Leishmania-speci®c T cells expressing interferon-gamma (IFN-g) and IL-10 upon

activation are expanded in individuals cured of visceral leishmaniasis

K. KEMP*, M. KEMP*, A. KHARAZMI*, A. ISMAIL*², J. A. L. KURTZHALS, L. HVIID* &

T. G. THEANDER* *Centre for Medical Parasitology at Laboratory Centre and RHIMA Centre, Copenhagen University Hospital

(Rigshospitalet), and Institute for Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark, and

²Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan

(Accepted for publication 19 February 1999)

SUMMARY

Peripheral blood mononuclear cells (PBMC) from patients who have recovered from visceral

leishmaniasis often respond to Leishmania antigens in vitro by production of both IL-4, IFN-g and

IL-10. In order to establish the cellular sources of these cytokines, we activated cells from individuals

with a history of visceral leishmaniasis with Leishmania antigen for 6 days in culture, and identi®ed

cytokine production at the single-cell level by ¯ow cytometry. The cytokines were only found in CD3�

cells and among these mainly within the CD4� subset. The percentage of cytokine-producing cells was

compared in Leishmania-activated PBMC cultures from the previous patients and from individuals

living in a village where leishmaniasis does not occur. The percentage of IL-10- and IFN-g-containing

cells was signi®cantly higher in the previous patients than in the controls, indicating that Leishmania-

speci®c T cells producing IL-10 and/or IFN-g had been expanded as a result of the infection. The

cytokine-producing cells in the previous patients could be divided into three types: (i) cells producing

IFN-g only; (ii) cells producing IL-4 only; and (iii) cells producing IFN-g and IL-10 simultaneously.

The ®rst and second group of cells can be described as Th1- and Th2-type cells, respectively. The third

group could be a regulatory subset of T cells important for maintaining a balance between Th1- and

Th2-type cells in these individuals.

Keywords leishmaniasis cytokines ¯ow cytometry

INTRODUCTION

Human leishmaniasis is caused by protozoan parasites of the genus

Leishmania, which infects host macrophages. The systemic fatal

disease visceral leishmaniasis (VL), which is caused by

L. donovani, is characterized by parasite invasion of lymphoid

tissues. Untreated the disease has a fatal outcome. However,

Leishmania parasites can infect humans without causing clinical

disease [1].

The interactions between Leishmania parasites and host

immune defences have been extensively studied in animal

models. Generally, in susceptible mouse strains experimental

infections with L. major result in a disseminating lethal disease

and the animals respond to the invading parasite by a Th2-type

response, characterized by IL-4, IL-5, and IL-10 [2]. In resistant

mouse strains a Th1-type response characterized by secretion of

IFN-g, IL-2, and lymphotoxin occurs and the animals recover

spontaneously. The response type is determined early during the

infection, and there is a dichotomy in the response, since the

response to Leishmania antigen in the animals polarizes into either

Th1 or Th2 type. The dichotomy is probably established because

the Th1- and Th2-type clones mutually down-regulate each other.

However, exceptions from this general rule exist [3]. Mouse strains

also vary in susceptibility to L. donovani infection, but the relation

between Th1/Th2 balance and susceptibility is less clear in this

model [4].

The relevance of the division of immune responses into Th1

and Th2 types in humans has been debated, partly because Th1-

and Th2-type cytokine production occur simultaneously in many

in¯ammatory and infectious conditions [5±8]. In humans,

measurement of cytokines in culture supernatants of Leishmania

antigen-activated peripheral blood mononuclear cells (PBMC) and

T cell clones has been used to classify the immune responses into

Th1 and Th2 types [5]. During visceral leishmaniasis in humans

the response is predominantly Th2 type, with absence of IFN-g in

Leishmania antigen-activated PBMC culture supernatants from

such patients [9,10]. Drug treatment induces a shift in the response

Clin Exp Immunol 1999; 116:500±504

500 q 1999 Blackwell Science

Correspondence: KaÊre Kemp, Department of Infectious Diseases

M7641, Rigshospitalet, Tagensvej 20, 2200 Copenhagen N, Denmark.

E-mail: kkcmp@rh.dk

so that individuals cured of VL often respond to Leishmania

antigen by production of both IFN-g and IL-4 [11], indicating

that the immunological response to Leishmania in these indivi-

duals does not polarize as in inbred mouse strains. The measure-

ment of cytokines in supernatants does not reveal the cellular

source of the cytokines, which may be produced simultaneously by

the same cells or by discrete Th1 and Th2 populations. In order to

identify the phenotypes of cytokine-producing cells as CD4� or

CD8� and possible co-expression of cytokines, we analysed the

intracellular expression of IFN-g, IL-4 and IL-10 at the single-cell

level in Leishmania antigen-activated PBMC cultures derived from

individuals with a history of VL and controls.

SUBJECTS AND METHODS

Subjects and cells

Heparinized peripheral blood (20 ml) was collected from nine

Kenyans cured of VL, as previously described [12,13], and from

six Sudanese individuals from an area non-endemic for VL. The

PBMC were isolated by Lymphoprep (Nyegaard, Oslo, Norway)

density centrifugation, cryopreserved, stored and transported in

liquid nitrogen [14]. Before use, the cells were rapidly thawed and

washed. The viability of the cells was ascertained by trypan

blue exclusion. The Kenyan donors were selected according to

the ability of their cells to proliferate in response to various

Leishmania antigen preparations in vitro [10].

Antigens

Sonicates of L. donovani parasites (LDS) were prepared as

described previously [15]. Puri®ed protein derivative of tuberculin

(PPD) was purchased from Statens Seruminstitut (Copenhagen,

Denmark).

Cultivation of PBMC

The cells were resuspended in RPMI 1640 supplemented with 15%

heat-inactivated pooled human serum, 20 U/ml penicillin and

20 mg/ml streptomycin (GIBCO, Paisley, UK), and seeded into 24-

well multidish plates (Nunc, Roskilde, Denmark). Each well

contained 1 ´ 106 PBMC in 1 ml of medium. The cells were

cultured for 6 days at 378C in a humidi®ed atmosphere with 5%

CO2 in the absence of antigens, or the presence of 12 mg/ml PPD, or

13´5 mg/ml LDS, which were found to be the optimal concen-

trations for proliferation. To allow detection of intracellular

cytokines, monensin (1´5 mM; Sigma, St Louis, MO), ionomycin

(1 mM) and phorbol myristate acetate (PMA; 50 mg/ml) were added

to the cultures 4 h prior to the end of the incubation period. Non-

adherent cells were then collected for analysis.

Detection of surface markers and intracellular cytokines

The method for intracellular staining was based on other studies

[16±18]. Following incubation, cells were harvested, washed in

PBS, resuspended in PBS containing 0´5% bovine serum albumin

(BSA) and 0´01% NaN3 (staining buffer), and labelled with anti-

body directed against cell surface markers (CD3, CD4 or CD8

from Dako, Glostrup, Denmark) (room temperature for 20 min).

The cells were then washed twice in PBS/BSA/NaN3, ®xed with

2% formaldehyde (Sigma) in staining buffer, washed twice in

staining buffer and twice in a freshly made saponin buffer (staining

buffer containing 0´1% (w/v) saponin (Sigma)), and ®nally

incubated with anti-cytokine (IFN-g, IL-4 and IL-10 from

Pharmingen, San Diego, CA) antibody for 30 min. Following

cytokine labelling, the cells were washed twice in saponin

buffer, twice in staining buffer, resuspended in the same buffer

and analysed on an EpicsXL-MCL ¯ow cytometer (Coulter,

Hialeah, FL).

The ¯ow cytometric data were analysed by Pc lysys II software

(Becton Dickinson, Mountain View, CA). Samples were live gated

on lymphocytes by forward and side scatter gates and limits

for ¯uorescence-positive cells were set using non-speci®c IgG

antibodies.

Statistical analysis

Comparison of means was done by Student's t-test or Mann±

Whitney rank sum test with Sigma Stat software.

RESULTS

Cellular sources of IFN-g and IL-4

PBMC were incubated with Leishmania antigens for 6 days and

restimulated with PMA and ionomycin in the presence of

monensin. The cytokines in the cultures from the donors who

had had VL were only detected in CD3� cells. Figure 1 shows a

typical example of intracellular staining for IFN-g and IL-4 and

surface labelling of CD3.

PBMC from ®ve individuals with a history of VL were selected

on the basis of having large populations of both IFN-g- and IL-4-

producing T cells. To investigate the expression of CD4 and CD8

among the cytokine-producing cells, a gate was set on IFN-g- and

IL-4-containing cells. Most of the IFN-g was found in CD4� cells,

as 75% ± 97% of the IFN-g-producing cells expressed CD4. The

remaining IFN-g-containing cells were either CD8� (between 2%

and 17%) or CD4±CD8± (0±8%). Similarly, most IL-4 was found

in CD4� cells, as 77±97% of IL-4-containing cells were CD4�.

CD8� cells constituted 3±20%, whereas 0±7% were CD4±CD8±

(Table 1). Thus IFN-g- and IL-4-producing cells were CD3�, and

among these the cytokines were mainly found in CD4� cells, rather

than in CD8� cells.

Frequency of cytokine-producing cells

We compared the frequency of cytokine-producing cells in

Leishmania antigen-activated PBMC cultures derived from indi-

viduals cured from VL and individuals living in a village where

leishmaniasis is not endemic (Fig. 2). The frequencies of IFN-g-

and IL-10-producing cells were signi®cantly higher in PBMC

Leishmania-speci®c T cells in individuals cured of VL 501

q 1999 Blackwell Science Ltd, Clinical and Experimental Immunology, 116:500±504

104

FL2\

CD

P

E

FL1\IFN-G FITC

(a)

100

103

102

101

100

102 103 104101

Fig. 1. Flow cytometry dot plots of intracellular cytokine and CD3 expression

by peripheral blood mononuclear cells (PBMC) incubated with Leishmania

donovani sonicate. Cells were obtained from an individual with a past

history of visceral leishmaniasis. (a) Intracellular expression of IFN-g and

surface labelling of CD3. (b) Intracellular expression of IL-4 and surface

labelling of CD3.

104

FL1\

CD

FI

TC

FL2\IL 4 PE

(b)

100

103

102

101

100

102 103 104101

cultures derived from individuals with a history of VL (P� 0´026

and P� 0´018, respectively). The frequency of IL-4-producing

cells was also higher, but the difference was not statistically

signi®cant (P� 0´054).

Co-expression of cytokines

PBMC from individuals with a history of VL responding to

Leishmania antigen with high frequencies of IFN-g-, IL-4- and

IL-10-producing cells were investigated for co-expression of these

cytokines. Simultaneous labelling of Leishmania antigen-

stimulated PBMC from six donors with antibodies against

IFN-g and IL-4 showed that these cytokines were not co-expressed,

and that two distinct populations of cells expressing either one or

the other of these cytokines could thus be identi®ed (Fig. 3a

shows a typical example). Co-expression of IFN-g and IL-10

was investigated in four donors. In contrast to the results obtained

after labelling of IFN-g and IL-4, IL-10 was exclusively produced

by cells that also produced IFN-g (see Fig. 3b for a typical

example). Based on these results the cytokine-producing cells

could be divided into three types (Table 2): (i) cells producing

IFN-g only; (ii) cells producing IL-4 only; and (iii) cells produ-

cing IFN-g and IL-10. Approximately half of the cells were of the

®rst type, whereas the rest were divided about equally between

the other two types.

DISCUSSION

It has been debated whether the division of T helper cells into Th1

and Th2 types is relevant and useful when analysing human

immune responses to microbial pathogens [5±8]. We and others

have analysed T cell responses to Leishmania parasites extensively

in individuals suffering from various forms of leishmaniasis,

individuals who have recovered from these diseases, and indivi-

duals never exposed to Leishmania parasites. Most individuals

have T cells recognizing Leishmania antigens even before they are

exposed to the parasite, probably as a consequence of cross-

activation by other microorganisms [19,20]. PBMC from such

individuals respond to Leishmania antigens by the production of

either IFN-g or IL-4 [15]. PBMC from individuals with cutaneous

leishmaniasis show a similar dichotomy in the response, and in

these patients the severity of the disease correlates to the response

type [21]. After patients have recovered from cutaneous leish-

maniasis, their response is dominated by IFN-g production, and IL-4

is rarely detected in supernatants of Leishmania antigen-activated

PBMC cultures from such individuals [22]. In the population

groups described above, the response type of an individual can

be classi®ed as either Th1 or Th2 based on the production of IL-4

or IFN-g in Leishmania antigen-activated PBMC cultures. How-

ever, problems arise when testing PBMC from individuals cured

from VL, because in these cultures IFN-g and IL-4 are often

produced simultaneously [11].

The present study was designed to determine the phenotype as

CD4� or CD8� and the cytokine production pattern of cells in

Leishmania-activated PBMC cultures derived from such indivi-

duals. We con®rm that IFN-g and IL-4 are often produced

502 K. Kemp et al.

q 1999 Blackwell Science Ltd, Clinical and Experimental Immunology, 116:500±504

Table 1. Cellular source of IFN-g and IL-4 production in Leishmania

antigen (LDS)-activated peripheral blood mononuclear cells (PBMC)

from individuals who have recovered from visceral leishmaniasis (VL)

IFN-g IL-4

CD4� CD8� CD4± CD8± CD4� CD8� CD4±CD8±

(%) (%) (%) (%) (%) (%)

VL1 95 5 0 96 3 1

VL2 90 8 2 91 9 0

VL3 81 15 4 76 20 4

VL4 86 11 3 83 10 7

VL5 75 17 8 77 19 4

IFN-γ

40

30

20

10

0

Per

cen

t cy

toki

ne-

pro

du

cin

g T

cel

ls(o

f to

tal T

cel

ls)

IL-4 IL-10

Fig. 2. Percentage of T cells producing cytokines in Leishmania antigen-

stimulated cultures of peripheral blood mononuclear cells (PBMC) from

individuals who had recovered from visceral leishmaniasis (W) or from

individuals living in a village non-endemic to L. donovani (K). All values

are net increment compared with cultures without antigen.

104

FL1\

IFN

-G F

ITC

FL2\IL 4 PE

(a)

100

103

102

101

100

102 103 104101

33·2 0·6

9·9

104

FL1\

IFN

-G F

ITC

FL2\IL 10 PE

(b)

100

103

102

101

100

102 103 104101

20·2 12

0·3

Fig. 3. Flow cytometry dot plots showing intracellular staining of periph-

eral blood mononuclear cells (PBMC) from a previous visceral leish-

maniasis patient incubated with Leishmania donovani sonicate. (a) The

cells were stained by antibodies against IFN-g and IL-4. (b) Cells were

stained for IFN-g and IL-10. The numbers indicate the percentage of cells

in each quadrant.

Table 2. Expression of IFN-g, IL-4 and IL-10 by T cells in Leishmania

antigen (LDS)-activated cultures of peripheral blood mononuclear cells

(PBMC) from patients cured of visceral leishmaniasis

Cytokine phenotype Donor 1 Donor 2 Donor 3 Donor 4

IFN-g�, IL-10±, IL-4± 47* 48 49 39

IFN-g�, IL-10�, IL-4± 29 15 23 23

IFN-g±, IL-10±, IL-4� 24 37 28 38

*Cells of the cytokine phenotype in percentage of all cytokine-contain-

ing cells.

simultaneously in these cultures, and identify the cells producing

IFN-g, IL-4, and IL-10 as CD3� T cells. Among these the

cytokines were mainly found in CD4� cells, although cytokines

were also detected in a low percentage of CD8� cells. When

examining the co-expression of cytokines by T cells in the cultures,

we found a dichotomy between IFN-g and IL-4, since no cells

contained both cytokines. This was in marked contrast to the

production of IL-10, which was con®ned to IFN-g-producing

cells. We could therefore divide the cytokine-producing cells

into (i) IFN-g producers, (ii) IL-4 producers, and (iii) IFN-g

and IL-10 producers. The ®rst group of cells ®ts the description

of Th1-type cells and the second group Th2-type cells, whereas the

group of cells producing IFN-g and IL-10 does not ®t into the

conventional description of Th0, Th1 and Th2 cells.

When comparing cell cultures derived from individuals cured

of VL with cultures derived from individuals who never had

leishmaniasis and were living in an African village where VL

does not occur, higher frequencies of cytokine-producing cells

were found in the ®rst group. The frequencies of IFN-g- and IL-10-

producing T cells were signi®cantly higher in Leishmania antigen-

activated PBMC cultures derived from individuals with a past

history of VL. Since IL-10 production was always associated with

IFN-g production, this indicates that a population of Leishmania

antigen-speci®c T cells co-producing IL-10 and IFN-g expand in

response to L. donovani infection.

Other studies have described human T cell populations co-

expressing IFN-g and IL-10 [23±25]. In these studies, T cell lines

producing IFN-g and IL-10 were generated after a long period of

in vitro cultivation. These cells were generated in the presence of

IL-12, whereas neutralizing anti-IL-12 MoAbs prevented the

generation of the IFN-g and IL-10 cell population.

Recently, a T cell subset secreting IFN-g, IL-10 and transform-

ing growth factor-beta (TGF-b) simultaneously has been identi®ed

in both humans and mice [26]. These cells, named T regulatory

cells (Tr1), conferred protection in adoptive transfer experiments

against colitis in IL-10-de®cient mice. The human Tr1 cells were

clones generated in vitro after allogeneic stimulation in the pre-

sence of IL-10. The physiological role of these cells is unknown.

Thus two sets of IFN-g- and IL-10-producing cells have been

described previously. Both of these types were generated in vitro

after long periods of incubation, one in the presence of IL-12, and

the other in the presence of IL-10. The IFN-g and IL-10-producing

cells described in the present study were found in short-term

PBMC cultures without addition of cytokines. They may have

expanded and acquired their cytokine pro®le during the infection,

either before treatment where IL-10 levels are high [27±30],

or during treatment where the ability for IL-12 production by

Leishmania antigen-stimulated PBMC is regained [9].

In previous VL patients there is a coexistence of Leishmania-

speci®c T cells of Th1 and Th2 type. These two subsets of cells can

down-regulate each other. In mice, infection with L. major results

in a polarized immune response that is either Th1 or Th2. Speci®c

T cells producing both IFN-g and IL-10 could function as a

regulatory subset that allows a balance between Th1- and Th2-

type cells to be maintained in the previous VL patients.

In conclusion, we found that CD4� T cells were the major

source of IFN-g, IL-4 and IL-10 in the investigated cultures. IFN-

g and IL-4 were never co-expressed by the same cells.

By contrast, a T cell population producing both IFN-g and

IL-10 but not IL-4 was demonstrated to exist besides conven-

tional Th1 and Th2 cell subsets.

ACKNOWLEDGMENTS

This work was supported by grants from the Danish International Devel-

opment Agency (RUF and the ENRECA programs), the Danish Bio-

technology Program and the Novo Nordisk Foundation. G. Pedersen and

M. Youse® are thanked for excellent technical assistance.

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