Vδ1-T-Cell Receptor+ T Cells Respond to B-Cell Lines and Lipopolysaccharides

+

Murine Epidermal Vy5N81-T-Cell Receptor

T Cells

Respond to B-Cell Lines and Lipopolysaccbarides Christopher L. Reardon, *§ Kent Heybome,:j: Moriya Tsuji,� Fidel Zava1a,� Robert E. Tigelaar, ** L. O'Brien, t§ and Willi K. Bornt§ Departments of * D ermatolo gy and tImmunology, University of Colorado Health Science s Cente r; *Dep artment of Reproductive Immunology, Swedish Medical Center, §Department of Medicine , NationalJewish Center for Immunology and Respiratory Medicine, Denver, Colorado; �Dep artment of Medical and Molecular Parasitology, New York University Medical Center, New York, New York; and ""Department of Derm ato lo gy , Yale University, New Haven, Connecticut, U.S.A.

Rebecca

The Vy5Nl>1 +-T-cell receptor (TCR)- bearing T-cell

When lipopolysaccharides (LPS) from Escherichi4

Upon stimulation, 2CBET-3 cells produce interleukin

presence of A20/1J cells, 2CBET -3 cells responded with increased cytokine production compared with the cy­

clone, 2CBET-3, was generated from

coli or S. typhimHriHm were added to 2CBET-3 cells in the

C57BLl6 mice.

(IL)...;3, granulocyte-m.acrophage colony-stimulating

factor, and tumor necrosis factor-a, but not ll..-2, ll..-4 ,

tokine production in the presence of A20/2J cells alone.

factor, or interferon-yo These cells were evaluated for

alone or to supernatants from

2CBET-3

ll..-S, ll..-6, ll..-8, ll..-10, macrophage colony-stimulating

cells by themselves did not respond to LPS

A20/2]

cells incubated

by a variety of murine cell

with LPS. Ulilike 2CBET-3, the epidermal T-cell hy­

tocytoma cells, and keratinocytes. The human B-Iym­

in the presence or absence ofLPS, suggesting a require­

kines up to several hundredfold above the control levels

This unique reactivity of

their ability to be stimulated

lines, including fibrobia:sts, trophoblasts, melanoma cells, embryonic carcinomas, B-cell lymphomas, mas­

bridoma 70BET-49, expressing a Vy5Nl>1-TCR identi­ cal to that of 2CBET-3, did not respond to A20/2J cells

ment for molecules other than the TCR for Vy5/Vl>1-

phoma cell line, Daudi, also was included in these studies. We found that 2CBET-3 cells produced cyto­ in response to the B-cell lines, Daudi, and

A20/2J,

not to the B-cellline 439.4.2. After fixation taraldehyde, Daudi and

this

A20/2J

TCR+ T-cell stimulation by the B-celllines and by LPS.

from that of

but

with glu­

tional specialization of to bacterial infections.

continued to stimulate

Yl> T -cell line. 2CBET-3 cells also responded to the

cyte. J Invest

keratinocyte line PAM212, but not to another, XB-2.

OSt murine

epidermal T cells, having a dendritic

morp holo gy in vivo, express a uniform T-cell

receptor (TCR) composed of a V'Y5 and a V81 gene product [1]. A minor population of e pider­ M mal T cells expr e ss es V'Y1/V86.3 TCRs and respond s to the heat shock protein HSP-60 [2]. However, V'Y5/ V8l-TCR + epidermal T cells do not resPQnd to HSP-60, and the ligands that they recognize have remained elusive. Investigation s thus far have shown that V'Y5/V81-TCR + epidermal T cells resp ond to fresh keratinocytes and to the keratinocyte cell line PAM2l2 [3], as well as to heat-shocked keratinocytes,tt but not to sple en cells and fibroblasts [3]. These data suggeste d that V'YS/V8l­ TCR + epidermal T-cell responsiveness is restricted to keratino­ cytes. However, an observed response of epidermal T-cell clones to the B-celllymphoma cell line A2012] (R. E. Ti gelaar , unpublished data) led us to investigate whether epidermal T cells Can respond to

yl>-TCR+ cells in the response Key words: T lymphocytelB lympho­

105:588- 61S, 19!J5

Because V'Y5/V8l-TCR+ epiderm al T cells are restric ted to the are theref ore likely to serve as a first line of de fense , they may respond to patho gens or to the products of patho gen s, such as lipopolysaccharides (LPS), a constituent of gram-neg a tive bacteria. Litde information exists as to the LPS respon siveness ofT cells. A characteristic that distinguishes B c ell s from T cells is that at least 30% ofB cells are stimulated by LPS, independen t ofT-cell help, whereas T cells as a rule do not respond to LPS (reviewed in [4]). However , a fe w studies have emerged suggesting that an LPS-reactive population of T cells exists. A clonal T-cell line, CT 6, p rolifer ates in respo nse to LPS without requiring the presence of other cells [5]. These investigators also found that a small popula­ tion (approximately 3%) of murine splenic T cells pr oliferates in res ponse to LPS. Similarly, gr am-nega tive bacteria-reactive T cells appear to r espond to the LPS portion of the bacteria [6]. None of epidermis and

aJ3-TCRs or 'Y8-TCRs. We show herein that V'Y5/V8l-TCR+ epidermal T cells respon d to LPS in the pre sence of A2012] cells.

to: Dr. Christopher L.. Reardon , Department of Der­ E-153, University of Colorado Health Sciences Center, Denver,

Reprint requests CO

Devmatol

Yl>-TCR+ cells is different

cells and may re:ft.ect a func­

these studies addressed whether the LPS-reactive T cells express

other cell types.

matology,

afJ-TCR+

MATERIALS AND METHODS

80262.

tt Lewis J, Tigelaar, RE. Recognition of an epideraml stress antigen by "118 dendritic epidermal T cells (DETC) (abstr). ] Invest Dennatol

Monoclonal Antibodies

96:538A,

1991.

0022-202X195/$09.50

•

SSDI0022-202X(95)00219-B

•

The following antibodies were used:

(anti-pan 'YB-TCR) [7], 1701 (anti-V')'5/V81),tt 145-2Cll (anti-CD3) [9].

murine

Copyright

58S

536

403A.10

(anti-V'Y5) [8],

© 1995 by The Society for Investigative Dermatology,

Inc.

and

VOL. lOS, NO.

1, SUPPLEMENT,

JULY 1995

B-CELL

10 ...

y3-TCR+ CELLS

EPIDERMAL

S9S

2CBET-3

n co "

AND LPS REACTIVE

DAUDI

0.1

2CBET-3 + DAUDI

0.Q1

2CBET·3 A20/2J IL·2

ILoS

IL-4

IL·s

IL·S

IL·B

IL-10

'l'IFN

M·CSF GM-CSF TNF..,

2CBET-3 + A20/2J

CYTOKINES +

Figure 1. Cytokine profile of the Vy5NB1-TCR

epidennal T-cell

a

clonal line 2CBET-3. Cytokines were measured by cytokine-specific

'000

2000

enzyme-linked immunosorbent assays, in which concentrations were deter­

mined

in ng/ml . 'Y-1FN, interferon-'Y; TNF-a,

3000

4000

5000

6000

7000

BOOO

CYTOKINES (Units I ml)

tumor necrosis factor-a.

2CBET-3. with Daudi or A2012J cells (1 X

Figure 2. Murine and human B-cell lines stimulate T-Cell Clonal Line Production and Analysis

Epidermal cells were

ME), y [10]. Briefly whole trunk or ear skin was trypsinized ovemight at 4°C. Epidermal cells were gendy separated from the dermis and

prepared from C57BL/6 mice (The Jackson Laboratory, Bar Harbor, as described previousl

,

trypsinized at 37°C to produce single-cell suspensions. After nylon wool

pass age to remove non-T cells, the cells were cultured in 24-well plates, coated with pan-an ti-'Yil TCR [7] together with 10 U/rul recom binant IL-2 (R and D Systems , Minneapolis, MN), for 72 h. Clonal T-cell lines were produced by limiting dilution. The epidermal T-cell clonal lines were stained with the ab ove monoclonal antibodies, followed by single-color cytofluorometric analysis to confirm that they expressed the V'Y5/Vlll­

TCR.

in microtiter wells. The culture medium was removed and

lOS/well)

assessed for the production ofIL-3 and GM-CSF using the DA-1 bioassay, as described in

experiments.

Materials and Methods. The data represent One of five replicate

as described below.

Of all

the cells tested,

2CBET-3

responded

strongly to only two cell lines: the murine B-cell lymphoma cell

A2012] and the human B-cell lymphoma Daudi (Fig 2). A 2CBET-l. re­ sponded similarly to A2012] and Daudi cells (data not shown). Fixation of A20/ 2] and Daudi cells did not interfere with their line

diiferent VyS/V81-TCR + epidennal T-cell clone,

Stimulator Cell Lines

2CBET-3 cells were

incubated with a variety of

murine cell lines, including fibroblast cell lines NIH3T3 and L929, tropho­ blast cell line TR-B, melanoma cell line B16FO, embryonic carcinomas

PCC3 and PCC4, B-celllymphomas 439.4.2 and A20/2J, keratin ocyte cell Jines XB-2 and PAM212, mastocytoma line P81S, and the human B­ lymphoma cell line Daudi. Cell Fixation

X 10· total), suspended in balanced salt

with glutaraldehyde (0.05%

v/v) for 30 seconds and

1:1 with 0.2 M lysine, pH 7.4, in balanced salt solution to stop the

reaction. Cells were washed further incubation with responder

ability to stimulate 2CBET-3 (Fig 3). Thus. the epidennal T-cell response was not due to cytokines made by the stimulator cells.

2CBET-3

T cells.

with

balanced salt solution before

Analysis ofEpidennal T-Cell Cytokine Production

Responses to the various

Because 2CBET-3 makes interleukin-3 (IL-3) and granulocyte-macrophage

colony-stimulating factor (GM-CSF) (Fig 1), culture supernatants from

the IL-3/GM-CSF-depen­

dent DA-l cell line, as des cribed previously [12]. One unit of these cytokines was defined as equivalent to 0.001 U of recombinant GM-CS F

Recombinant

the fibroblast

melanoma cell line B16FO. embryonic carcinomas PCC3 and the mastocytoma line P81S (Fig 4). 2CBET-3 also responded, albeit to a lesser degree, to the murine

Cytokines were

cell lines or to LPS were detected by cytokine production by 2CBET-3.

experiments involving 2CBET-3 were tested on

439.2.4,

and L929. the trophoblast cell line TR-B , the

PCC4. and

10000

DNAX Research Institute, as described previously [11]. Assessment ofEpidennal T-Cell Reactivity

3T3

cell lines

identifie d by specific enzyme-linked immunosorbent assay techniques at the

(Genzyme).

cells were not or were only weakly stimulated by other

cell lines. including the murine B-ce11line

Stimulator cells (5

solution, were fixed mixed

2CBET-3 cells (3 X 105/well) were mixed

GM-CSF was u se d as a standard in

all assays .

Units of IL-2 in the culture supernatants of experiments involving the

epidermal T-cell hybridoma 70BET-49 [2] were measured using the cytokine-dependent HT-2 cell line, as described p reviously [12].

......

E

� c::

1000

2C/) w z 52

@C,.)

100

10

LPS LPS from Escherichia coli serotype 05 5 :B5 and S. typhimllrium SL1181 Re mutant (Sigma Chemical Co., St. Louis, MO) were added to A2012J

in further experiments. 2CBET-3 was then tested for responses to a variety of cell lines.

2.5

5

10

STIMULATOR CELL CONCENTRATION 5 (X 10 CELL I WELL)

RESULTS AND DISCUSSION The VyS/V81-TCR + epidennal T-cell clone 2CBET-3 was tested to detennine which cytokines are produced after activation of these T c ells . When stimulated by TCR cross-linking. 2CB ET-3 pro­ duced IL-3. GM-CSF. and tumor necrosis factor-a: (Fig 1). No detectable production of IL-2, IL-4, IL-S, IL-6. IL-8. IL-I0. or interferon-y was found. Therefore. we used an IL-3- and GM­ CSF-dependent cell line. DA-l, to assess stimulation of 2CBET-3

1.2

0.6

cells and to 2CBET-3 at the start of the culture.

Figure 3. 2CBET-3 responds to fixed B-cell lines. 2CBET-3 cells lOs/well) were mixed with A2012J

cells that were fixed

Methods.

with

(closed squares)

and Daudi

glutaraldehyde, as described

(3 X

(open squares)

in Materials and

The culture medium was removed and assessed for

the production

of IL-3 and GM-CSF using the DA-1 bioassay. Greater concentrations of

the fixed B-cell lines were required for maximal responses to occur, compared

with

assays using live cells. The

replicate experiments.

data represent one of three

60S

REARDON ET AL

THE JOURNAL OF

300

2000 250

E � ."

1500

200

2en w z

150

52 0

�

�

100

!;: ()

c:

coli LPS

1000

500

en W

0

�U

1500

Z 52 NONE

3T3

L929

TAB

B16FO

PCC3

PCC4 439.4.2

P815

XB2

PAM212

CELL LINES

Figure

E.

DERMATOLOGY

708ET-49 d)

2C8ET-3 b)

a)

2-

50

INVESTIGATIVE

•

e)

•

•

.Ai A ..eo A ......

•

f)

g)

h)

S. typhlmurium LPS

1000

4. Various cell lines do not stimulate 2CBET-3. 2CBET-3

cells (3 X 1 05/well) were mixed with a variety of murine cell lines (1 X 5 10 /well) in microtiter wells, including fib roblast cell lines NIH3T3 and

L929,

trophoblas t cell line TR-B, melanoma cell line B16FO, embryonic

o

carcinomas PCC3 and PCC4, B-cell lymphoma 439.4.2, ma s to cyto ma line

PSIS, and the keratinocyte cell lines XB-2 and PAM2 12 . The culture

IL-3 and GM-CSF using the DA-l bioassay. Open bars, cytokine by the cell lines alone; closed bars, cytokine production by 2CBET-3 cells mixed with the cell lines. The data represent one of three production

replicate experiments.

kera tinocyte

line

PAM212, but showed little reactivity with the XB-2 (Fig 4). The re activity to PAM212 is consistent with the data of Havran et al [3], in which the epidermal T-cell line 7-17 was found to respon d stron g ly to PAM212. Dllferences appeared to exis t between variousV1S/V61TCR + epidermal T-cell clones in their reactivity to A20/2J and PAM212 cells, with some clones responding preferentially to the murine keratinocyte line

3

9

o

1

3

9

0 ·1

3

9

o

LIPOPOLYSACCHARIDE CONCENTRATION

medium was removed from these mixtures and assessed for the p roduction of

1

3

9

(1l9/ ml)

2CBET-3 is stimulated by bacterial LPS in the presence E. coli (a,b,e,d) or S. typhimurium (eJ,g,IJ) were added at the start ofthe culture to 2CBET-3 5 cells (3 X 105/well) mixed withA2012] cells (1 X 10 /well), as before. The data reflect LPS resp on ses of2CBET-3 alone (a,e; open squares) and A20/2J cells alone (a,e; open circles), of 2CBET-3 cells incubated with culture supernatants from A2012] cells that had been incubated with LPS for 24 h (bJ; closed circles), of2 CBET-3 together with A20/2] cells (e,g; closed squares), and of the V-yS/VIlI-TCR + hybridoma 70BET-49 (d,h) together with (closed triangles) and without (open triangles) A2012] cells. The culture medium was removed from these mixtures and assessed for the production ofIL-3 and GM-CSF using the DA-l bioassay . Cytokine values shown near 100 U/ml refel ct less than 30 U/ml. These data represent one of three

Figure 5.

of A20/2] cells. Various concentrations ofLPS from either

replicate experiments.

B-cell lines and others to keratinocytes (R. E. Tigelaar, unpublished data). However, 2CBET-3 responses to A2012j sidera bly in s trength ,

as

cells

varied con­

can be seen by comparing the responses

shown in

Fig 2 and Fig 5. The reason for the varia bility of responses to A2012j is not clear, but could be related to cell densities at which the 2CBET-3 (data not shown) and other V1S/V61-TCR+ epidermal T-cell clones (R. E. Tig ela ar, unpub­ lished data) were maintained before their incubation with A2012j cells, such th at cell cultures maintained at higher densities displayed better responses. Because all of these responding cells expressed the same TCR and similar levels of TCR expression (data not shown), the variable reactivity may reflect dllferences in expression of accessory or adhesion molecules or alternative lig ands on the V1S/V61-TCR+ epidermal T-cell clones. In contrast to the re­ sponses

to A2012j cells, the responses of2CBET-3 to PAM212 cells

have been consistently small. Because murine V"S/V61-TCR+ epidermal both a mouse

T cells respond to and a human B-cell line , they may recognize a highly

conserved ligand. Similarly, V11 -TCR+ cells respond to both murine and human trophoblast cells and also may recognize a highly conserved ligand TCR + epidermal T cell,

as

[13]. The ligand recognized byV1S/V61-. cells may not be unique to a p articular type of

the same epidermal T cells respond to keratinocytes. This

view contrasts with that of Havran et al [3], who propose d that V1S/V81-TCR + epidermal T cells recognize

a ligand specific to cells may recognize not a sin gle ligand but rather a set of relate d lig ands expres sed on dllferent cells. It is inte restin g that V1S/V61-TCR+ epidermal T cells do not respond to all keratinocyte and B-cell lines. This findin g is reminiscent of the polyclonal responses of human V-y9/ V62-TCR + T-cell clones, which res pond only to certain B-cell lines, particular ly the Burkitt's lymphoma line Daudi (14]. Such a variable capability of B-cell lines to stimulate 16 T cells could reBect variable ligand expression or the expression of accessory keratinocytes. Alternatively, epidermal T

molecules

such as the integrins or the co stimula tory B7 molecules

by the B-cell lines (reviewed in

[1S,16]). Because of the likely exposure of epidermal T cells to pathogens from the external environment, 2CBET-3 was assessed for its ability to respond to bacterial LPS. 2CBET-3 cells responded to LPS in the presence of A20/2j cells, but not in their absence (Fig 5). They responded similarly to LPS from two dllferent bac terial spe cies, E. coli and s. typhimurium. By themselves, the A2012j cells incu bated with LPS did not produce cytokine s stimulatory for DA-l cells (Fig 5). Because the 2CBET-3 response could be due to cytokines produced by LPS-activated A2 01 2j cells, we tested th e ability of culture supernatants from A20/2j cells incubated with LPS for 24 h to stimulate theV1S/V61-TCR+ cells in the absence of A20/ 2j cells. No response was detected (Fig 5). Moreover, the necessity for A2012j cells for the 2CBET-3 response to LPS is underscored by the finding thatV1SIVB1-TCR+ epi dermal T cells do not re spond to LPS adherent to tissue culture plastic (data not shown). We also note that although the 2CBET-3 cells responded weakly to keratinocyte lines, evaluation of a response to LPS in the presence of these cell lines is clouded by the ability of the keratinocyte lines to produce IL-3 in response to LPS (personal observation). In contrast to the LPS response by the V1S/VB1-TCR+ clone 2CBET-3, the V1S/V61-TCR+ hyb ridoma 70BET-49 did not respon d to LPS under similar conditions, even up to concentrations of 1 00 ILg/mI (data not shown), or to A2 0/ 2j cells alone (Fig 5). Nevertheless, this cell does produce cytokines in response to TCR cross-linking with anti-TCR antibodies, sug ges ting that 70BET-49 lacks qualities other than V1S/V61-TCR expression that are essential for the responses to LPS and to the B-celllines. However, the hybridoma fusion partner, BWS1 47 , is known to suppress the T-cell surface molecule CDS [17], and therefore it also might

VOL.

105, NO.

1,

SUPPLEMENT, JULY 1995

suppress other accessory molecules required for

B-CELL AND

V'YS/V61-TCR+

cell responses. The observation that V'YS/V61-TCR+ epidermal T cells are able to respond to bacterial LPS in the presence of A20/2] cells distinguishes V'YS/V61-TCR+ epidermal T cells from the majority ofT lymphocytes, whic� do not respond to LPS [S]. However, LPS

responsiveness does not appear to be unique to epidermal

T

cells,

as

murine 'Y6-TCR+ cell lines

V'YS/V61-TCR+

LPS stimulation represents an important difference between

these cells and the

two T-cell

at3-TCR +

cells, and likely reflects different roles of

subsets in vivo. Possibly, stimulation of

'Y6-TCR+

cells by LPS (and perhaps by other bacterial products) could initiate

a cascade of events that affect the host and the pathogen in a

scenario comparable to TCR+ cell responses.

superantigen-induced

Several possibilities exist

polyclonal

at3-

mechanism of stimulation of epidermal T cells by LPS in association with A2012] cells. 2CBET-3 cells either respond to LPS "presented" in some form by A2012] cells or respond to another ligand on A20/2] cells whose stimula­ for

the

LPS

This project

the

A2012] cells may CD14, CDII/CD18, or thus the V'YS/V61-TCR+

such as

80-kDa LPS-binding protein, and epidermal T cells could recognize receptor-bound LPS via a member of the CDII/CD18 LPS receptor family (reviewed in

[18]).

cell-surface interactions that lead to epidermal T-cell activation by

A20/2] cells. Therefore, one might expect that other cells expressing LPS receptors similar to those on A20/2] cells, e.g., macrophages, would be able to function in place of A20/2] cells. Alternatively, LPS may act on either the epidermal T cells or the A20/2J cells to induce or increase the expression of accessory molecules important to the response, such as adhesion molecules.

2.

endogenous ligands for

V'YS/V61-TCR+ epidermal T cells [1], LPS recently to induce expression of HSP-70 in

m acrophages after LPS administration in vivo [19]. Perhaps LPS

induces expression of

HSP-70 or other heat shock proteins on A20/2] cells, to which the 2CBET-3 cells respond. The ligand on A20/2] cells that stimulates V'YS/V61-TCR+ epidermal T cells remains unknown at the present time. However, the response to LPS in association with A20/2] cells may offer clues as to the nature of interactions not only between 'Y6 T cells and bacterial pathogens, but also between 'Y6 T cells and other cells of the host. That V'YS/V61-TCR+ epidermal T cells are activated by some B-cell lines in vitro may reflect the ability of these epidermal T cells to interact with B lymphocytes in vivo, and such intera c tions ,

DM,

Kuziel WA,

Institutes

of

Bonyhad i M, T i gelaar RE, Tucker PW, Allison]p:

diversity of -y1l antigen receptor genes of Thy-l + de ndritic epidennal

cells . Cell 55:837-847, 1988 Reardon CL, Vollmer M, Cran6.ll R, murine epidennal V-y5IVB6-TCR +

O'Brien RL. Born WK: Respon se of a hybridoma to heat shock protein, HSP-60.

] Inve$l Dermaloll03:544-546. 1994 3.

4.

Chi en Y-H. AllisonJP: Recognition ofse1fantigens by skin-derived with invariant -y1l antigen receptors. Sdence 252:1430-1432. 1991

Havran WL, T cells

Morrison D. Ryan JL: Bacterial endotoxins and host immune

response. Adv

Immlmol 28:294-431. 1979

5.

Vogel SN. Hilfiker ML. Caulfield MJ: Endotoxin-induced T lymphocyte prolif­

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Jiri1lo E, De Simone

eration.] ImmunoI130:1774-1779, 1983 LPS-mediated gram-negative 7.

C. Covelli

triggering of T

V, Kiyono

H.

McGhee

JR,

Antonaci S:

lymphocytes in immune re sponse against

bacteria. Adv Exp Med Bioi 256:417-425. 1990 Kanagawa 0, Kubo R, Ton e gawa S:

Itoha.. S. Nakanishi N.

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murine T-cell receptor -y1l: Analysis of -y1l T cells

during thymic ontogeny and in peripheral lymphoid organs.

Proc Nail Acad Sci

USA 86:5094-5098, 1989

8.

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KruisbeekAM, O 'Brien RL. Bom W. Tige1aar RE, Allison]p: Limited diversity ofTCR oy-chain expression of murine Thy-l + dendritic epidermal cells revealed by V-y3-specific mono­ clonal antibody. Proc Natl Acad Sci USA 86:4185-4189. 1989 Leo O. Foo M. Sachs DH, Samelson LE. Bluestone JA: Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc NaIl Acad Sci Havran WL. GrellS. Duwe G.KimuraJ. WilsonA.

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the

Also, considering the suggestions of heat shock proteins as possible

Asamow

Limited

In either case, LPS may serve only to enhance other

has been shown

National

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