Vβ8+CD4−CD8− subpopulation induced by staphylococcal enterotoxin B

ELSEVIER

Immunology Letters 55 (1997) 85-91

VP 8 + CD4CD8-

Miao-Chen

subpopulation induced by staphylococcal enterotoxin B

Chou I, Shiour-Ching

Lee, Yee-Shin

Lin, Huan-Yao

Lei *

Deparlment qf Microbiology and Immunology, College of Medicine. National Cheng Kung University,. Tainan. Taiwan. ROC Received

1 December

1996: received

in revised

form 23 December

1996: accepted

26 December

1996

Abstract Injection of staphylococcal enterotoxin B (SEB) into BALB/c mice induced the depletion of V,,8+T-cells which were either V,,8 +CD4 ’ or V,]8 + CD8 + cells. The CD8 molecule on V,8+CD8 + cells was found to decrease at 72 h after SEB treatment while the V,{8 molecule on V,8+ cells or CD4 molecule on V,j8 + CD4 + cells was not affected. Furthermore, a subpopulation of V,,8 + CD4CD8. T-cells was also induced after SEB-priming. This subpopulation can be found in spleen or lymph nodes. It

was small in size and constituted the major part of V,8+ cells in lymph nodes at 72 h after SEB-priming. Some of the V,{8 + CD4_CD8- T-cells might be autoreactive because they could be stimulated to proliferative by syngenic mitomycin C-treated splenocytes. 0 1997 Elsevier Science B.V. Keywords:

Staphylococcal

enterotoxin

B: Double-negative

V,j8 T-cells; Autoreactive

1. Introduction Staphylococcal enterotoxin B (SEB) is a superantigen that stimulates T-cells bearing V,)7, 8.1-3 T-cell receptors [l-4]. Injection of SEB into naive mice induced specific tolerance associated with a selective deletion of peripheral V,]S cells by apoptosis [5-91. The SEB-induced deletion of V,]8 + T-cells was a bimodal response with an early deletion of V, 8 + CD4 + and V, 8 + CD8 + cells at 12 h after SEB injection. Following a transient expansion of V,$8 + cells, a second wave of cell deletion occurred at day 4. Moreover, the remaining SEB-specific T-cells became anergic and were hyporesponsive to SEB restimulation. SEB also caused apoptosis of immature thymocytes [lo]. The outcome of SEB-induced

* Corresponding author. Fax: + 86 6 2082705. ’ Present address: Department of Medical Technology, Foo Yin Junior College of Nursing and Medical Technology, Kaohsiung, Taiwan, Republic of China. 0165-2478!97,$17.00 Q 1997 Eisevier Science B.V. All rights PII SOl65-2478(97)02689-8

reserved

T-cell responses, such as activation, anergy, or apoptosis, seems to depend on the subset or status of the T-cells. In the present study, we reported that SEB priming also induced a subpopulation of V,j8 + CD4 CD8- T-cells which might be autoreactive.

2. Materials

and methods

2.1. Mice Breeder mice of BALB/c strain were purchased from The Jackson Laboratory, Bar Harbor, ME or Charles River Japan (Atsugi, Japan). They were maintained on standard laboratory chow and water ad libitum in the animal facility of the Medical College, National Cheng Kung University, Tainan, Taiwan. The animals were raised and cared for following the guidelines set up by the National Science Council of Republic of China. Six to twelve week old mice were used in all experiments.

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86

2.2. Reagents

SEB was purchased from Toxin Technologies, Sarasots, FL. Rat anti-mouse V,8.1, 8.2-FITC conjugate and rat anti-mouse CDB-PE conjugate were purchased from PharMingen, San Diego, CA. Rat anti-mouse CD4-TRI-COLOR was purchased from Caltag Laboratories, San Francisco, CA. 2.3. In vivo treatment of SEB Groups of four mice were injected subcutaneously at the base of tail and flank with 50 ~1 of an emulsion containing 20 pg of SEB in complete Freund’s adjuvant (CFA) (Difco Laboratories, Detroit, MI) [l 11. Fourteen days later, the mice were challenged with 20 ,ug of SEB intravenously (i.v.). Naive mice were used as a control. 2.4. In vitro T-cell proliferation Single cell suspension (5 x lo6 cells/ml) from spleen was seeded in 96-well flat-bottom microtitre plate (Falcon, Becton Dickinson Labware, Oxnard, CA) in 0.2 ml of RPM1 1640 medium containing 5 x 10 - 5 M of 2-mercaptoethanol and 10% fetal bovine serum. CD4 + and CD8 + lymphocytes were depleted by anti-CD4 (GK1.5) and anti-CD8 (3.155) cocktail with rabbit complement. The depletion was greater than 95% as confirmed by FACScan analysis. These cells were cultured with syngenic mitomycin C-treated splenocytes for 3 days and pulsed with 1 &i of [3H]thymidine for the final 24 h. The [3H]thymidine uptake was determined by direct B-counters (Matrix 9600, Packard Instrument Co, Meriden, CT). 2.5. Immunojluorescence

Letters 55 (1997) 85-91

the kinetic study to follow the change of V,8 + cells after SEB treatment, experiments were repeated three times with the same pattern and one set of representative data is shown.

3. Results 3.1. VR8 + CD4_CD8- subpopulation induced by SEB

The V,8+ subpopulation was monitored by flow cytometry with three color stains of FITC-V,B/PECDB/TRI-COLOR-CD4. In naive mice, the number of V,8+ cells decreased about 50% from 95 x lo6 to 52 x lo6 cells/mouse at 12 h, increased back to background level at 24 h and decreased again to half at 72 h after i.v. SEB injection (Fig. l(a)). Among the changes of V,8 + cells, V,]8 + CD4 + CD8- cells were the major cell type that contributed to the SEB-induced V,8+ cell fluctuation (Fig. I(b) and (c)). Interestingly, a subpopulation of V,]8 + CD4 - CD8- cells was manifested. This subpopulation was decreased at 12 h, expanded at 24 h and decreased again at 72 h, which was Q

Naive + i.v. SEB

--0 --

SEBKFA

+ i.v. SEB

1b

V88+, CD4+, CD8-

analysis H

Fifty microliters of lymphocytes (2 x 107/ml) were suspended in HBSS containing 2% FCS and 0.1% NaN,. Cells were then incubated with anti-V88.1, 8.2FITC, anti-CDB-PE, and anti-CDCTRI-COLOR [12]. After incubation for 30-45 min on ice, the mixture was washed twice with ice-cold HBSS and the cells were resuspended and adjusted in HBSS containing 2% FCS and 0.1% NaN, to approximately 1 x lo6 cells/ml. Stained cells were analyzed by flow cytometry (FACScan, Becton-Dickinson, Mountain View, CA) with excitation set at 488 nm. The V,8+ subpopulation was monitored by three color stains of FITC-V, 8/PE-CDB/TRI-COLOR-CD4 at 12, 24 and 72 h after SEB i.v. injection. The V,8+ cells were gated and their CD4 + or CD8 + percentage was calculated. The cells from four mice per group were pooled. The total number of splenocytes or lymph node cells multiplied by the percentage of the subpopulation gave the absolute numbers of that subpopulation. In

1c

Vg8+, CD4-, CD8+

Id

VB8+, CD4-, CD8-

Tj40p

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2

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60

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Fig. 1. SEB-induced changes of V,8 + CD4 + CD8 - /V,8 + CD4 CD8cells in spleen. Groups of four BALB/c mice of naive or SEBjCFA sensitized 14 days before were given 20 pg of SEB i.v. 12, 24 or 72 h after SEB injection, the pooled splenocytes were stained with anti-V/,8.1, 8.2-FITC, anti-CDI-PE and anti-CD4-TRICOLOR. The V,8 + cells and their subpopulation were analyzed and the cell numbers of different subpopulations per mouse were expressed. Experiments were repeated three times with the same pattern and one set of representative data is shown.

M.-C. Chou et al. /Immunology

Letters 55 (1997) 85-91

87

Vp8+ T cells 7501

+ - -*

350.

0

40

20

60

Naive + i.v. SEB --

SEBICFA + i.v. SEB

60

H

Vg8+CD4+ T cells

Vg8+CD8+ T

,.J”

cells

760

700

700

650

650

600 i

600 550 500

350 I I

H Fig. 2. Fluorescence or CD8 on V,8+.

0

20

40

60

intensity of V,8. CD4 or CD8 molecules on V,8 + cells after SEB treatment. The mean fluorescence V,)8+CD4+, or V,{8+CD8+ cells. respectively, was determined from the data of Fig. 1.

similar to the change of V,8 +CD4+ CDS- cells (Fig. l(d)). The pattern of fluctuation of V,8 + cells in SEB/CFA-sensitized mice after i.v. SEB injection was different from that of naive mice. The number of V,8 + cells increased gradually till 24 h, which was mostly due to the increased number of V,8 + CD4CD8- cells. The fluctuation of V,{8 + CD4 + CD8- cells in SEBjCFA sensitized mice was similar to that in naive mice. The changes of V,j8 + CD4CD8 + cells were not as dramatic as those of V,{8+ CD4+CD8- cells. The mean fluorescence intensity of CD4 or CD8 molecules was calculated in V, 8 + CD4 + or VP8 + CD8 + cells, respectively. The fluorescence intensity of CD8 molecule on V,8 -+ CD8+ cells was significantly decreased at 72 h after SEB injection in both naive and SEBjCFA sensitized mice (Fig. 2). On the other hand, change in fluorescence intensity was not observed for either the VP8 molecules on V,8 + T-cells or the CD4 molecules on V,8 + CD4 + T-cells. The dynamic change of V,8+ cells in lymph nodes showed a slightly different fluctuation pattern. In naive

3

H intensity

of V,,8. CD4.

mice, the number of VP8 + cells gradually reduced from 16 x lo6 cells to 10 x lo6 per mouse by 24 h, then increased back to 23 x lo6 cells at 72 h after SEB i.v. injection (Fig. 3(a)). The depletion of V,/ 8 + CD4 + CD8cells in lymph nodes was the primary change while V,8 + CD4CD8 + cells were only slightly affected by SEB (Fig. 3(b) and (c)). V,]8 + CD4CD8- cells were also found in the lymph node and they (14 x IO6 cells) constituted 60% of the lymph node V,]8 + (23 x 106) cells at 72 h after SEB injection (Fig. 3(d)). In SEB/ CFA-primed mice, there was a transient increase in the number of V,8+ lymph node cells by 24 h, which attributed to the increase in V,{8 + CD4 + CD8- or V,,8 + CD4 - CD8 - cells. The SEB-induced change of V,8 + CD4 ~ CD8 - cells in SEB/CFA-primed mice was much weaker than that in naive mice. The fluorescence intensity of CD8, not V,8, molecules in V,j8 + CD8 + lymph node cells was also significantly decreased at 72 h (data not shown). We have consistently sought these kinetic changes of V,8+ cells of different subpopulations in three experiments and one representative data is shown.

88

M.-C. Chou et al. /Immunology

3.2. Characteristics of SEB-induced subpopulation

V,S+ CD4_CD8-

V,8 + lymph node cells were analyzed based on their size. Either small or regular size-V/,8+ cells bore CD4 or CD8 markers. Seventy-two hours after SEB iv. injection into naive mice, the number of small-size lymphocytes increased, and the small V,{8 + lymphocytes lost CD4 or CD8 molecules and became CD4CD8- (the percentage of CD4_CD8- cells increased from 2 to 83%) (Fig. 4(a)). The regular sizelymphocytes, however, still bore the CD4+ or CD8+ marker (only 26% of cells were CD4-CD8). Apparently, the size of V,8 + CD4CD8. cells was smaller than that of V,8 + CD4 + or V,,8 + CD8 + cells. In SEB/ CFA-primed mice, small V,8 + CD4 - CD8 - cells were already present (88% of small V,8 + cells) and their percentages were not changed 72 h after SEB injection. In the regular size of V,8 + lymphocytes, 49954% were also CD4CD8 - (Fig. 4(b)). The CD4CD8- cells were obtained by treating lymph node cells with anti-CD4, anti-CD8 antibodies and complement. These double +

Naive + i.v. SEB

- 0

3b

-

SEBICFA + i.v. SEB

Vg8+, CD4+, CD8‘

14’

0

20

40

60

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3c v$3+,CM, CM+ 12 10 6

I---_

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-0

0

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20

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80

0

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80

H

Fig. 3. SEB-induced changes of Vp8+CD4+CD8-/V,8+CD4CD8 - cells in lymph nodes, Groups of four BALB/c mice of naive or SEBjCFA sensitized 14 days before were given 20 pg of SEB i.v. 12, 24 or 72 h after SEB injection, the pooled lymph node cells were stained with anti-V,8.1, 8.2-FITC, anti-CDI-PE and anti-CDCTRICOLOR. The V,8 + cells and their subpopulation were analyzed and the cell numbers of different subpopulations per mouse were expressed. Experiments were repeated three times with the same pattern and one set of representative data is shown.

Letters

5.5 (1997) 85-91

negative cells derived from naive or SEB/CFA-primed mice could be stimulated to proliferate by syngenic mitomycin C-treated splenocytes (Fig. 5). This suggested that some of the V,j8+CD4CD8cells were autoreactive. Taken together, we demonstrated that SEB stimulated the generation of V,8 + CD4CD8- and some of them were autoreactive.

4. Discussion The injection of SEB into naive mice caused depletion, anergy and apoptosis of SEB-reactive lymphocytes such as the V,8 + cells. In this study, we demonstrated that the SEB priming also induced a subpopulation of V, 8 + CD4-CD8 ~ V,]8 + CD4CD8cells. These lymphocytes were small in size and constituted the major part of the V,]8 + cells in lymphoid organs, especially in lymph nodes after SEB-priming. The V,8+ CD4CD8- cells were autoreactive and might be involved in the regulation of SEB-induced responses. Injection of SEB into naive mice induced selective deletion of peripheral V,8 + cells via apoptosis [7,8]. In addition, bimodal deletion of V,8 + T-cells after SEBtreatment was reported [13]. In this study, we also found that the first deletion of V,8+ T-cells occurred at 12 h and targeted the V,8 + CD4 + and V,8 + CD8 + cells. The second deletion occurred at day 4 and was preceded by a transient expansion of V,j 8 + CD4 + CD8. or V,8+CD4CD8T-cells at 24-48 h. Moreover, the remaining SEB-specific T-cells became anergy and were hyporesponsive to SEB-restimulation (unpublished observation). However, the early depletion of V,8 + Tcells was not observed in SEB/CFA-primed mice after SEB injection, this might be due to previous depletion of some SEB-reactive V,{8 cells during the priming process. The majority of V,j8 + cells depleting was CD4 + CD8- (Figs. 1 and 3) or CD4 + CD45RB + cells (unpublished observation). Whether this reflects the resistance of V,]8 + CD45RB cells (or V,8 + CD45RO + equivalent) to SEB-induced deletion needs further investigation. TCR&CD4CD8T-cells, a novel subset of T-cells, was reported to be present in normal tissues or in autoimmune diseases of man and mouse [14,15]. The TCR repertoire of these double negative T-cells was restricted. T-cells of this phenotype were immunocompetent and could be activated via the CD3 pathway. In murine splenic and pulmonary CD4CD8‘ cells, TCRV,]8 + genes were preferentially expressed. Some cloned murine splenic TCR$,CD4_CDS ~ T-cell lines recognized syngenic and allogenic dendritic cells in an MHCunrestricted way [16]. Purified murine splenic CD4_CD8- T-cells proliferated specifically in response to syngeneic stimulator cells. Splenic TCRsCD4CD8 T-cells from normal mice showed MHC class II-re-

fluorcxmcc 2

CD8

V,8.1,8.2

Fluomrmn

2

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V,8.1, 8.2

M.-C. Chou et al. /Immunology Letters 55 (1991) 85-91

90

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stricted autoreactivity [ 17,181. Furthermore, reported the switching of CD8+ T-cells tolytic CD4CD8cells and their synthesis

Erard et al. to non-cyof Th2 cy-

tokines [19]. The TCR,fpCD4CD8T-cells may represent a unique developmental pathway of a separate lymphoid lineage [20]. Alternatively, they may

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Letters 55 (1997) 85-91

Acknowledgements

5a

This work was supported BOO6050 from the National

by grant, NSC84-2331Science Council, ROC.

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