ability to regulate inflammation under some
Effector: target cell ratio
conditions [21].
Fig. 5. Effect of Lactobacillus casei Shirota (LcS) on natural killer It is suggested that cell-surface components of the bacteria
(NK) cell activity in peripheral blood mononuclear cells (PBMCs) play a central role in inducing immune responses [35,39]. An
and monocyte-depleted (MD)-PBMCs. PBMCs or MD-PBMCs were in vitro study showed that the effects of cell-surface compo-
incubated with medium or live LcS at 106 colony-forming units nents obtained by sonication of B. longum strains replicated
(CFU)/ml for 24 h. NK cell activity represented by percentage of
the stimulation of PBMCs with live cells, indicating that
specific lysis of target cells was assessed in freshly prepared PBMCs
these components are important determinants of the immu-
(a), in PBMCs cultured with medium or LcS (b) and in MD-PBMCs
nomodulatory activity of B. longum. On the other hand,
cultured with medium or LcS (c) for 24 h. Data are shown as
mean ⫾ standard deviation of five subjects. *P < 0·05, **P < 0·01, cell-free culture supernatants of the studied strains do not
***P < 0·001 compared with non-stimulated cultures of PBMC or tend to induce production of TNF-a, indicating that surface
MD-PBMC. structures are important in determining the immunomodu-
latory activity of probiotic bacteria [39].
These surface structures are generally called pathogen-
duction of IL-1b, IL-6 and TNF-a, and also significantly associated molecular patterns (PAMPs), and are recognized
induces production of IL-12 and IL-10. These results are by pattern recognition receptors (PRRs), which include
consistent with an in vitro study, which showed that heat- TLRs [43]. Activation of different TLRs results in induction
killed LcS stimulated human PBMC to secrete IL-12, IFN-g, of different cytokines. For example, TLR-4 is the key target
TNF-a and IL-10 [21]. In another in vitro study using for LPS-induced signalling which results in IL-10 produc-
murine monocyte/macrophage cell line J774A.1, LcS was tion, while TLR-2 recognizes lipoprotein/lipopeptide, lipote-
shown to stimulate markedly secretion of IL-12 and TNF-a, ichoic acid, glycoinositol-phospholipids and peptidoglycan,
but had no effect on IL-10 production [34]. This study also which are present in Gram-positive and/or Gram-negative
showed that live bacteria stimulated higher levels of IL-12 bacteria cell walls, which results in secretion of proinflam-
and TNF-a than heat-killed preparations. matory cytokines and anti-inflammatory IL-10 [44].

© 2010 British Society for Immunology, Clinical and Experimental Immunology, 161: 378–388 385
H. Dong et al.

toxic T lymphocytes (CD8+ T cells), possibly via induction of

Role of monocytes on LcS-induced
proinflammatory cytokines. This is the first time such an
immunomodulation
effect of LcS has been shown, and suggests that LcS could
In the current study, LcS induced preferential activation of potentiate the destruction of infected cells in the body.
cytotoxic cells (both CD8+ and CD56+). It has been reported Further research to investigate mechanisms is required and
that early production of IL-12 by macrophages contributes human trials are needed to confirm these immunomodula-
to the maturation of both NK cells and CD8+ T cells, leading tory effects of LcS in vivo.
to a T helper type 1 (Th1)-biased response [45]. In an in vitro
study, LcS was shown to be phagocytosed by monocytes and
stimulated them directly to secrete not only proinflamma- Acknowledgements
tory cytokines, such as IL-12 and TNF-a, but also the anti-
This research was sponsored by a Dorothy Hodgkin Post-
inflammatory cytokine, IL-10 [21]. It was therefore proposed
graduate Award and Yakult UK.
that monocytes play an important role in LcS-induced
immunomodulation. The current study demonstrates that
monocyte depletion reduced significantly the activation of T Disclosure
cells and NK cells, as well as the production of IL-1b, IL-6,
TNF-a and IL-12, and totally blocked the secretion of IL-10. None declared.
This suggests that monocytes are critical for the induction of
IL-10 by LcS, which is supported by another study in which
removal of the monocytes abrogated IL-10 production upon References
interaction with lactic acid bacteria [28] or LPS [46], and are 1 Corthesy B, Gaskins HR, Mercenier A. Cross-talk between probi-
important (but not critical) for the production of other otic bacteria and the host immune system. J Nutr 2007; 137:781S–
cytokines and induction of activation markers. Other studies 790S.
have reported that L. johnsonii induces CD25 expression 2 Galdeano CM, de LeBlanc AD, Vinderola G, Bonet AEB, Perdigon
directly on purified NK cells [45], and mRNA for the p35 G. Proposed model: mechanisms of immunomodulation induced
and p40 subunits of IL-12 was still induced in monocyte- by probiotic bacteria. Clin Vaccine Immunol 2007; 14:485–92.
3 de Vrese M, Winkler P, Rautenberg P et al. Effect of Lactobacillus
depleted cultures [46], which indicates that LcS could
gasseri PA 16/8, Bifidobacterium longum SP 07/3, B-bifidum MF
modify immune function via pathways not involving
20/5 on common cold episodes: a double blind, randomized, con-
monocytes. trolled trial. Clin Nutr 2005; 24:481–91.
Our results show that the magnitude of the increase of NK 4 Fukushima YMS, Yamano T, Kaburagi T, Iino H, Ushida K, Sato K.
cell activity in PBMC was greater than that in MD-PBMC Improvement of nutritional status and incidence of infection in
after 24 h incubation with LcS, suggesting that monocytes hospitalized enterally fed elderly by feeding of fermented milk
potentiate the effect of the LcS-induced increase in NK cell containing probiotic Lactobacillus johnsonii La1(NCC533). Br J
activity. This is consistent with the study of Shida et al. [21], Nutr 2007; 98:969–77.
which reported that monocytes play a role in LcS-induced NK 5 Olivares M, Diaz-Ropero MP, Sierra S et al. Oral intake of Lacto-
cell activity. In the current study, the greater NK cell activity in bacillus fermentum CECT5716 enhances the effects of influenza
MD-PBMC than in PBMC after 24 h incubation may be due vaccination. Nutrition 2007; 23:254–60.
6 Arunachalam K, Gill HS, Chandra RK. Enhancement of natural
partly to a relatively higher proportion of NK cells in
immune function by dietary consumption of Bifidobacterium lactis
MD-PBMC than in PBMC (as monocyte depletion by nature
(HN019). Eur J Clin Nutr 2000; 54:263–7.
will result in a higher proportion of NK cells). Moreover, 7 Bunout D, Barrera G, Hirsch S et al. Effects of a nutritional supple-
some other studies support the observation that monocytes ment on the immune response and cytokine production in free-
might suppress NK cell activity [46–48]. It is possible that living Chilean elderly. J Parenter Enteral Nutr 2004; 28:348–54.
increased NK cell activity in monocyte-depleted cultures is a 8 Gill HS, Rutherfurd KJ, Cross ML. Dietary probiotic supplementa-
consequence of the reduced/inhibited IL-10 secretion, as tion enhances natural killer cell activity in the elderly: an investi-
there is evidence that IL-10 plays a part in the regulation of gation of age-related immunological changes. J Clin Immunol
NK cell function. For example, Goodier [46] found that 2001; 21:264–71.
cytolytic activity, IFN-g production, proliferation and expan- 9 Klein A, Friedrich U, Vogelsang H, Jahreis G. Lactobacillus acido-
sion of human peripheral blood CD3– CD56+ cells (NK cells) philus 74-2 and Bifidobacterium animalis subsp lactis DGCC 420
modulate unspecific cellular immune response in healthy adults.
induced by LPS were enhanced in the presence of anti-IL-10
Eur J Clin Nutr 2008; 62:584–93.
receptor-blocking antibodies or on the removal of CD14+
10 Sheih YH, Chiang BL, Wang LH, Liao CK, Gill HS. Systemic
cells (monocytes) from the cultures. IL-10 was lost from the immunity-enhancing effects in healthy subjects following dietary
culture supernatants of CD14-depleted PBMC and rIL-10 consumption of the lactic acid bacterium Lactobacillus rhamnosus
reversed the effect of this depletion. HN001. J Am Coll Nutr 2001; 20:149–56.
In conclusion, in the absence of mitogenic stimulation, 11 Gill HS, Rutherfurd KJ. Probiotic supplementation to enhance
LcS enhances immune function, particularly that of cyto- natural immunity in the elderly: effects of a newly characterized

386 © 2010 British Society for Immunology, Clinical and Experimental Immunology, 161: 378–388
 Probiotics and immune function

immunostimulatory strain Lactobacillus rhamnosus HN001 (DR20 27 Castellazzi AM, Valsecchi C, Montagna L et al. In vitro activation of
(TM)) on leucocyte phagocytosis. Nutr Res 2001; 21:183–9. mononuclear cells by two probiotics: Lactobacillus paracasei I 1688,
12 Ozkan TB, Sahin E, Erdemir G, Budak F. Effect of Saccharomyces Lactobacillus salivarius I 1794, and their mixture (PSMIX).
boulardii in children with acute gastroenteritis and its relationship Immunol Invest 2007; 36:413–21.
to the immune response. J Int Med Res 2007; 35:201–12. 28 Haller D, Blum S, Bode C, Hammes WP, Schiffrin EJ. Activation of
13 Takeda K, Okumura K. Effects of a fermented milk drink contain- human peripheral blood mononuclear cells by nonpathogenic bac-
ing Lactobacillus casei strain shirota on the human NK-cell activity. teria in vitro: evidence of NK cells as primary targets. Infect Immun
J Nutr 2007; 137:791S–3S. 2000; 68:752–9.
14 Matsumoto K, Takada T, Shimizu K et al. The effects of a probiotic 29 Meyer AL, Micksche M, Herbacek I, Elmadfa I. Daily intake of
milk product containing Lactobacillus casei strain Shirota on the probiotic as well as conventional yogurt has a stimulating effect on
defecation frequency and the intestinal microflora of sub-optimal cellular immunity in young healthy women. Ann Nutr Metab 2006;
health state volunteers: a randomized placebo-controlled cross- 50:282–9.
over study. Biosci Microflora 2006; 25:39–48. 30 Olivares M, Diaz-Ropero MP, Gomez N et al. The consumption of
15 Aso Y, Akaza H, Kotake T, Tsukamoto T, Imai K, Naito S. Preven- two new probiotic strains, Lactobacillus gasseri CECT 5714 and
tive effect of a Lactobacillus-casei preparation on the recurrence of Lactobacillus coryniformis CECT 5711, boosts the immune system
superficial bladder-cancer in a double-blind trial. Eur Urol 1995; of healthy humans. Int Microbiol 2006; 9:47–52.
27:104–9. 31 Arunachalam KGH, Chandra RK. Enhancement of natural
16 Ishikawa H, Akedo I, Otani T et al. Randomized trial of dietary immune function by dietary consumption of Bifidobacterium lactis
fiber and Lactobacillus casei administration for prevention of col- HN019. Eur J Clin Nutr 2000; 54:263–7.
orectal tumors. Int J Cancer 2005; 116:762–7. 32 Zanini K, Marzotto M, Castellazzi A, Borsari A, Dellaglio F, Torri-
17 Koebnick C, Wagner I, Leitzmann P, Stern U, Zunft HJF. Probiotic ani S. The effects of fermented milks with simple and complex
beverage containing Lactobacillus casei Shirota improves gas- probiotic mixtures on the intestinal microblota and immune
trointestinal symptoms in patients with chronic constipation. Can response of healthy adults and children. Int Dairy J 2007; 17:1332–
J Gastroenterol 2003; 17:655–9. 43.
18 Nagao F, Nakayama M, Muto T, Okumura K. Effects of a fermented 33 Borchers AT, Selmi C, Meyers FJ, Keen CL, Gershwin ME. Probi-
milk drink containing Lactobacillus casei strain Shirota on the otics and immunity. J Gastroenterol 2009; 44:26–46.
immune system in healthy human subjects. Biosci Biotechnol 34 Cross ML, Ganner A, Teilab D, Fray LM. Patterns of cytokine
Biochem 2000; 64:2706–8. induction by Gram-positive and Gram-negative probiotic bacteria.
19 Ivory K, Chambers SJ, Pin C, Prieto E, Arques JL, Nicoletti C. Oral FEMS Immunol Med Microbiol 2004; 42:173–80.
delivery of Lactobacillus casei Shirota modifies allergen-induced 35 Helwig U, Lammers KM, Rizzello F et al. Lactobacilli, bifidobacte-
immune responses in allergic rhinitis. Clin Exp Allergy 2008; ria and E-coli nissle induce pro- an anti-inflammatory cytokines in
38:1282–9. peripheral blood mononuclear cells. World J Gastroenterol 2006;
20 Takeda K, Suzuki T, Shimada SI, Shida K, Nanno M, Okumura K. 12:5978–86.
Interleukin-12 is involved in the enhancement of human natural 36 Hessle C, Hanson LA, Wold AE. Lactobacilli from human gas-
killer cell activity by Lactobacillus casei Shirota. Clin Exp Immunol trointestinal mucosa are strong stimulators of IL-12 production.
2006; 146:109–15. Clin Exp Immunol 1999; 116:276–82.
21 Shida K, Suzuki T, Kiyoshima-Shibata J, Shimada S, Nanno M. 37 Zeuthen LH, Christensen HR, Frokiaer H. Lactic acid bacteria
Essential roles of monocytes in stimulating human peripheral inducing a weak interleukin-12 and tumor necrosis factor alpha
blood mononuclear cells with Lactobacillus casei to produce cytok- response in human dendritic cells inhibit strongly stimulating
ines and augment natural killer cell activity. Clin Vaccine Immunol lactic acid bacteria but act synergistically with Gram-negative
2006; 13:997–1003. bacteria. Clin Vaccine Immunol 2006; 13:365–75.
22 Rutella S, Rumi C, Lucia MB et al. Induction of CD69 antigen on 38 Mohamadzadeh M, Olson S, Kalina WV et al. Lactobacilli activate
normal CD4(+) and CD8(+) lymphocyte subsets and its relation- human dendritic cells that skew T cells toward T helper 1
ship with the phenotype of responding T-cells. Cytometry 1999; polarization. Proc Natl Acad Sci USA 2005; 102:2880–5.
38:95–101. 39 Medina M, Izquierdo E, Ennahar S, Sanz Y. Differential immuno-
23 Lindsey WB, Lowdell MW, Marti GE et al. CD69 expression as an modulatory properties of Bifidobacterium logum strains: relevance
index of T-cell function: assay standardization, validation and use to probiotic selection and clinical applications. Clin Exp Immunol
in monitoring immune recovery. Cytotherapy 2007; 9:123– 2007; 150:531–8.
32. 40 Lammers KM, Brigidi P, Vitali B et al. Immunomodulatory effects
24 Antas PRZ, Oliveira EB, Milagres AS et al. Kinetics of T cell- of probiotic bacteria DNA: IL-1 and IL-10 response in human
activation molecules in response to Mycobacterium tuberculosis peripheral blood mononuclear cells. FEMS Immunol Med Micro-
antigens. Mem Inst Oswaldo Cruz 2002; 97:1097–9. biol 2003; 38:165–72.
25 Foote MR, Nonnecke BJ, Fowler MA, Miller BL, Beitz DC, Waters 41 Hart AL, Lammers K, Brigidi P et al. Modulation of human den-
WR. Effects of age and nutrition on expression of CD25, CD44, and dritic cell phenotype and function by probiotic bacteria. Gut 2004;
L-selectin (CD62L) on T-cells from neonatal calves. J Dairy Sci 53:1602–9.
2005; 88:2718–29. 42 Yaqoob P, Newsholme EA, Calder PC. Comparison of cytokine
26 Clausen J, Vergeiner B, Enk M, Petzer AL, Gastl G, Gunsilius E. production in cultures of whole human blood and purified mono-
Functional significance of the activation-associated receptors nuclear cells. Cytokine 1999; 11:600–5.
CD25 and CD69 on human NK-cells and NK-like T-cells. Immu- 43 Medzhitov R. Toll-like receptors and innate immunity. Nat Rev
nobiology 2003; 207:85–93. Immunol 2001; 1:135–45.

© 2010 British Society for Immunology, Clinical and Experimental Immunology, 161: 378–388 387
H. Dong et al.

44 Bauer S, Hangel D, Yu P. Immunobiology of Toll-like receptors in human natural killer cells responding to Mycobacterium bovis
allergic disease. Immunobiology 2007; 212:521–33. bacille Calmette–Guerin. Immunology 2004; 112:143–52.
45 Niers LEM, Timmerman HM, Rijkers GT et al. Identification of 48 Higuchi K, Aoki K, Kimbara T, Hosoi N, Yamamoto T, Okada H.
strong interleukin-10 inducing lactic acid bacteria which down- Suppression of natural-killer-cell activity by monocytes follwing
regulate T helper type 2 cytokines. Clin Exp Allergy 2005; immunotherapy for recurrent spontaneous aborters. Am J Reprod
35:1481–9. Immunol 1995; 33:221–7.
46 Goodier MR, Londei M. Lipopolysaccharide stimulates the prolif- 49 Berman SH, Eichelsdoerfer P, Yim D, Elmer GW, Wenner CA.
eration of human CD56(+)CD3(-) NK cells: a regulatory role of Daily ingestion of a nutritional probiotic supplement enhances
monocytes and IL-10. J Immunol 2000; 165:139–47. innate immune function in healthy adults. Nutr Res 2006;
47 Esin S, Batoni G, Pardini M et al. Functional characterization of 26:454–9.

388 © 2010 British Society for Immunology, Clinical and Experimental Immunology, 161: 378–388