Leukotriene B4 activates T cells that inhibit B-cell proliferation in EBV-infected cord...

doi:10.1182/blood-2007-08-102319Prepublished online December 19, 2007;

Anquan Liu, Hans-Erik Claesson, Yilmaz Mahshid, George Klein and Eva Klein infected cord blood derived mononuclear cell culturesLeukotriene B4 activates T cells which inhibit B cell proliferation in EBV

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Leukotriene B4 activates T cells which inhibit B cell proliferation in EBV infected cord

blood derived mononuclear cell cultures

Anquan Liu1, Hans-Erik Claesson2,3, Yilmaz Mahshid2, George Klein1, and Eva Klein1

1Department of Microbiology, Tumor and Cell Biology, 2Department of Medical

Biochemistry and Biophysics, Karolinska Institute, 171 77 Stockholm, 3Biolipox AB,

Berzelius vag 3, 171 65 Solna, Sweden.

Corresponding author: Anquan Liu

Department of Microbiology, Tumor and Cell Biology, Karolinska Institute;

Nobels väg 16, 171 77 Stockholm; Sweden

Tel.: +46-8-5248 6761

Fax: +46-8-330498

E-mail: [email protected]

Running title: LTB4 can activate T cells in EBV infected CBMC

1

Blood First Edition Paper, prepublished online December 19, 2007; DOI 10.1182/blood-2007-08-102319

Copyright © 2007 American Society of Hematology


mailto:[email protected]



Abstract

EBV specific cellular memory is not transferred from mother to child. Therefore, EBV

induced B cell proliferation in in vitro infected cord blood mononuclear cell cultures is not

inhibited. However, by addition of immunomodulators, PSK or Trx80 that activate

monocytes, EBV specific T cell response could be generated in such cultures. Presently we

demonstrate that leukotriene B4 (LTB4) is involved in the effect of the immunomodulators.

LTB4 was detected in the medium, and T cell activation was compromised by addition of

leukotriene biosynthesis inhibitors. Moreover, we found that LTB4 added to infected cultures,

which did not receive the immunomodulators, induced functional activation of the T cells.

LTB4 activated the monocytes and acted directly on the T cells. In consequence, addition of

LTB4 inhibited the EBV induced proliferation of B lymphocytes. Specific cytotoxicity could

be generated by restimulation of the T cells. The experiments showed successive stages of T

cell activation in acquisition of their immunological effector function. This is orchestrated by

complex cellular interactions, and autocrine loops mediated by soluble factors - here IFN-γ,

IL-15, IL-12 and LTB4. Importantly, the results indicate that endogenous LTB4 can induce T

cell activation that inhibits the EBV induced proliferation of B lymphocytes.

2




Introduction

Leukotriene B4 (LTB4) is a potent fast acting lipid mediator with a wide range of biological

effects.1 It is produced mainly by granulocytes, monocytes and B lymphocytes,1-3 LTB4 binds

to BLT1, a high affinity cell membrane receptor.4 Several studies have been directed to the

function of LTB4 and indicated its regulatory role in both natural and adaptive specific and

non-specific immune responses.1,5-7

Epstein-Barr virus (EBV) is carried by the majority of human adults. Primary infection of

adolescents occurs either without characteristic symptoms or leads to a benign disease of

variable severity, infectious mononucleosis.8 Regardless of the clinical picture, EBV infection

is followed by a lifelong carrier state that can be easily revealed by EBV specific humoral and

cellular immunity. B lymphocyte is the main target of EBV. In vitro infected B lymphocytes

are induced to proliferate.9,10 However the virus carrier state is harmless due to the finely

tuned immune response against virally encoded proteins that leads to the recognition and

elimination of EBV transformed B cells. Several of the EBV encoded proteins that function in

transformation are immunogenic. The costimulatory proteins expressed by the transformed

cells secure their recognition by immune effector cells.11-13 Survival and proliferation of EBV

infected B cells is controlled by innate immunity, IFN-γ and NK cells, and also by EBV

specific cytotoxic T lymphocytes (CTL).14 The details of these immune responses have been

extensively studied in the in vitro B cell transformation system. Specific immune memory can

be detected by inhibition of B cell transformation.13-15

EBV specific cellular immunity is not transferred from mother to child. Therefore B

lymphocytes in cord blood mononuclear cell (CBMC) cultures infected in vitro are efficiently

transformed. We have established an experimental system that permits the study of relevant

cellular interactions leading to EBV specific cell mediated immune responses without the

need for substantial cell separation steps.16,17

In this system of in vitro infected CBMC cultures we found that addition of the

immunomodulators (PSK and Trx80) can induce inhibition of EBV transformed B cell

3




growth.17 We showed that the T cells were activated in such cultures by the EBV infected B

cells. For proper function, however, the T and NK cells required the assistance of activated

monocytes. The monocytes were activated by the added PSK or Trx80, and in the presence of

activated T cells they produced IL-15 and IL-12, respectively. In their turn, the activated T

cells could respond to the lymphokines. They became functionally active and inhibited the

EBV induced B lymphocyte proliferation.

We show now that LTB4 can act as immunomodulator in EBV infected cord blood

lymphocyte cultures. It activates the monocytes and also the T cells that by the encounter of

EBV infected B lymphocytes enter in a responsive state. The activated T cells then

compromise the EBV induced B lymphocyte proliferation.

4




Materials and Methods

Reagents and Antibodies

Ficoll-paque was purchased from Pharmacia Biotec, Uppsala, Sweden; PSK, from Kureha

Chemical Ind. Co., Tokyo; purified recombinant human Trx80 was the kind gift of A.

Holmgren, Department of Medical Biochemistry and Biophysics, Karolinska Institutet.18

Synthetic LTB4, LTC4, LTD4 and 5(S),12(S)-DiHETE were obtained from Biomol

(Plymouth, PA); BWA4C was a kind gift from L. Garland (Wellcome Research Laboratories,

Beckenham, United Kingdom) and MK-886 from Merck Research Laboratories, Rahway, NJ.

Anti-CD19 Ab-conjugated beads (Dynabeads M-450); Dynabeads Goat anti-Mouse IgG, from

Dynal, Oslo, Norway. 3H-thymidine from Amersham Pharmacia Biotech, Uppsala, Sweden.

Monoclonal mouse anti-human CD3; mouse monoclonal fluorescein isothiocyanate (FITC)-

conjugated anti-CD3, anti-CD19; phycoerythrin (PE)-conjugated anti-CD4 and anti-CD8

antibodies from Dako, Denmark. PE-Cy5-conjugated anti-CD56; peridinin chlorophyll

protein (PerCP)-conjugated anti-CD3; neutralization mAb of anti-human IL12 (p40/p70)

(C8.6), from BD PharMingen, CA. PE-conjugated anti-CD69, from Immuno Tools, Germany.

Anti-BLT1 antibody (7B1 FITC) was raised in-house.19 Monoclonal mouse anti-CCR5, from

Diaclone, France; FITC-conjugated anti-CXCR4; monoclonal mouse anti-CXCR3; IL15,

IL12 and IFN-γ enzyme-linked immunosorbent assay (ELISA) antibodies; IL18 ELISA kit;

Neutralization mAb of mouse anti-human IL15 (MAB647), from R&D System, MN, USA.

Neutralization mAb of mouse anti-human IL18 (125-2H), from MBL, Japan. The mAb anti-

HLA class I (mAb W6/32) or II (mAb CR3/43) from Dako; Affinity purified rabbit anti-SAP

antiserum was the kind gift of J. Sümegi, Cincinnati Medical Center, Ohio, USA. HRP-

conjugated donkey anti-rabbit Ig antibody; ECL+Plus detection reagent; Hyperfilm-ECL film,

from Amersham, Arlington Heights, IL.

5




Preparation and culture of cells

Cord blood samples were obtained from the Department of Obstetrics and Gynecology,

Karolinska University Hospital. The study was approved by the Ethics Committee of the

Karolinska Institutet and the Karolinska Hospital.

Mononuclear cells were isolated from heparinized cord blood by Ficoll–paque density

centrifugation.

For isolation of B cells, cord blood mononuclear cells (CBMC) were incubated with anti-

CD19 Ab-conjugated beads. The attached cells were recovered from the beads using

DETACHaBEADs (Dynal).

Monocytes were isolated on gelatin/ plasma coated dishes as described earlier.17

For T cell depletion, CBMC were incubated with mouse anti-human CD3 mAb, followed by

goat anti-mouse IgG conjugated beads. The fluent cell suspension was collected.

The cells were cultured at a density of 106/ml in RPMI 1640 supplemented with 10% FCS,

2mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin at 37 °C, 5% CO2.

EBV infection

CBMC or separated B cells were exposed to B95-8 virus containing supernatant for 1.5 hrs in

a humidified 37 °C, 5% CO2 incubator. The cells were washed and resuspended in complete

RPMI at 1×106/ml concentration.

Proliferation of EBV infected B cells

Proliferation of EBV infected B cells was analyzed on the 6th day of culture by 3H-thymidine

incorporation assay. The infected CBMC cells were seeded in 2×105 per well/200 μl, in 96-

well plates. Each sample was represented by triplicate cultures. On the 6th day 1 μCi 3H-

thymidine was added and 14 hrs later the cells were harvested onto glass fiber filters.

6




Radioactivity was measured in a liquid scintillation counter. At this time the results reflect the

number of B cells induced to proliferate by EBV.16,20

EBV-infected B cells were seeded in 1×105 per well/200 μl in 96-well plates. Each sample

was represented by triplicates. 1μCi 3H-thymidine was present during the final 14 hrs.

Flow cytometric analysis

The subset specific cell surface markers CD3, CD4, CD8, CD14, CD19, CD56; the

lymphocyte activation marker CD69; LTB4 receptor BLT1; the chemokine receptor CXCR4

were detected with FITC-, PE-, PE-Cy5- or PerCP-conjugated mouse anti-human monoclonal

antibodies. CXCR3 and CCR5 were detected by indirect immunofluorescence. The cells were

washed in cold PBS/1% FCS and then exposed to the relevant antibodies, washed and

thereafter incubated with the FITC–conjugated secondary antibody. All incubations were

performed for 30 min at 4°C. Cells were then washed once with PBS/1% FCS and

resuspended in 500 µL PBS. Ten thousand events were collected on a FACScan flow

cytometer, and the results were analyzed using Cell Quest software (Becton Dickinson).

Detection of Leukotriene B4

The cell populations were cultured in 24-well plates at a density of 1×106/ml. Supernatants of

24 hrs old cultures were collected and diluted in EIA-buffer, then added to an antibody coated

96-well plate. The enzyme immunoassay (EIA) was performed according to manufacturer’s

protocol. The detection limit was 4 pg/ml.

Detection of cytokines in the culture medium

The cell populations were cultured in 24-well plates. Supernatants of the cultures were

collected 3 days later, and analyzed in duplicate wells using sandwich ELISA for IL-15, IL-

12, IL-18 and IFN-γ according to the manufacturer's instructions. The sandwich ELISA for

7




IL-12 detected the IL-12 p70 heterodimer. The detection limit of the IL-15, IL-12 and IL-18

was 10 pg/ml. That of IFN-γ was 30 pg/ml.

SAP detected by immunoblotting

The cells were lysed directly in SDS gel-loading buffer. Aliquots corresponding to 1.5×105

cells were electrophoresed on 12% SDS-PAGE gel and transferred to PVDF membranes at 75

V for 2 hrs. After blocking for 1 hr with 5% non-fat dried milk in PBS-Tween 20, the

membranes were incubated with the anti-SAP antibody overnight at 4 °C. The blots were then

incubated with HRP-conjugated donkey anti-rabbit Ig antibody and developed with ECL+Plus

detection reagent. Hyperfilm-ECL film was exposed to the developed membranes for

visualization.

Cytotoxic function generated in the cultures

EBV infected cultures were stimulated on the 7th and on the 14th day, at 10:1 ratio, with

irradiated (50Gy) autologous EBV infected B cells. At each stimulation half of the medium

was replaced with fresh medium. From the 9th day 20 U/ml IL-2 was added every third day.

On the 20th day, an aliquot of cells from cultures were used for analysis of cell composition by

flow cytometry. B cells were depleted from the remaining cells using anti-CD19 Ab-

conjugated beads and the residual cells were used as effectors in 51Cr-release cytotoxicity

assay. Autologous LCL (autologous EBV infected B cells were cultured for 3 weeks),

activated autologous B cells (autologous B cells were cultured with irradiated (50Gy) CD40L-

L cells and IL-4 (5ng/ml) for 7 days), allogeneic LCLs and K562 were used as targets.

Autologous target cells were used also after 30 min incubation with the mAb W6/32 (anti-

HLA class I) or with the mAb CR3/43 (anti-HLA class II).

The specific lysis was calculated as follows: specific release= [(experimental release-

spontaneous release)/(maximum release-spontaneous release)]×100.

8




Statistical analysis

Statistical significance was determined using paired two-tailed Student’s t test. Significance is

presented for individual experiments (* P< 0.05, ** P< 0.01).

9




Results

1. Involvement of LTB4 in T and NK cell activation and B cell growth inhibition in EBV

infected cord blood cell cultures.

We have shown earlier that T and NK cells were activated upon addition of the

immunomodulators, PSK or Trx80 to EBV infected CBMC cultures and they could inhibit B

cell proliferation.16,17 Activation of the T and NK cells was detected in the cell mixture by an

increase of SAP protein seen in immunoblots. The SAP protein of the cell population was

contributed by T and NK cells and the level of SAP reflected their activation status. The blots

were performed with lysates prepared from a fixed number of cells. Unless inhibited, the B

lymphocytes are induced to proliferate in the infected cultures and they do not express SAP.

Thus, the relative increase of B cells lower the SAP content in the samples representing a

fixed number of cells.

1a. SAP expression

In accordance with our earlier results, the samples of the EBV infected cultures containing

PSK or Trx80 showed an increase of the SAP protein (Figure 1A).17 Lysates prepared from

LCL (lymphoblastoid cell line), were included as controls. The lack of SAP band in the LCL

samples confirms that EBV infected B cells do not express SAP.

The SAP level increase induced by the immunomodulators was prevented by the leukotriene

biosynthesis inhibitors MK886 or BWA4C. Addition of LTB4 (100nM) to such cultures

restored the intensity of the SAP bands. Inhibition of the endogenous LTB4 production could

thus be counteracted by introduction of the leukotriene from the outside, confirming the

contribution of LTB4 to the activation of T and NK cells.

1b. Production of IL-15 and IL-12

In accordance with our earlier findings, the supernatants of the PSK and Trx80 containing

cultures contained IL-15 and IL-12 respectively. Addition of the leukotriene biosynthesis

inhibitors MK886 or BWA4C reduced the production of these cytokines (Figure 1B). The IL-

15 content was reduced from 334 ± 142 pg/ml to 156 ± 72 pg/ml and to 79 ± 16 pg/ml

10




(inhibition 54 % and 74 %) respectively. The corresponding values for IL-12 in the Trx80

containing cultures were 378 ± 120 pg/ml and 132 ± 51 pg/ml, 155 ± 50 pg/ml (inhibition

66% and 58%). Cytokine production was lower in presence of both inhibitors. Similarly to the

reestablishment of SAP expression in the cells, the cytokine levels were restored to the control

values when LTB4 was added to the cultures.

1c. B cell proliferation

EBV induced B cell proliferation was inhibited in the cultures containing the

immunomodulators. The inhibition was shown by the composition of the cell populations and

by thymidine incorporation measured on day 6 of culture (Table 1 and Figure 1C).16 The

mean inhibition in three experiments was 57 ± 10% and 50 ± 11% for PSK and Trx80

respectively. The leukotriene biosynthesis inhibitors MK886 or BWA4C reduced the growth

inhibitory effects of PSK to 21 ± 5%, 19 ± 7%, and to 27 ± 4%, 23 ± 6% in Trx80 containing

cultures. In the presence of both inhibitors, thymidine incorporation was similar to that of the

control, thus the inhibition was completely eliminated.

These results confirm our earlier findings that activation of T and NK cells in the EBV

infected CBMC cultures can be detected by SAP expression, by the production of cytokines

and by the inhibition of B cell proliferation. The new aspect that emerges from the present

experiments is the essential role of LTB4 in the activation of the effector cells.

2. LTB4 and IFN-γ production in the EBV infected cultures

In line with the reduction of the immunomodulator mediated effects by LTB4 synthesis

inhibitors, LTB4 was detected in the EBV infected cultures that received the

immunomodulators and it required the presence of monocytes (Figure 2A). In five

experiments the mean concentrations were 182 ± 108 pg/ml, (range: 63-353) in the PSK and

117 ± 105 pg/ml (range: 13-329) in the Trx80 containing cultures. This is consisting with the

identification of monocytes as the main LTB4 producing cells under our culture conditions.

Granulocytes are strong LTB4 producers, but they were not present in our experiments. The

11




results showed that activation of monocytes by the immunomodulators was essential for LTB4

production, as shown earlier for the production of IL-15 and IL-12.

IFN-γ was produced in the infected cell population and its level increased in the cultures

containing the immunomodulators (Figure 2B). Figure 2C shows that LTB4 production was

considerably reduced when any one of the cytokines IFN-γ, IL-15 or IL-12 were neutralised

by specific antibodies.

These results show the activation circuit between monocytes, NK and T cells. The latter cells

assist in the activation of monocytes for LTB4 production with provision of IFN-γ, but they

require IL-15 or IL-12 produced by the monocytes.

3. Expression of the LTB4 and chemokine receptors in the cell subsets of EBV infected

cultures.

The cellular composition of the ex vivo cells and cultures at day 6 is shown in Table 1. In the

PSK or Trx80 containing cultures, the enrichment of B cells was lower (19% or 22%

respectively vs 41% in the absence of the immunomodulators), reflecting the inhibition of

EBV induced B lymphocyte proliferation.

T and NK cells were activated in the infected cultures. A low proportion of T (CD3+) and NK

(CD56+) cells expressed CD69 (activation marker) in the ex vivo samples. Their frequency

increased in the virus infected, and it increased further in the immunomodulator containing

cultures. Although the frequencies of positive cells were lower, expression of BLT1 on the T

cells, showed a similar increase in these two cultures, including its both subsets, CD4 and

CD8 (Figures 3A, B).

In accordance with earlier reports, ex vivo T cells did not express BLT1.4,21 Interestingly, the

activated NK cells did not express BLT1.

12




BLT1 is known to be expressed on B lymphocytes.21 We detected 50% BLT1 positive cells in

the CD19 subset of the ex vivo population while in the cultures this subset did not express the

receptor.

The majority of ex vivo T cells expressed the chemokine receptor CXCR4, it was

downregulated in the infected cultures (Table 1). Rare ex vivo T cells expressed CXCR3 and

CCR5, they increased to 51 and 54 % respectively in the infected cultures.

4. LTB4 mediated potentiation of T cell activation in the EBV infected cultures

The production of LTB4 in the immunomodulator containing cultures and the restoration of

lymphocyte activation by addition of exogenous LTB4 following inhibition of its endogenous

synthesis prompted us to test the effect of LTB4 added to the cultures, in the absence of PSK

and Trx80.

4a. SAP expression

The experiment shown in Figure 4A was designed in the same way as the previous ones,

except that the immunomodulators were omitted and LTB4 was added to the EBV infected

cultures. The results in Figure 4A and 4B show a dose dependent increase of SAP expression.

Specificity controls included addition of LTC4, LTD4 and 5 (S),12 (S)-DiHETE which were

inactive at a final concentration of 100nM. LTB4 did not increase SAP expression in the

uninfected cultures. Thus LTB4 has a similar effect as the immunomodulators, PSK and

Trx80.

4b. B cell proliferation

The B cells increased and the T cells decreased proportionally in the EBV infected cultures.

In the presence of LTB4, however, the B cell enrichment was impaired (Figure 4C). The

proportion of NK cells remained unchanged.

Proliferation of the B lymphocytes, were in line with the composition of the cell populations

in that the values were lower in the cultures containing LTB4 (Figure 4D). Inhibition of B cell

13




proliferation mediated by 10nM, 50nM and 100nM LTB4 was 23 ± 9%, 50 ± 7% and 61 ±

12%, respectively.

The growth inhibition in the cultures could be attributed to the activated effector cells,

because LTB4 did not influence the proliferation of separated B lymphocytes infected by the

virus (Figure 4E).

Taken together, these data suggest that addition of LTB4 activated the T cells in the cultures

and they inhibited EBV induced B cell proliferation.

4c. Lymphokine production

Similarly to the PSK and Trx80 treated cultures lymphokine production was induced by

LTB4. It elevated the production of IFN-γ. Monocyte-depleted cultures contained less IFN-γ

(Figure 5A). Interestingly the assortment of T and NK cell activating lymphokines IL-15, IL-

12 and IL-18 differed from that induced by PSK and Trx80, in that IL-18 dominated (Figure

5B-D). These required also the presence of monocytes.

5. LTB4 added to the culture stimulated monocytes and activated T cells.

A fraction of T cells expressed BLT1 in the infected cultures (Figure 3). Therefore the added

LTB4 could activate directly the T cells, and induced elevation of SAP expression even in the

absence of monocytes (Figure 6A). Comparison of the dose responses indicated, however,

that the SAP inducing capacity of LTB4 was about tenfold in the presence of monocytes. This

effect was in part due to the autocrine effect of LTB4 for monocytes, that has been reported

earlier.22

Inhibition of B lymphocyte proliferation showed the functional aspect of the LTB4 treatment

in that the cell proliferation was also inhibited in this culture (Figure 6B).

Separated monocytes could be activated by LTB4. When such cells were returned to the

infected, monocyte depleted residual cell population their SAP expression increased. Added

LTB4 to these cultures further increased SAP expression (Figure 6C). Inhibition of LTB4

14




biosynthesis with MK886 or BWA4C to these reconstituted cultures abolished the effect on

SAP expression (Figure 6D).

The LTB4 induced activation of monocytes can produce the T and NK cell activating

lymphokines IL-18, IL-15 and IL-12. These lymphokines contributed to the effect of LTB4

treated monocytes as shown by the reduction of the intensity of SAP band in the immunoblot

when they were neutralised by antibodies (Figure 6E). Neutralisation of the lymphokines

decreased the function of the T cells, in that the inhibition of B cell proliferation was

abolished by anti IL-18 and decreased by the mixture of anti IL-15 and anti IL-12 reagents

(Figure 6F). This was in line with the detection of these lymphokines in the supernatants and

showed that LTB4 induced relatively higher amount of IL-18 (Figure 5).

Addition of IL-18 to the infected cultures elevated SAP expression even if the cultures did not

contain monocytes (Figure 6G).

We can conclude thus that LTB4 could activate monocytes and these produced LTB4, IL-18,

IL-15 and IL-12 which all could contribute to the functional activation of T cells and the

lymphokines could act on NK cells.

6. Activated T- but not NK cells respond to LTB4

We have shown earlier that SAP can be expressed both by activated T and NK cells. Next, we

posed the question whether both cell types contribute to the increase of SAP. To this end,

LTB4 was added to monocyte depleted cultures and this induced SAP expression, however the

lysates prepared after T cells removal did not show the increase of SAP band intensity,

indicating that T cells were responsible for the increase of SAP in the treated culture (Figure

6Ha). Apparently, in the remaining NK cells LTB4 did not elevate SAP expression. This is in

line with the absence of the LTB4 receptor on the NK cells.

When monocytes and T cells were removed prior to infection and LTB4 administration, the

SAP bands in the lysates of the remaining B and NK cells did not show the increased intensity

(Figure 6Hb).

15




In conclusion, LTB4 added to the EBV infected CBMC culture could activate both the

monocytes and the T cells. Thus, in these cultures T cells can be activated both by the added

LTB4 and by the products of the activated monocytes.

7. Generation of EBV-specific cytotoxic cells.

We have shown earlier that the immunomodulator treated cultures could be restimulated by

autologous EBV infected B cells to yield specific cytotoxicity.16 Next, we tested whether this

was also possible when LTB4 was used as immunomodulator. The cultures were stimulated

twice with autologous EBV infected B cells, on the 7th and 14th days. The composition of the

cell population in the 20 day old culture is shown in Table 2. CD19 positive B cells

dominated the culture that did not receive LTB4. In contrast, the LTB4 treated culture

contained mainly CD4 T cells. After removal of B lymphocytes by anti-CD19 Ab-conjugated

beads, the residual cells in the culture exhibited appreciable cytotoxicity affecting autologous

EBV infected B cells, 52% at 30:1 E/T ratio (Figure 7). In accordance with the preponderance

of CD4 T cells in the culture the lytic effect was reduced by HLA class II mAbs CR3/43 (to

20% at 30:1 E/T ratio). The effect seemed to be specific, because activated autologous B

cells, K562 or allogeneic LCLs (CBM1 and LCL2996) were not damaged.17,23

16




Discussion

We have reported earlier that the immunomodulators, PSK and Trx80 initiate a stimulatory

circuit between various cell subsets in EBV infected CMBC cultures. T and NK cells were

recruited into the activation pathway by encounter of EBV infected B lymphocytes and

monocytes were activated by the immunomodulators.17 The present study showed that the fast

acting lipid mediator LTB4, produced by the activated monocytes is an essential part of this

circuit. LTB4 was detected in the supernatants of the cultures in which T cell activation was

induced and inhibition of LTB4 synthesis substantially reduced the T cell activation.

Transformation of B lymphocytes by EBV is the first step that initiates the events in the virus

infected cultures by activation of T and NK cells. Our results did not reveal activation of

monocytes by EBV, that was reported to occur under certain conditions.24,25

We found that LTB4 could initiate the response against the EBV infected B lymphocytes. The

events were similar to what was seen in the PSK or Trx80 treated cultures, with the difference

that monocytes were required for the effect of PSK or Trx80, but LTB4 could stimulate the T

cells even in absence of monocytes. This was due to the expression of BLT1, the specific

LTB4 receptor on the activated T cells. When the culture contained monocytes, however, the

added LTB4 was more efficient because it activated also the monocytes and these provided

additional LTB4 and other lymphokines.

The three monocyte activating compounds used probably differ in their signals. PSK may

activate a Toll-like receptor (Dr. T. Ando, Kureha Corporation, Japan, personal

communication). The receptor of Trx80 is unknown, while the specific receptor of LTB4 is

well defined.4 Using these activators, the lymphokine profiles differed in these short term

experiments. PSK treated monocytes produced predominantly IL-15, Trx80 induced IL-12

while LTB4 induced relatively higher amount of IL-18. Whether these differences are related

to the differences in the activation signals remains to be seen. These lymphokines can activate

T and NK cells.26-28 It is certain that additional monocyte derived factors contribute as well,

however this experimental strategy could show that innate immunological mechanisms

17




operate in the mononuclear cell population and LTB4 could influence the outcome of EBV

infection in vitro.29,30

The results obtained in the primary infection of mononuclear cell cultures are consistent with

the immunological analysis of acute infectious mononucleosis, such as activation of B and T

lymphocytes, NK cells and monocytes, and the presence of lymphokines e.g. IFN-γ, IL-18 in

the serum.31,32

The CD4 T cell response occurs in in vivo the primary infection as well. CD4 T cells with

specificity for lytic and latent EBV encoded proteins were detected in the initial phase of the

disease and the early CD8 T cells were found to recognize lytic proteins.33 The BLT1

expression on the T cells in the 6 day old cultures is in good accordance with the findings of

Islam et al.34 In parallel with the intensity of blast transformation. BLT1 expression peaked on

the 6th day on T cells stimulated by allogeneic dendritic cells.

It is noteworthy that activated, CD69 positive NK cells in the infected cultures did not acquire

BLT1. Consequently NK cells were not influenced by LTB4 directly, but they could be

activated by the lymphokines IL-18, IL-15 and IL-12 contributed by the activated monocytes.

The difference between T and NK cells with regard to BLT1 expression is noteworthy and is

one of the questions that emerge from this experiment system.

The expression of three chemokine receptors in the cells of the culture is in line with the

finding on T cells in the blood of mononucleosis patients. The frequency of CXCR3 and

CCR5 positive T cells increased in parallel with the degree of T cell activation while the

frequency of CXCR4 positive cells decreased.34

In accordance with earlier reports, we detected BLT1 positive B lymphocytes in the ex vivo

population, but not in the infected cultures.21 The loss of BLT1 on the CD19 B cells is in line

with earlier functional results on B lymphocytes, in that LTB4 enhanced the effect of growth

and differentiation inducing factors in resting, high density, but only marginally or not at all in

activated, low density B cells.35,36 The lack of response by activated cells was confirmed with

SAC activated B cells. BLT1 has not been characterized yet at the time of these reports. Our

18




findings suggest that the cells could not be activated by LTB4, because concomitant with

activation BLT1 was downregulated in these early experiments.

NK cells are required in the initiation of the events in the cultures when PSK and Trx80 were

used as activators. Provision of IFN-γ was found to be the main function of the NK cells as

shown by administration of IFN-γ that corrected their depletion (to be published).

In the EBV infected mononuclear cultures derived from cord blood, T cells that lysed EBV

infected autologous B cells could be generated. The first step of this event can be ascribed to

mobilisation of innate immunity. Once the T lymphocytes were activated, selection could be

applied for cells which recognize autologous EBV infected B cells. The functional cells were

CD4 T cells. We found that the initial activation of the effector T cells was essential, since

EBV specific cytotoxicity was obtained also without restimulation in the enlarged population

(to be published).

Several studies have shown that LTB4 is involved in the host defence against bacterial and

viral infections.37-39 EBV infection can trigger innate immunity and LTB4 seems to be an

essential contributor. EBV has unique characteristics, because its main target, the B

lymphocyte is activated by the virus, and activated B cells initiate cell interactions within the

immune system.

Thus, LTB4 acted as immunoactivator in our short term experiments. The immune parameters

studied corresponded well to those observed in mononucleosis. Keeping in mind that the cord

blood leukocyte population differs from the peripheral blood leukocyte population in children

and adults, the model is still useful for analysis of LTB4 induced modification of the

interaction between EBV infected B cells and the innate and adaptive immune system.

LTB4 is a well known physiological effector with multiple actions. The detailed knowledge

and the availability of drugs that inhibit its synthesis is already exploited in therapy.2,40

19




In the in vitro experiments the stimulatory circle between monocytes and T cells could be

interrupted by the inhibition of LTB4 synthesis of the monocytes, neutralisation of their

lymphokine products and by neutralisation of NK and T cell derived IFN-γ.

We anticipate that the experimental strategy used with the cells derived from cord blood can

be used for study of the kinetics and the specificities of T cells as they emerge during the

primary infection. Various aspects of the response to EBV infection have been extensively

studied using mononuclear cell populations collected from seronegative and seropositive

individuals. The EBV encoded proteins that serve as targets for cytotoxic CD8 T cells

selected from seropositive individuals have been identified in several studies.14,41,42 In cultures

with mononuclear cells from seronegative individuals and from cord blood, the effector cells

were CD4 cells.43,44 Further studies are needed for identification of the EBV encoded proteins

that serve as targets in the cord blood system.

Primary EBV infection may be silent or it may induce the symptoms of mononucleosis.8,9 The

severity of the symptoms is highly variable. Sustained immunity is a regular consequence of

both the silent infection and of the overt disease. Therefore the development of lifelong

immunity does not seem to require dramatic activation of the immune system. Conceivably

severe cases could be mollified by reducing the extensive immune activation.

LTB4 may be used to strengthen the immune response to avoid EBV induced B cell

proliferation. Transplant patients receiving immunosuppressive treatment have often elevated

EBV load and high risk on the developing EBV positive B cell malignancies. Administration

of in vitro educated T cells for the recognition of EBV positive B lymphocytes showed good

therapeutical effects in such cases. In experiments with CMV infected mice administration of

LTB4 reduced the viral load but it did not compromise the efficiency of T cell therapy.39

These considerations motivate detailed analysis of the role of LTB4 in the development of

cellular immune responses and the possibility for its modification by LTB4 directed measures.

20




Acknowledgments

We thank the Department of Obstetrics and Gynecology, Karolinska University Hospital, for

the provision of cord blood samples. We thank Dr. Arne Holmgren, Medical Nobel Institute

for Biochemistry, Karolinska Institute for continuing collaboration and providing the Trx80

preparations for this study. We thank Dr. Peter Krammer, Deutsches Krebsforschungszentrum

for critical reading of the manuscript and valuable suggestions. This work was supported by

the Swedish Cancer Society and by the Cancer Research Institute (New York)/Concern

Foundation (Los Angeles).

Author’s contribution

A.L. designed research, performed experiments, analyzed the results and wrote the text;

H.E.C. designed research and analyzed the results; Y.M. performed the LT determinations;

G.K. discussed the results and wrote the text; E.K. designed research, analyzed the results and

wrote the text.

The authors declare no competing financial interests.

21




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26




Table 1. Composition of the cell populations, expression of CD69, BLT1, CXCR4, CXCR3 and CCR5 in ex vivo CBMC and 6 day old cultures

Uninfected CBMC, EBV infected CBMC without, with PSK (25 μg/ml), with Trx80 (100nM), were cultured for 6 days. Expression of CD3, CD56, CD19, CD69, BLT1, CXCR4, CXCR3 and CCR5 were registered by flow cytometry, double staining. Results represent Mean ± SD of 4 experiments.

Cells ex vivo CBMC CBMC+EBV CBMC+EBV+PSK CBMC+EBV+Trx80 CD3 T 66 ± 9 59 ± 11 44 ± 7 61 ± 6 59 ± 6 CD56 NK 12 ± 7 13 ± 5 15 ± 2 19 ± 4 13 ± 3CD19 B 14 ± 5 15 ± 4 41 ± 6 19 ± 7 22 ± 4CD69+CD3+/CD3 3 ± 2 8 ± 4 26 ± 5 71 ± 18 68 ± 16CD69+CD56+/CD56 8 ± 2 16 ± 3 44 ± 10 69 ± 8 71 ± 14BLT1+CD3+/CD3 0 1 33 ± 8 37 ± 6 41 ± 10 BLT1+CD56+/CD56 0 0 0 0 0BLT1+CD19/CD19 51 ± 9 11 ± 4 7 ± 2 8 ± 6 9 ± 4CXCR4+CD3+/CD3 86 ± 6 16 ± 9 5 ± 2 3 ± 1 3 ± 3CXCR3+CD3+/CD3 3 ± 1 2 ± 2 51 ± 11 57 ± 14 56 ± 6CCR5+CD3+/CD3 4 ± 3 3 ± 2 54 ± 5 56 ± 7 61 ± 11

27




Table 2. Composition of the cell populations in 20 day old cultures initiated with EBV infected CBMC (%)

The cultures were restimulated twice, on the 7th and 14th days with irradiated (50Gy) autologous EBV infected B cells. Half of the culture medium was replaced with fresh medium. From the 9th day 20 U/ml IL2 was added every third day. Results represent Mean ± SD of 3 experiments.* LTB4 was added at the initiation of the culture

Cells CBMC+EBV+LTB4* CBMC+EBV

CD19 B 11 ± 6 89 ± 7 CD4 T 71 ± 7 3 ± 3 CD8 T 6 ± 5 2 ± 1 CD56 NK 6 ± 3 4 ± 2 CD3 T 79 ± 8 5 ± 3

28




Figure legends

Figure 1. The involvement of LTB4 in T and NK cell activation in the EBV infected PSK

and Trx80 containing cultures. (A) SAP expression. Immunoblot of lysates corresponding

to 1.5×105 cells, harvested on the 6th day of culture. EBV infected CBMC were cultured with

PSK (25 μg/ml) or Trx80 (100nM). MK886 (1μM), BWA4C (100nM) and LTB4 (100nM)

were added as indicated. Jurkat cells and LCL were used as positive and negative controls.

The results show 1 representative of 3 experiments. (B) IL-15 and IL-12 in the supernatant of

3 day old cultures were tested by ELISA. The results represent the Mean ± SD of 3

independent experiments. * P< 0.05 and ** P< 0.01 compared with PSK or Trx80 treated

cultures. (C) Inhibition of B cell proliferation induced by EBV. 3H-thymidine incorporation

was tested on the 6th day of culture. 3H-thymidine was present during the final 14 hrs. The

cultures were initiated with 2×105 cells. Mean ± SD of 3 independent experiments. * P< 0.05

compared with untreated cultures.

Figure 2. Production of LTB4 and IFN-γ in the EBV infected PSK and Trx80 containing

cultures. (A) LTB4 in the supernatant of 24 hrs old cultures was detected by EIA. Cultures

were initiated with 1×106/ml EBV infected CBMC, total and monocyte (Mo) depleted, and

uninfected CBMC. The results represent the Mean ± SEM of 5 independent experiments. (B)

IFN-γ in the supernatant of 3 day old cultures was tested by ELISA. The results represent the

Mean ± SD of 4 independent experiments. ** P< 0.01 compared with untreated cultures. (C)

LTB4 production in cultures containing the indicated antibodies added at the initiation of

cultures. The results represent the Mean ± SEM of 3 independent experiments.

Figure 3. Expression of CD69, BLT1 (LTB4 receptor) on T and NK cells and BLT1 on B

cells. Expression of CD69 and BLT1 were registered by flow cytometry, using double

staining. The numbers denote the percentage of positive cells in the indicated cell categories.

29




Uninfected CBMC; EBV infected CBMC were cultured for 6 days. (A) CD69 expression on

CD3+ T cells and CD56+ NK cells. BLT1 expression on CD3+ T cells, CD56+ NK cells and

CD19+ B cells. The results show 1 representative of 4 independent experiments. The cell

composition of the cultures is given in Table 1. (B) BLT1 expression on CD3+, CD4+, CD8+ T

cells.

Figure 4. Added LTB4 induced SAP expression, and inhibition of B cell proliferation in

the EBV infected cord blood cell cultures. (A) SAP expression. Immunoblot as in Figure

1A. EBV infected CBMC cultured for 6 days with LTB4, or LTC4, LTD4, 5(S),12(S)-DiHETE

at 100nM concentration. The results show 1 of 3 experiments. (B) Dose response of the added

LTB4, 1 representative experiment of 4 performed. (C) Cell composition in EBV infected

CBMC cultured with LTB4 for 6 days. The numbers denote the percentage of CD3, CD56,

CD19 positive cells. (D) EBV induced B cell proliferation in the LTB4 containing cultures.

3H-thymidine incorporation was measured on the 6th day of culture, 3H-thymidine was

present during the final 14 hrs. The cultures were initiated with 2×105 cells. The results

represent the Mean ± SD of 4 independent experiments. * P< 0.05 and ** P< 0.01 compared

with untreated cultures. (E) Isolated B cells were infected with EBV and cultured with LTB4.

The cultures were initiated with 1×105 cells. Thymidine incorporation was measured on the

6th day of culture. The results represent the Mean ± SD of 4 independent experiments.

Figure 5. Cytokine production in the LTB4 containing EBV infected cultures.

IFN-γ, IL-15, IL-12 and IL-18 in the supernatants of 3 day old cultures were tested by ELISA.

Dose response of the added LTB4 in total and in monocyte depleted cultures. (A) IFN-γ. (B)

IL-15. (C) IL-12. (D) IL-18. The results represent Mean ± SD of 4 independent experiments.

* P< 0.05 and ** P< 0.01 compared with untreated cultures.

30




Figure 6. Contribution of monocytes (Mo) and T lymphocytes to the SAP expression

induced by LTB4 in the EBV infected cultures. SAP expression was tested on the 6th day in

all cultures. (A) EBV infected CBMC, total and monocyte (Mo) depleted populations were

cultured with 1nM, 10nM and 100nM LTB4. 1 representative experiment of 4 performed. (B)

B cell proliferation. Total and monocyte (Mo)-depleted populations infected with EBV, and

cultured without or with 100nM LTB4. 3H-thymidine incorporation was measured on the 6th

day of culture. The results represent the Mean ± SD of 3 independent experiments. ** P< 0.01

compared with untreated cultures. (C, D, E) Monocyte depleted populations were infected

with EBV. The separated monocytes were exposed to LTB4 for 20hrs, and thereafter

reintroduced to the depleted population. The cultures were kept for further 5 days. (C) SAP

expression. The separated monocytes were treated with LTB4 (100nM), PSK (25 μg/ml) and

Trx80 (100nM). LTB4 was added to the cultures as indicated. The results show 1

representative of 3 experiments. (D) LTB4 (10nM or 100nM) treated monocytes were

reintroduced to the cultures. MK886 (1 μΜ) and BWA4C (100nM) were added as indicated.

(E) LTB4 (100nM) treated monocytes were reintroduced to the cultures. Anti-IL-18 (2 μg/ml),

anti-IL-15 (10 μg/ml) and anti-IL-12 (10 μg/ml) reagents were added as indicated. (F) Cell

proliferation. EBV infected CBMC cultures without and with LTB4. To parallel samples

antibodies anti-IL-18 (2 μg/ml), anti-IL-15 (10 μg/ml) and anti-IL-12 (10 μg/ml) were added

as indicated. 3H-thymidine incorporation was measured on the 6th day of culture. The results

represent the Mean ± SD of 3 independent experiments. * P< 0.05 and ** P< 0.01 compared

with untreated EBV-infected cultures. (G) SAP expression. EBV infected CBMC, total and

monocyte (Mo) depleted populations were cultured with 10 ng/ml IL-18. The results show 1

representative of 3 experiments. (H) SAP expression in monocyte and T cell depleted

cultures. a, Monocyte depleted cultures were infected with EBV and cultured without or with

LTB4 (100nM). On the 6th day of culture the cells were collected and the T cells were

depleted before preparation of the lysates, which were then tested for SAP expression. b, Cell

31




population depleted of Mo and T cells were infected with EBV and cultured without or with

LTB4. The results show 1 representative of 3 experiments.

Figure 7. Cytotoxic cells could be generated from the EBV infected LTB4 containing

cultures. EBV infected CBMC were cultured with LTB4 (100nM) and restimulated twice (on

7th and 14th day of culture) with irradiated autologous EBV infected B cells at a ratio of 10:1.

Starting on the 9th day of culture IL-2 (20U/ml) was added every 3rd day. The 20th day old

cultures were depleted of B cells, the cytotoxic effect of the residual cells was tested against:

Autologous EBV infected B cells , preincubated with mAb W6/32 , preincubated

with the mAb CR3/43 ; Autologous B cells activated with CD40L and IL4 ; K562

cells ; and allogeneic LCLs: CBM1 × , LCL2996 ◊ .

32




Figure 1

A

B

Actin

SAPex

viv

o

Jurk

at

LC

L

EB

V

----

MK

BW

-------------------------EBV+ PSK

MK

+BW

MK

+BW

+LT

B4

ex v

ivo

Jurk

at

LC

L

EB

V

----

MK

BW

-------------------------EBV+ Trx80

MK

+BW

MK

+BW

+LT

B4

0123456

D 3

IL-1

2 (1

02 pg/

ml)

--

EBV+ Trx80

MK

BW

MK

+BW

MK

+BW

+LTB

4

----------------------EBV

* *

0123456

IL-1

5 (1

02 pg/

ml)

--

EBV+ PSK

MK

BW

MK

+BW

MK

+BW

+LTB

4

---------------------EBV

*

**

C

0

20

40

60

80

100

2 10

cpm

x10

3

--

EBV+ PSK

MK

BW

MK

+BW

MK

+BW

+LTB

4

----------------------

EBV

* *

0

20

40

60

80

100

cpm

x10

3

--

EBV+ Trx80

MK

BW

MK

+BW

MK

+BW

+LTB

4

---------------------

EBV

* *




Figure 2

A B C

0

1

2

3

LTB 4

(10

2 pg

/ml)

--

CBMC+EBV--------------

PSK

Trx

80

PSK

Trx

80

PSK

Trx

80

---------------

-- --

-------------CBMCCBMC-Mo

+EBV

0

1

2

3

4

5

D 3

IFN

- γ

(ng/

ml)

--CBMC+EBV-----------------

PSK

Trx

80

****

0

1

2

3

4

LTB4

(10

2 pg

/ml)

--

CBMC+EBV-------------------------------------

PSK

Trx

80

PSK

Trx

80

PSK

Trx

80

---------α-IFN-γ

α-IL

-15

α-IL

-12




Figure 3

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104CD69 PE

100 101 102 103 104CD69 PE

100 101 102 103 104CD69 PE

100 101 102 103 104CD69 PE

100 101 102 103 104CD69 PE

100 101 102 103 104CD69 PE

100 101 102 103 104CD69 PE

100 101 102 103 104CD69 PE

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

10 724323

751150

000

ex vivo

CD3

+EBV +EBV+Trx80

Day 6

---

CD56

2 8 21 69

BLT1 -FITC

CD56

CD19

0

CD3453610

CD69 -PE

A

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

100 101 102 103 104BLT1 FITC

373100

44420

ex vivo +EBV +EBV+Trx80

Day 6

---

BLT1 -FITC

CD4

CD8

0

CD3

403800

100 101 102 103 104BLT1 FITC

CD1952 8 1 3

B




Figure 4

A B

ex v

ivo

Jurk

at

LC

L ----

-----------------------------CBMC+EBV

LT

B4

100n

M

LT

C4

100n

M

LT

D4

100n

M

DiH

ET

E 1

00nM

CB

MC

day

6

Actin

SAP

ex v

ivo

---

-----------------------

LT

B4

10nM

LT

B4

100n

M

LT

B4

1nM

CBMC+EBV

---

-------------------------

LT

B4

10nM

LT

B4

100n

M

LT

B4

1nM

CBMC

Actin

SAP

100 101 102 103 104CD19 FITC

100 101 102 103 104CD19 FITC

100 101 102 103 104CD19 FITC

100 101 102 103 104CD19 FITC

100 101 102 103 104CD56 Cy5

100 101 102 103 104CD56 Cy5

100 101 102 103 104CD56 Cy5

100 101 102 103 104CD56 Cy5

100 101 102 103 104CD3 PerCP

100 101 102 103 104CD3 PerCP

100 101 102 103 104CD3 PerCP

100 101 102 103 104CD3 PerCP

-- 1 10 100

LTB4 (nM)------------------

010203040506070

cpm

x10

3

-- 1 10 50 100

LTB4 (nM)----------------------

0

20

40

60

80

100

120

cpm

x 1

03

****

*

D

E

C

47 32 2116

12 13 17 10

ex vivo +EBV +EBV+LTB4 10nM

Day 6

+EBV+LTB4 100nM

CD3

CD56

CD19

68 41 52 63




Figure 5

012

345

IFN

- γ (

ng/m

l)

-------------Monocyte

-- 1 10 50 100 -- 10 100

LTB4 (nM)--------------------------------

**

**

B

0

1

2

3

IL-1

5 (1

02 pg

/ml)

-- 1 10 50 100 -- 1 10 50 100

LTB4 (nM)----------------------------------------

-Monocyte---------------

A

0

1

2

3

IL-1

2 (1

02 pg/

ml)

-- 1 10 50 100 -- 1 10 50 100

LTB4 (nM)----------------------------------------

-Monocyte---------------

0

1

2

3

4

5

6

7

IL-1

8 (1

02 pg/

ml)

-- 1 10 50 100 -- 1 10 50 100

LTB4 (nM)----------------------------------------

-Monocyte----------------

**

**

*

D

C




Figure 6 A-D

ex v

ivo

---

----------------------L

TB

410

nM

LT

B4

100n

M

LT

B4

1nM

CBMC-Mo+EBV

---

--------------------

LT

B4

10nM

LT

B4

100n

M

LT

B4

1nM

CBMC+EBV

Actin

SAP

A

0

20

40

60

80

100

cpm

x10

3

****

B

---

----------

LTB

410

0nM

CBMC+EBV

---

----------

LTB

410

0nM

CBMC-Mo+EBV

D

Mo

LTB

4 10

nM

--------------------------------M

o LT

B4

100n

M

Mo

LTB

4 10

0nM

CBMC-Mo+EBV

Mo

LTB

4 10

nM

ex v

ivo

Jurk

at

LC

L

Mo

----

MK+BW -- -- -- -- -- -- -- + +

Actin

SAP

C

Mo

LTB

4

-----------------------------------

Mo

PSK

+Trx

80

LT

B4

CBMC-Mo+EBV

Mo

LTB

4 +L

TB

4

ex v

ivo

Jurk

at

LC

L

Mo

----

Actin

SAP




Figure 6 E-H

E

Mo

LTB

4 10

0nM

--------------------------------M

o LT

B4

100n

M

CBMC-Mo+EBVM

o L

TB4

100n

M

ex v

ivo

Jurk

at

LC

L

Mo

----

α-IL-15+α-IL-12 -- -- -- -- -- -- -- + α-IL-18 -- -- -- -- -- -- + --

Actin

SAP

0102030405060

cpm

x10

3

F

** *

α-IL-15+α-IL-12 -- -- + -- -- +α-IL-18 -- + -- -- + --LTB4 -- -- -- + + +

CBMC+EBV

G

ex v

ivo

CBMC-Mo

+EBV

---

------------

IL-1

8

CBMC+EBV

Jurk

at

LC

L ---

------------

IL-1

8

Actin

SAP

H

---------------------L

TB

4

LT

B4

CBMC-Mo+EBV

---

ex v

ivo

Jurk

at

LC

L

----

-T c

ell

LT

B4

-T c

ell

----------------CBMC-Mo-T+EBV

a b-------------------- -------------Actin

SAP




Figure 7

E/T ratio

010203040506070

30 10 3

% s

peci

fic ly

sis




Date post:	13-May-2023
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