Constant TCR triggering suggests that the TCRexpressed on intestinal intraepithelial cd T cells isfunctional in vivo

Frano H. Malinarich1,2, Elena Grabski3, Tim Worbs1,

Vijaykumar Chennupati1, Jan D. Haas1, Susanne Schmitz1,

Enzo Candia2, Rodrigo Quera4,5, Bernard Malissen6, Reinhold Forster1,

Marcela Hermoso2 and Immo Prinz1

1 Hannover Medical School, Institute for Immunology, Hannover, Germany2 Disciplinary Program of Immunology, Institute of Biomedical Sciences, Faculty of Medicine,

University of Chile, Santiago, Chile3 Twincore Center for Experimental and Clinical Infection Research, Hannover, Germany4 Gastroenterology Unit, Internal Medicine Department, Clinic Hospital, University of Chile

Santiago, Chile5 Gastroenterology Unit, Las Condes Clinic, Lo Fontecilla, Santiago, Chile6 Centre d’Immunologie de Marseille-Luminy, Universite de la Mediterranee, Marseille, France

Intestinal intraepithelial lymphocytes carrying the cd TCR (cd iIEL) are involved in the

maintenance of epithelial integrity. cd iIEL have an activated phenotype, characterized by

CD69 expression and increased cell size compared with systemic T lymphocytes. As an

additional activation marker, the majority of cd iIEL express the CD8aa homodimer.

However, our knowledge about cognate ligands for most cd TCR remains fragmentary and

recent advances show that cd T cells including iIEL may be directly activated by cytokines

or through NK-receptors, TLR and other pattern recognition receptors. We therefore asked

whether the TCR of cd iIEL was functional beyond its role during thymic selection. Using

TcrdH2BeGFP (Tcrd, T-cell receptor d locus; H2B, histone 2B) reporter mice to identify cd T

cells, we measured their intracellular free calcium concentration in response to TCR-

crosslinking. In contrast to systemic cd T cells, CD8aa1 cd iIEL showed high basal calcium

levels and were refractory to TCR-dependent calcium-flux induction; however, they readily

produced CC chemokine ligand 4 (CCL4) and IFN-c upon TCR triggering in vitro. Notably,

in vivo blocking of the cd TCR with specific mAb led to a decrease of basal calcium levels

in CD8aa1 cd iIEL. This suggests that the cd TCR of CD8aa1 cd iIEL is constantly being

triggered and therefore functional in vivo.

Key words: Ca21-flux . CCL4 . gd intestinal intraepithelial lymphocytes . gd T cells . IFN-g

Supporting Information available online

Correspondence: Dr. Immo Prinze-mail: Prinz.Immo@mh-hannover.de

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

DOI 10.1002/eji.201040727 Eur. J. Immunol. 2010. 40: 3378–3388Frano H. Malinarich et al.3378

Introduction

Heterodimers of the gd TCR are shared by diverse T-lymphocyte

populations comprising motile gd T cells that migrate in blood

and secondary lymphoid organs as well as tissue-specific and

tissue-resident subsets that do not exchange with other gd T-cell

populations [1, 2]. A prototype for the latter is the compartment

of intestinal intraepithelial lymphocytes carrying the gd TCR (gdiIEL), composed of gdCD8aa and gdCD8�CD4� double negative

(DN) populations. There is increasing evidence that the primary

role of gd iIEL and other tissue-resident gd T cells is immune

surveillance of their habitat and the maintenance of epithelial

integrity [3–8]. It is assumed that gd iIEL screen gut epithelial

cells for the presence of self-derived and external danger signals

and respond by the secretion of inflammatory cytokines [9, 10],

tissue repair factors [3, 11] or induction of cytolytic activity [12].

Although there are notable exceptions [13–18], however, cognate

ligands of most human and mouse gd TCR still remain unknown.

Moreover, there have been convincing reports of alternative ways

of gd T-cell activation through either NK-receptors (C-type

lectins) such as NKG2D [7] or via pattern recognition receptors

such as TLR or aryl-hydrocarbon receptor [19, 20]. Finally, it is

known that subsets of gd T cells can directly produce the

effector cytokines IL-17A or IFN-g in response to stimulation with

IL-23 or IL-12/IL-18, respectively [21, 22]. Therefore, it seems

tempting to speculate that the gd TCR may actually be

dispensable for the in vivo function of gd T cells, which would

make it a receptor molecule ‘without a job’ [23], or that it might

instead exhibit yet unidentified functions other than T-cell

activation.

gd iIEL as well as other iIEL carrying an ab TCR (ab iIEL)

differ from T-lymphocyte subsets found in secondary lymphoid

organs in that they show an ‘activated yet resting’ phenotype

characterized by high basal MAP2K activity, high expression of

chemokine and granzyme mRNA, and are hyporeactive to TCR

stimulation and do not proliferate in response to TCR-triggering.

Accordingly, gd iIEL and ab iIEL can display on their surface

T-cell activation markers such as CD69 and approximately 75%

express the CD8aa homodimer [24–28]. Together, this implies

that iIEL are being constantly activated in vivo through signals

from their specific environment [29, 30]. However, it is not

clear whether or to what extent the gd TCR is involved in this

process.

In this study, we investigated the functionality of gd and abTCR expressed on freshly isolated systemic T lymphocytes and

iIEL by measuring the increase of intracellular free calcium

concentration ([Ca21]i) levels after TCR stimulation on a single

cell basis. Of note, we found that gd and ab iIEL had high levels of

basal [Ca21]i. Furthermore, we detected elevated basal [Ca21]i

levels in CD8aa1 when compared with [Ca21]i in CD8aa� gd(DN) iIEL. These elevated basal [Ca21]i levels correlated with

lower responsiveness to TCR-specific stimulation. Furthermore,

we were able to tune down basal [Ca21]i levels of gd CD8aa1 iIEL

in vivo through the systemic administration of specific anti-gdTCR mAb. Irrespective of the mechanism, this effect implied that

diminished TCR signaling capacity resulted in lower basal [Ca21]i

levels and thus provided evidence that the gd TCR was indeed

functional and likely to be constantly triggered in vivo. Addi-

tional, albeit indirect support for a functional TCR in iIEL was

offered by ex vivo stimulation assays demonstrating that TCR

ligation of some gd and ab iIEL populations led to more effective

chemokine and cytokine production compared with unspecific

stimulation with PMA/ionomycin. Taken together, we describe

here the short-term (seconds) and medium-term (hours)

outcome of TCR-stimulation of various iIEL populations. We

conclude that their TCR, at least in gd iIEL, must be functional

in vivo.

Results

Different basal intracellular Ca21 levels in systemicand intestinal cd T cells

Monitoring of [Ca21]i increase in the cytoplasm of T cells after

TCR ligation is an established experimental system to quantify

TCR responsiveness on a single-cell basis [31, 32]. For gd T cells,

this was so far difficult, because the identification of bona fide gdT cells depended on staining with mAb directed against the gdTCR. In order to directly measure intracellular Ca21 levels of gdT cells in response to stimulation of their TCR, we thus made use

of TcrdH2BeGFP (Tcrd, T-cell receptor d locus; H2B, histone 2B)

reporter mice [33]. More precisely, we used F1 C57BL/6-

Tcra�/��TcrdH2BeGFP double heterozygous mice (gd reporter

mice) in which expression of the reporter H2BeGFP unambigu-

ously identifies gd T cells without touching their TCR. This system

was chosen to avoid any false-positive GFP1 cells that could be

found in the homozygous TcrdH2BeGFP reporter mice due to

mono-allelic rearrangements of the Tcra/Tcrd locus. By co-

staining with anti-CD8a, five populations of either systemic

T cells or iIEL were defined (Fig. 1A). In the systemic T-cell

compartment, CD8a expression identified abCD81 T cells (CD81

p-ab) while GFP expression identified gdDN T cells (CD8� p-gd).

In iIEL preparations, GFP1 gd T cells were divided into CD8a�

(CD8� i-gd, approximately 20% of all gd T cells, corresponding to

gdDN iIEL) or CD8a1 (CD81 i-gd, approximately 80% of all gdT cells, corresponding to gdCD8aa1 iIEL). Finally, we gated

GFP�CD8a1 cells (CD81 i-ab), representing ab CD8a1 iIEL. As a

general observation, the iIEL compartment showed substantially

higher basal [Ca21]i levels than systemic T cells (Fig. 1B). The

systemic populations had equal basal [Ca21]i levels, though 50%

less in relation to iIEL populations (Fig. 1B). In spite of these

differences, all five T-cell populations showed robust ionomycin-

induced Ca21-fluxes (Fig. 1C). However, Ca21 response ampli-

tudes were higher in CD81 p-ab and CD8� p-gd representing

systemic T cells.

Next, we studied the Ca21-flux of isolated iIEL or systemic

T cells from gd reporter mice after TCR-clustering with anti-

bodies. For this, we applied an anti-gd TCR mAb clone (GL3) and

an anti-CD3e clone (145-2C11, here 2C11) and subsequently

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clustered them on the cell surface with secondary goat anti-

hamster antibody. This procedure induced robust anti-CD3-

induced Ca21-fluxes in the systemic populations CD81 p-ab and

CD8� p-gd (Fig. 1D). Similarly, clustering with anti-gd TCR mAb

specifically induced Ca21-flux of systemic CD8� p-gd cells

(Fig. 1D). However, in the iIEL compartment, we observed

discrete Ca21-fluxes in response to anti-CD3 or anti-gd TCR mAb

only in CD8� i-gd but not in CD81 i-gd (Fig. 1E). This suggested

that high basal [Ca21]i levels in gdCD8aa1iIEL correlated with

TCR-unresponsiveness. Taken together, we found that systemic

ab and gd T cells showed comparable Ca21-flux responses to TCR

ligation, whereas CD8aa1 ab and gd iIEL were presumably pre-

activated and thus refractory to further stimulation of the TCR

complex and displayed high intrinsic [Ca21]i levels. These results

suggest a chronic stimulation of CD8a1 iIEL in vivo.

TCR stimulation and intracellular Ca21-flux induceCCL4 and production by iIEL

Next, we sought to investigate the outcome of ab- and gd-specific

TCR stimulation on isolated iIEL in ex vivo stimulation assays.

Since systemic gd T cells in lymph nodes, spleen and circulation

[19, 21, 34] as well as intraepithelial gd T cells in the skin [35]

have been described to be biased to produce IL-17A, we tested

whether this pro-inflammatory cytokine was produced by

intestinal gd iIEL. We found that, irrespective of CD8a expression,

gd iIEL did not produce IL-17A upon stimulation with anti-

TCR mAb or PMA/ionomycin (Fig. 2). This is in accordance with

a recent report showing that intestinal gd IEL are not ‘pre-

wired’ toward a specific lineage [36]. Therefore, we focused in

this study on the well-established gd IEL effector molecules CC

Figure 1. gd iIEL and systemic gd T cells differ in basal intracellular [Ca21] levels and Ca21-flux responses to CD3/gd TCR stimulation.(A) Representative FACS plots show the identification of specific T-cell subpopulations according to FSC/SSC (left column) and the surface markerCD8a versus gd TCR reporter fluorescence (right column) within systemic lymphocytes (upper panel) and iIEL (lower panel) derived from F1 C57BL/6-Tcra�/��TcrdH2BeGFP reporter mice. Within the systemic T-cell compartment, CD81 p-ab (pink) and CD8� p-gd (light blue) were designated. Withinthe iIEL population, CD8� i-gd (green), CD81 i-gd (red) and CD81 i-ab (blue) were gated. (B) Histogram overlay showing basal [Ca21]i levels of T-cellpopulations as gated in (A) over time (left panel). Summary plot of the basal [Ca21]i level values of T-cell populations (right panel), columns showmean7SEM, n 5 3 independent experiments. (C) Overlay of representative ionomycin-induced Ca21-flux responses of systemic and iIELcompartment T-cell subpopulations. Color coding of populations is as described in (A). (D) Representative Ca21-flux kinetics of Indo-1AM-labeledCD81 p-ab (pink) and CD8� p-gd (light blue) induced by the addition of anti-CD3 (clone 2C11, upper panel) or anti-gd TCR (clone GL3, lower panel)monoclonal antibodies, followed by cross-linking with polyclonal anti-Hamster-Ab at the indicated time points. (E) Representative Ca21-fluxkinetics of CD8� i-gd (green), CD81 i-gd (red) and CD81 i-ab (blue) subpopulations of Indo-1AM-labeled iIEL after addition of the same antibodycombinations as described in (D). Data shown in panels (C–E) are representative for at least three independent experiments.

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chemokine ligand 4 (CCL4) and IFN-g. Chemokine and cytokine

production of ab, gd and total iIEL from WT mice was monitored

by stimulation with plate-bound anti-gd TCR (GL3 and GL4),

anti-ab TCR (H57-597, called H57) and anti-CD3 (2C11),

respectively, followed by cytokine measurement in the super-

natants. Here, ab or gd TCR triggering induced similar

concentrations of CCL4 (Fig. 3A, upper panel), whereas higher

amounts of IFN-g were produced through anti-ab TCR stimula-

tion (Fig. 3A, lower panel). In addition, matching results were

obtained in different iIEL populations from WT mice by

stimulation with plate-bound anti-CD3 (2C11), anti-ab TCR

(H57) and anti-gd TCR (GL3) followed by intracellular staining.

TCR engagement induced CCL4 production in both ab and gdiIEL populations (Fig. 3B, left panel), whereas more ab iIEL than

gd iIEL produced IFN-g (Fig. 3B, right panel). These results

clearly showed that iIEL were not anergic in these assays and that

the TCR in ab and gd iIEL was functional. These findings were

also in line with previous reports [37, 38] that showed

cytokine production by iIEL during TCR complex activation.

Moreover, downstream of TCR engagement, activation of the

cells with the Ca21 ionophore ionomycin showed that gd iIEL

populations had a better capacity to produce CCL4 (Fig. 3C, left

panel) and ab iIEL populations a better ability to produce IFN-gin response to ionomycin-induced Ca21-flux (Fig. 3C, right

panel). Interestingly, direct comparison revealed that mAb-

mediated TCR stimulation was significantly more efficient than

PMA/ionomycin incubation in inducing CCL4 and IFN-g produc-

tion in gdCD8aa1 iIEL (Fig. 3D). In contrast to gd iIEL, ab iIEL

populations showed similar activation behavior either with PMA/

ionomycin or TCR stimulation (Fig. 3E); however, ab1CD41 iIEL

produced IFN-g more efficiently after PMA/ionomycin stimula-

tion than via TCR complex triggering. These findings show the

diverse responsiveness of each iIEL population upon the TCR

complex activation and underline the role of the intracellular

Ca21 increase in the activation process. On the other hand, the

importance of the gd TCR, especially in gdCD8aa1 iIEL

population, highlights a central role of this receptor for the

function of gd iIEL.

In vivo anti-cd TCR mAb treatment decreases ½Ca21�ilevels in iIEL

We hypothesized that the high basal [Ca21]i levels observed in gdiIEL (Fig. 1B) might be due to continuous TCR stimulation in situ.

Taking into account that the anti-gd TCR mAb clone GL3 could

specifically activate gd iIEL ex vivo and down-regulate surface gdTCR complex levels in vivo [39], we tested the effect of in vivo TCR

modulation on basal [Ca21]i levels of gd iIEL. Therefore, reporter

mice were treated with a regimen of three consecutive injections of

200mg anti-gd TCR mAb (GL3) at day �6, day �4 and day �2

before analysis. First, in vivo gd TCR modulation induced down-

modulation of CD3 and gd TCR surface levels of gd iIEL (Fig. 4A,

upper panel), similar to what we showed previously [39]. However,

this protocol of repeated high-dose injection of anti-gd TCR mAb

did not alter the expression level of CD8a on the targeted gd iIEL

(Fig. 4A, upper panel) or the frequency of CD8a1 cells among all gdiIEL (data not shown); neither did it significantly modulate the

chronically activated phenotype of the gd iIEL as assessed by

surface activation markers (Fig. 4A, lower panel). Similarly, the

activation status, as well as ab TCR complex and CD8a expression

on ab iIEL (Fig. 4B), was not influenced by this regimen.

Importantly, basal [Ca21]i levels and amplitudes of ionomycin-

induced Ca21-fluxes were significantly decreased in CD8a1 iIEL

derived from mice injected with GL3 compared with those from

mock-treated animals (Fig. 4C, D). However, not only gdCD8aa1

iIEL but also abCD8a1 iIEL cells showed a basal [Ca21]i decrease.

This was unlikely to be a direct effect of the GL3 mAb on ab iIEL but

may be due to changes in the composition of abCD8a1 iIEL,

e.g. through attraction of systemic ab1CD81 cells with lower

basal [Ca21]i levels into the gut epithelium [40]. In contrast, basal

[Ca21]i levels of neither systemic CD8� p-gd nor CD8� i-gd were

altered by GL3-treatment (Fig. 4C and D). These data suggest that

the observed high basal [Ca21]i levels of gdCD8aa1 iIEL reflect a

constant TCR-specific activation in vivo, which could be partially

blocked by anti-gd TCR mAb treatment.

In vivo anti-cd TCR mAb treatment impairs the TCRresponsiveness of cd iIEL

Next, we investigated how gd T cells from GL3-treated gd reporter

mice responded to TCR stimulation. As shown in Fig. 4A, the TCR

complex was down-regulated but still present at residual levels

control anti-γ δ TCR PMA/iono66.5 4.27 74.3 0.23105 72.4 0.049

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0.1127.4

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IL-17A

Figure 2. TCR-ligation is a potent inducer of IFN-g but not IL-17Aproduction by CD8aa1 intestinal intraepithelial gd T cells. Intracellularcytokine staining of intestinal intraepithelial gd T cells gated asCD8b�CD4�TCR-b�H2B-eGFP1 as detailed in Supporting InformationFig. 1. Isolated iIEL were cultured for 4 h either in medium (control), onplate-bound anti-gd TCR mAb or stimulated by PMA/ionomycin beforeextra- and intracellular staining. Upper panels show intracellular IFN-gversus CD8a surface staining. Lower panels show intracellular IL-17Aversus CD8a surface staining. Numbers in quadrants indicate thepercentage of cells in each. Data are representative of three independentexperiments. Statistics for IFN-g production are shown in Fig. 3.

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on the cell surface of these gd T cells. We found that anti-CD3 and

anti-gd TCR mAb clustering still elicited Ca21-fluxes in CD8� p-gdand CD8� i-gd from mice injected with GL3, albeit with lower or

almost flat amplitudes compared with those from mock-treated

animals. The iIEL populations CD81 i-gd and CD81 i-ab only

showed a decrease of basal [Ca21]i, without evident mAb-

induced Ca21-flux neither in PBS nor in GL3 treated mice (Fig.

5A). The quantification of these changes, displayed as fold of

basal [Ca21]i levels after anti-CD3 and anti-gd TCR mAb

clustering, showed that CD8� p-gd and CD8� i-gd were affected

by the GL3 treatment (Fig. 5B). In addition, iIEL from PBS- and

GL3-treated gd reporter mice were analyzed for responsiveness to

Figure 3. CCL4 and IFN-g production by iIEL populations depends on TCR complex stimulation and correlates with ionomycin-inducedintracellular [Ca21] increase. Freshly isolated iIEL suspensions were stimulated in vitro. Subsequently, various T-cell populations were gatedaccording to expression of TCR ab or TCR gd as well as co-receptors CD8a, CD8b, and CD4 and analyzed by intracellular cytokine staining asdetailed in Supporting Information Fig. 2 and 3. (A) Representative quantification of CCL4 (upper panel) and IFN-g (lower panel) in supernatants ofiIEL stimulated by plate-bound anti-gd TCR (clones GL3 and GL4), anti-ab TCR (clone H57-597), anti-CD3 (clone 145-2C11) measured by cytokinebead array. N/S: no stimulation. (B) Intracellular FACS analysis of CCL4 (left panel) and IFN-g (right panel) production in iIEL populations incubatedon plates coated with anti-gd TCR (clone GL3), anti-ab TCR (clone H57), anti-CD3 (clone 2C11). Columns show mean7SEM, n 5 3 independentexperiments. (C) Intracellular FACS analysis of CCL4 (left panel) and IFN-g (right panel) production in the presence (black bars) or absence (whitebars) of ionomycin (2 mg/mL) in the same iIEL populations. Columns show mean7SEM, n 5 3 independent experiments. (D) Direct comparison ofCCL4 (left panel) and IFN-g (right panel) production after stimulation with PMA/ionomycein ionomycin (black), anti-CD3 (red) or anti-gd TCR(green) of gd1CD8aa1 and gd1DN iIEL populations by intracellular FACS analysis. N/S: no stimulation. Columns show mean7SEM, n 5 3independent experiments. (E) Analysis as in (D) for ab1CD41, ab1CD41CD8a1, ab1CD8ab1 and ab1CD8aa1 iIEL populations. All experiments werecarried out with cells derived from WT C57BL/6 mice.

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ex vivo stimulation with GL3 and GL4, a different anti-gd TCR

mAb. In vivo treatment with GL3 reduced the TCR-dependent

CCL4 and IFN-g production of gd iIEL (Fig. 5C). Surprisingly, the

CCL4 and IFN-g production capability of gb iIEL from GL3-treated

gd reporter mice stimulated ex vivo with the anti-ab TCR (H57)

was increased (Fig. 5D). In conclusion, gd iIEL suffered a loss of

function in response to TCR stimuli when their TCR was

modulated by GL3 treatment for 6 days. Together, this suggests

that the iIEL do not become exhausted and do not change their

activated phenotype with repeated high-dose anti-gd TCR

treatment. However, the down-modulation of their surface TCR

in combination with the decoration of residual surface gd TCR is

likely to be the reason for the diminished TCR responsiveness and

cytokine production. This further implies a role for the TCR in the

physiology of gd T cells. However, it is at present not clear to

what extent the responsiveness of gd T cells to other stimuli, e.g.

engagement of other receptors such as NKG2D or TLR, may be

also altered by TCR modulation.

Discussion

The question whether, after thymic selection, the TCR on gd T

cells had a physiological role at all was not unanticipated [19,

23]. Our knowledge about cognate ligands of the gd TCR remains

limited and gd T cells are equipped with a variety of receptors

that can mediate T-cell activation and cytokine release. Here we

provide evidence that the gd TCR on gd iIEL is functional in a

normal mouse. We found that its down-modulation led to lower

basal [Ca21]i levels suggesting the gd TCR on gd iIEL to be

constantly triggered in vivo.

The experiments carried out in the gd reporter mice were an

improvement to previous Ca21-flux studies on gd T cells [32,

41–44] because bona fide gd T cells could be easily identified by

their intrinsic fluorescence without the use of specific mAb directed

against the gd TCR. Still, we cannot formally rule out that iIEL were

however activated by stressed epithelial cells during the purification

process. Nevertheless, we obtained unchanged results for systemic

Figure 4. Treatment with anti-gd TCR mAb decreases ex vivo basal intracellular [Ca21] levels in iIEL. (A and B) Representative histograms showingTCR, CD3, CD8a, CD69, CD44 and CD62L surface expression of gd iIEL (A) and ab iIEL (B), (gated as depicted in Supporting Information Fig. 1) fromPBS (dotted line) and GL3 (black line) treated gd reporter mice. Fluorescence minus one (FMO) control is shown as gray-filled histogram, all surfacemarkers were similarly revealed with Streptavidin-PerCP. (C) Representative basal [Ca21]i levels (left column) of iIEL in PBS (upper panel) or GL3-treated (lower panel) gd reporter mice and corresponding ionomycin-Ca21-flux induction kinetics (right column). The iIEL populations are shownas CD8� i-gd (black line), CD81 i-gd (gray line) and CD81 i-ab (dotted line). (D) Comparison of the basal [Ca21]i level values of PBS (black bars) or GL3(white bars) treated gd reporter mice. Columns show mean7SEM, n 5 3 independent experiments.

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T cells irrespective of whether they were prepared by simple

mashing through a nylon sieve or processed similar to iIEL by an

adapted protocol including incubation and shaking of the cells in

supplemented medium (without EDTA) and subsequent Percoll

gradient purification (data not shown). A striking result was that

TCR-mediated Ca21-fluxes in CD8a1 iIEL compartments were

hardly detectable, possibly due to high basal [Ca21]i levels in these

cells. This was observed for both ab iIEL and gd iIEL. In contrast,

CD8a� gd DN iIEL, which had lower basal [Ca21]i levels, showed a

sizeable Ca21-flux. The reason for this dichotomy of CD8a1 and

CD8a� gd iIEL is not clear. It is possible that the CD8aa homodimer

directly modulates the iIEL’s Ca21 responses by direct interaction

with the TCR. More likely, the interaction of CD8aa and thymus

leukemia antigen expressed by intestinal epithelial cells could

induce a higher iIEL activation level and thereby decrease TCR

sensitivity [30, 45]. It is to date not clear whether CD8a� cells are

the precursors of CD8a1 gd iIEL or whether CD8a1 and CD8a� gdiIEL represent largely unrelated populations that co-exist in the

intestinal epithelium.

The observed intrinsically high basal [Ca21]i levels in iIEL and

the fact that these cells were refractory to TCR stimulation were

reminiscent of former reports suggesting that T cells from the

lamina propria were continuously stimulated in vivo because they

displayed high levels of CD69 and higher basal [Ca21]i levels

compared with autologous systemic blood lymphocytes [29].

High basal [Ca21]i levels were equally found in ab and gd iIEL

thus raising the questioning whether both types of TCR experi-

enced antigen-specific stimulation. Certainly, other factors may

contribute to the activated phenotype of iIEL [46]; however both

ab and gd iIEL showed constitutive cytolytic activity in response

to TCR engagement [46]. In addition, it is likely that the TCR of

abCD8aa1 iIEL recognizes self-antigens [47, 48]. Moreover,

diminished Ca21-fluxes in response to TCR stimulation were

previously reported for memory CD41 T cells compared with

naıve T cells [49, 50]. Collectively, it emerges that gdCD8aa1

iIEL, which had high basal [Ca21]i levels, are chronically acti-

vated by their specific environment. Such continuous activation

should at least in part be mediated by TCR triggering, because

Figure 5. Treatment with anti-gdTCR mAb impairs the TCR responses of gd iIEL. (A) Ca21-flux in PBS (gray line) or GL3 (black line) treated gdreporter mice induced by the antibodies anti-CD3 (clone 2C11, left column) or anti-gd TCR (clone GL3, right column). (B) Maximal fold increase of[Ca21]i to average basal levels induced by anti-CD3 (clone 2C11, left panel) or by anti-gd TCR (clone GL3, right panel) in the indicated T-cellpopulations of PBS (black bars) or GL3 (white bars) treated gd reporter mice. Columns show mean7SEM, n 5 3 independent experiments. (C and D)CCL4 (left panel) and IFN-g (right panel) measured by cytokine bead array in supernatants of iIEL from PBS (black bars) or GL3 (white bars) treatedWT C57BL/6 mice. iIEL isolated from these two groups were either stimulated by plate-bound anti-gd TCR clones GL3 and GL4 (C) or with anti-abTCR clone H57 (D). N/S: no stimulation. (C and D) One representative of three individual experiments is shown.

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TCR modulation with anti-gd TCR mAb reduced the high basal

[Ca21]i levels in CD8a1 gd iIEL.

Administration of anti-gd TCR was formerly used to ‘deplete’

gd T cells in many experimental models for human disease.

Several studies have reported profound effects of gd TCR

modulation in vivo thereby highlighting an important beneficial

role for gd iIEL in the protection of epithelial tissues under

inflammatory conditions [3, 51–55]. By investigating the effects

of the commonly used clones GL3 and UC7-13D5 on gd T cells in

TcrdH2BeGFP reporter mice we had previously reported that

there is no depletion but that binding of anti-gd TCR mAb

rendered the target cells ‘invisible’ for further detection based on

anti-gd TCR mAb [39]. However, at that time it was not further

investigated what effect mAb treatment would have on gd T-cell

function in vivo. We favor a scenario where docking of the anti-

bodies would presumably induce a limited initial activation

of the gd T cells and later would lead to a sustained down-

regulation of the TCR from the cell surface. This in turn would

probably inhibit or compromise TCR triggering as suggested by

the reduced basal [Ca21]i levels in gdCD8aa1 iIEL from GL3-

treated mice. This has technical implications for experimental in

vivo administration of anti-gd TCR antibody to block the biolog-

ical functions of gd iIEL. It appears that signaling through the TCR

of gd cells in repeated high-dose GL3-treated mice is at least

partially blocked in vivo. Since the cells are clearly not depleted

or diminished in numbers and do not lose their activated

phenotype as determined by the expression of surface activation

markers this implies that biological differences observed in other

studies of anti-gd TCR-treated mice further highlight the

physiological role of the TCR in gd T cells [3, 51–56]. Potential

future therapeutic approaches to block gd TCR signaling in

humans may thus represent promising intervention strategies. In

conclusion, the TcrdH2BeGFP reporter system enabled us to

measure dynamic [Ca21]i levels of gd T cells in normal mice. Not

ignoring the presence of NK-receptors or pattern recognition

receptors expressed on gd T cells we propose that the gd TCR of

CD8aa1 gd iIEL is functional because it is constantly being trig-

gered in vivo, most likely by ligands expressed on intestinal

epithelial cells.

Materials and methods

Mice

F1 C57BL/6-Tcra�/�� TcrdH2BeGFP reporter mice were obtained

from crossbreeding Tcra�/� [57] and TcrdH2BeGFP [33]. Both

strains were either backcrossed to or generated on a C57BL/6

genetic background, respectively. WT C57BL/6 mice were

purchased from Charles River Laboratories, Sulzfeld, Germany.

Mice were used with 6–12 wk of age. Animals were housed under

specific pathogen-free conditions in individually ventilated cages

at the Hannover Medical School animal facility. All animal

experiments were performed according to institutional guidelines

approved by the Niedersachsisches Landesamt fur Verbrau-

cherschutz und Lebensmittelsicherheit.

Antibodies

The mAb used for ex vivo iIEL stimulation directed against gd TCR

(clone GL3), CD3 (clone 145-2C11), ab TCR (clone H57-597) (all

Armenian hamster) were purified from hybridoma supernatants

and gd TCR (clone GL4) was a gift from Dr. Leo Lefranc-ois. For

Ca21-flux studies anti-gdTCR (clone GL3), CD3 (clone 145-2C11)

and goat anti-Armenian hamster (anti-Hamster, Jackson Immuno-

Reasearch) were applied. For the analysis of T-cell populations by

FACS the following mAb were used: gdTCR-FITC (clone GL3),

gdTCR-biotin (clone GL3) and CD3-biotin (clone 145-2C11),

CD8a-Cy5 or CD8a-biotin (clone Rm CD8), CD8b-Pacific

Orange (clone Rm CD8-2), CD4-Pacific Blue (clone GK1.5),

CD62L-biotin (clone MEL-14) and Fc receptor (clone 2.4G2)

were purified from hybridoma supernatants; anti- CD69-biotin

(clone H1.2F3) and Streptavidin-PerCP were obtained from BD

Bioscience, CD44-biotin (clone IM7) from Caltag and ab TCR-

APC-AlexaFluor 750 (clone H57-597) from eBiosciences. For

measurement of intracellular cytokines, we used polyclonal goat

anti-mouse CCL4 (R&D Systems), polyclonal F(ab0)2 Donkey

anti-goat IgG-PE (Jackson ImmunoReasearch), ChromPure goat

IgG (Jackson ImmunoReasearch) or anti-IL-17A-PE (clone

ebio17B7, eBiosciences) and anti-IFN-g-PE (clone XMG1.2,

Caltag).

Isolation of iIEL and systemic T cells

iIEL were isolated according to a modification of a previously

published method [39]. Briefly, the small intestines were flushed

with cold PBS 3% FBS, connective tissue and Peyer’s patches were

removed and the intestines opened longitudinally. Next, the small

intestines were incubated two times for 15 min in a HBSS 10%

FBS 2 mM EDTA at 371C, shaken vigorously for 10 s and cell

suspensions were collected and pooled. The cell suspension was

filtered through a nylon mesh and centrifuged at 678� g, 20 min

at room temperature, in a 40%/70% Percoll (Amersham)

gradient. The iIEL were recovered from the interphase and were

washed with PBS 10% FBS. Systemic T cells were isolated from

systemic lymphocytes of spleens and systemic lymph nodes from

gd reporter mice (F1 C57BL/6-Tcra�/�� TcrdH2BeGFP), mashed

in nylon filters, both mixed and subjected to erythrocytes lysis.

Next, the cell suspension was washed with PBS 3% FBS, filtered

through a nylon mesh and resuspended in RPMI 1640 10% FBS

for further analysis.

In vivo cd T-cell modulation

gd reporter mice were treated with a regime of three consecutive

intraperitoneal injections of purified anti-gd TCR mAb at day �6,

Eur. J. Immunol. 2010. 40: 3378–3388 Cellular immune response 3385

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

day �4 and day �2 before analysis (clone GL3, 200mg/mouse).

Control groups received mock injections with PBS.

Ca21-flux measurements

iIEL and systemic T cells from gd reporter mice were prepared for

Ca21-flux cytometry as described with minor modifications [58]. In

order to avoid a bias in the Ca21-flux measurements, all the

procedures were carried out at room temperature, without EDTA

and with a final cell viability Z92% determined by Trypan blue

prior to Indo-1AM loading of the cells. Cells were incubated at a

concentration of 0.5� 107 per mL with 5mM Indo-1AM (Invitrogen,

Molecular Probes) for 60 min at 371C, stained with anti-CD8a-PE for

10 min and left at room temperature in the dark. The viability of

cells after Indo-1AM loading was 490% as assessed by propidium

iodide staining gated on the lymphocyte FSC/SSC population. Prior

to data acquisition, the cell suspensions were warmed to 371C in the

dark for 10 min and then aliquoted in 200mL, then CaCl2 was added

to a final concentration of 1 mM and Ca21-flux was measured with a

LSRII (BD) cytometer equipped with a 355 nm UV laser at 371C

using a custom-built heating device adapted to cytometer tubes.

After acquisition of the baseline levels for 60 s, anti-CD3 or anti-gdTCR mAb was added and the cross-linking anti-Hamster Ab were

added at second 90. The following concentrations of mAb were

used: systemic T-cell compartment, 100mg/mL of anti-CD3 (clone

145-2C11) with 180mg/mL of anti-hamster and 100mg/mL of anti-

gd TCR (clone GL3) with 180mg/mL of anti-hamster final

concentrations; iIEL compartment, 200mg/mL of anti-CD3 with

180mg/mL anti-hamster and 100mg/mL of anti-gd TCR (clone GL3)

with 360mg/mL of anti-hamster final concentrations. After the

stimulation, the cells were acquired for additional 3 min. Ionomycin

was used as a positive control for Ca21-flux (2mg/mL). The kinetic

Ca21 changes were analyzed in FlowJo software (Version 8.8.2,

Treestar).

Cytokine measurements

For cytokine quantification, C57BL/6 iIEL were incubated in 96-

well plates coated either with 10 mg/mL of anti-gd TCR (clone

GL3 and GL4), anti-ab TCR (clone H57-597) or anti-CD3 (clone

145-2C11) for a period of 24 h and the supernatants were

analyzed for CCL4 and IFN-g by cytometric bead array (CBA, BD

Biosciences) according to the manufacturer’s instructions. For

intracellular cytokine detection in iIEL populations, WT C57BL/6

iIEL were incubated in a 24-well plate coated with 10 mg/mL of

anti-gd TCR (clone GL3 or GL4), anti-ab TCR (clone H57-597),

anti-CD3 (clone 145-2C11) or in presence of PMA (10 ng/mL)

and ionomycin (2 mg/mL), for 4 h. Brefeldin A (10 mg/mL) was

added for the last 3 h. The cells were stained with surface marker

and intracellular cytokine antibodies for FACS analysis of CCL4,

IL-17A and IFN-g. FACS experiments were performed on an LSRII

flow cytometer (BD Biosciences) and the data were analyzed by

FlowJo software (Version 8.8.2, Treestar).

Statistical analysis

All bar graphs are presented as mean7SEM and were made using

GraphPad Prism software (Version 4.03). Fold changes of Violet/

Blue ratio were obtained by dividing the peak values (after

antibody Ca21-flux induction either with clones 145-2C11 or

GL3) with the mean baseline levels (before antibody Ca21-flux

induction). These values obtained from iIEL or systemic T cells in

PBS (control group) and anti-gd TCR (GL3 group) treated mice

conditions were compared using unpaired one-tailed t test.

Values o0.05 were considered as significant (�).

Acknowledgements: This work was supported by grants from

the Chilean government FONDECYT 1070954 (R.Q.) and

Scholarship for Postgraduate Studies 21050679 (F.M.) and by

grants of the Deutsche Forschungsgemeinschaft DFG-PR 727/3-1

(I.P.) and SFB621-A14 (I.P.). The authors thank Andreas Krueger

and Nadja Bakocevic for critically reading the manuscript and

Mathias Herberg for animal care.

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

References

1 Itohara, S., Farr, A. G., Lafaille, J. J., Bonneville, M., Takagaki, Y., Haas, W.

and Tonegawa, S., Homing of a gamma delta thymocyte subset with

homogeneous T-cell receptors to mucosal epithelia. Nature 1990. 343:

754–757.

2 Stankovic, S., Zhan, Y. and Harrison, L. C., Homeostatic proliferation of

intestinal intraepithelial lymphocytes precedes their migration to extra-

intestinal sites. Eur. J. Immunol. 2007. 37: 2226–2233.

3 Chen, Y., Chou, K., Fuchs, E., Havran, W. L. and Boismenu, R., Protection

of the intestinal mucosa by intraepithelial gamma delta T cells. Proc. Natl.

Acad. Sci. USA 2002. 99: 14338–14343.

4 Girardi, M., Immunosurveillance and immunoregulation by gammadelta

T cells. J. Invest. Dermatol. 2006. 126: 25–31.

5 Groh, V., Steinle, A., Bauer, S. and Spies, T., Recognition of stress-induced

MHC molecules by intestinal epithelial gammadelta T cells. Science 1998.

279: 1737–1740.

6 Hayday, A. and Tigelaar, R., Immunoregulation in the tissues by

gammadelta T cells. Nat. Rev. Immunol. 2003. 3: 233–242.

7 Hayday, A. C., Gammadelta T cells and the lymphoid stress-surveillance

response. Immunity 2009. 31: 184–196.

8 Russano, A. M., Bassotti, G., Agea, E., Bistoni, O., Mazzocchi, A., Morelli,

A., Porcelli, S. A. and Spinozzi, F., CD1-restricted recognition of

exogenous and self-lipid antigens by duodenal gammadelta1 T lympho-

cytes. J. Immunol. 2007. 178: 3620–3626.

9 Culshaw, R. J., Bancroft, G. J. and McDonald, V., Gut intraepithelial

lymphocytes induce immunity against Cryptosporidium infection

through a mechanism involving gamma interferon production. Infect.

Immun. 1997. 65: 3074–3079.

Eur. J. Immunol. 2010. 40: 3378–3388Frano H. Malinarich et al.3386

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

10 Olivares-Villagomez, D., Mendez-Fernandez, Y. V., Parekh, V. V., Lalani,

S., Vincent, T. L., Cheroutre, H. and Van Kaer, L., Thymus leukemia

antigen controls intraepithelial lymphocyte function and inflammatory

bowel disease. Proc. Natl. Acad. Sci. USA 2008. 105: 17931–17936.

11 Ismail, A. S., Behrendt, C. L. and Hooper, L. V., Reciprocal interactions

between commensal bacteria and gamma delta intraepithelial lympho-

cytes during mucosal injury. J. Immunol. 2009. 182: 3047–3054.

12 Kawaguchi, M., Nanno, M., Umesaki, Y., Matsumoto, S., Okada, Y., Cai,

Z., Shimamura, T. et al., Cytolytic activity of intestinal intraepithelial

lymphocytes in germ-free mice is strain dependent and determined by T

cells expressing gamma delta T-cell antigen receptors. Proc. Natl. Acad. Sci.

USA 1993. 90: 8591–8594.

13 Boyden, L. M., Lewis, J. M., Barbee, S. D., Bas, A., Girardi, M., Hayday,

A. C., Tigelaar, R. E. and Lifton, R. P., Skint1, the prototype of a newly

identified immunoglobulin superfamily gene cluster, positively selects

epidermal gammadelta T cells. Nat. Genet. 2008. 40: 656–662.

14 Bukowski, J. F., Morita, C. T. and Brenner, M. B., Human gamma

delta T cells recognize alkylamines derived from microbes, edible

plants, and tea: implications for innate immunity. Immunity 1999. 11:

57–65.

15 Puan, K. J., Jin, C., Wang, H., Sarikonda, G., Raker, A. M., Lee, H. K.,

Samuelson, M. I. et al., Preferential recognition of a microbial metabolite

by human Vgamma2Vdelta2 T cells. Int. Immunol. 2007. 19: 657–673.

16 Scotet, E., Martinez, L. O., Grant, E., Barbaras, R., Jeno, P., Guiraud, M.,

Monsarrat, B. et al., Tumor recognition following Vgamma9Vdelta2 T cell

receptor interactions with a surface F1-ATPase-related structure and

apolipoprotein A-I. Immunity 2005. 22: 71–80.

17 Shin, S., El-Diwany, R., Schaffert, S., Adams, E. J., Garcia, K. C., Pereira, P.

and Chien, Y. H., Antigen recognition determinants of gammadelta T cell

receptors. Science 2005. 308: 252–255.

18 Zhang, L., Jin, N., Nakayama, M., O’Brien, R. L., Eisenbarth, G. S. and

Born, W. K., Gamma delta T cell receptors confer autonomous respon-

siveness to the insulin-peptide B:9–23. J. Autoimmun. 2010. 34: 478–484.

19 Martin, B., Hirota, K., Cua, D. J., Stockinger, B. and Veldhoen, M.,

Interleukin-17-producing gammadelta T cells selectively expand in

response to pathogen products and environmental signals. Immunity

2009. 31: 321–330.

20 Pietschmann, K., Beetz, S., Welte, S., Martens, I., Gruen, J., Oberg, H. H.,

Wesch, D. and Kabelitz, D., Toll-like receptor expression and function in

subsets of human gammadelta T lymphocytes. Scand. J. Immunol. 2009. 70:

245–255.

21 Haas, J. D., Gonzalez, F. H., Schmitz, S., Chennupati, V., Fohse, L.,

Kremmer, E., Forster, R. and Prinz, I., CCR6 and NK1.1 distinguish

between IL-17A and IFN-gamma-producing gammadelta effector T cells.

Eur. J. Immunol. 2009. 39: 3488–3497.

22 Kreslavsky, T., Savage, A. K., Hobbs, R., Gounari, F., Bronson, R., Pereira,

P., Pandolfi, P. P. et al., TCR-inducible PLZF transcription factor required

for innate phenotype of a subset of gammadelta T cells with restricted

TCR diversity. Proc. Natl. Acad. Sci. USA 2009. 106: 12453–12458.

23 Kapsenberg, M. L., Gammadelta T cell receptors without a job. Immunity

2009. 31: 181–183.

24 Fahrer, A. M., Konigshofer, Y., Kerr, E. M., Ghandour, G., Mack, D. H.,

Davis, M. M. and Chien, Y. H., Attributes of gammadelta intraepithelial

lymphocytes as suggested by their transcriptional profile. Proc. Natl. Acad.

Sci. USA 2001. 98: 10261–10266.

25 Kawaguchi-Miyashita, M., Shimada, S., Matsuoka, Y., Ohwaki, M. and

Nanno, M., Activation of T-cell receptor-gammadelta1 cells in the

intestinal epithelia of KN6 transgenic mice. Immunology 2000. 101: 38–45.

26 Mosley, R. L., Whetsell, M. and Klein, J. R., Proliferative properties of

murine intestinal intraepithelial lymphocytes (IEL): IEL expressing TCR

alpha beta or TCR tau delta are largely unresponsive to proliferative

signals mediated via conventional stimulation of the CD3-TCR complex.

Int. Immunol. 1991. 3: 563–569.

27 Shires, J., Theodoridis, E. and Hayday, A. C., Biological insights into

TCRgammadelta1 and TCRalphabeta1 intraepithelial lymphocytes

provided by serial analysis of gene expression (SAGE). Immunity 2001.

15: 419–434.

28 Sydora, B. C., Mixter, P. F., Holcombe, H. R., Eghtesady, P., Williams, K.,

Amaral, M. C., Nel, A. and Kronenberg, M., Intestinal intraepithelial

lymphocytes are activated and cytolytic but do not proliferate as well as

other T cells in response to mitogenic signals. J. Immunol. 1993. 150:

2179–2191.

29 De Maria, R., Fais, S., Silvestri, M., Frati, L., Pallone, F., Santoni, A. and

Testi, R., Continuous in vivo activation and transient hyporesponsiveness

to TcR/CD3 triggering of human gut lamina propria lymphocytes. Eur. J.

Immunol. 1993. 23: 3104–3108.

30 Hayday, A., Theodoridis, E., Ramsburg, E. and Shires, J., Intraepithelial

lymphocytes: exploring the Third Way in immunology. Nat. Immunol.

2001. 2: 997–1003.

31 Finkel, T. H., Marrack, P., Kappler, J. W., Kubo, R. T. and Cambier, J. C.,

Alpha beta T cell receptor and CD3 transduce different signals in

immature T cells. Implications for selection and tolerance. J. Immunol.

1989. 142: 3006–3012.

32 Hayes, S. M. and Love, P. E., Distinct structure and signaling potential of

the gamma delta TCR complex. Immunity 2002. 16: 827–838.

33 Prinz, I., Sansoni, A., Kissenpfennig, A., Ardouin, L., Malissen, M. and

Malissen, B., Visualization of the earliest steps of gammadelta T cell

development in the adult thymus. Nat. Immunol. 2006. 7: 995–1003.

34 Wakita, D., Sumida, K., Iwakura, Y., Nishikawa, H., Ohkuri, T., Chamoto,

K., Kitamura, H. and Nishimura, T., Tumor-infiltrating IL-17-producing

gammadelta T cells support the progression of tumor by promoting

angiogenesis. Eur. J. Immunol. 2010. 40: 1927–1937.

35 Cho, J. S., Pietras, E. M., Garcia, N. C., Ramos, R. I., Farzam, D. M., Monroe,

H. R., Magorien, J. E. et al., IL-17 is essential for host defense against

cutaneous Staphylococcus aureus infection in mice. J. Clin. Invest. 2010.

120: 1762–1773.

36 Jensen, K. D., Shin, S. and Chien, Y. H., Cutting edge: Gammadelta

intraepithelial lymphocytes of the small intestine are not biased toward

thymic antigens. J. Immunol. 2009. 182: 7348–7351.

37 Gramzinski, R. A., Adams, E., Gross, J. A., Goodman, T. G., Allison, J. P.

and Lefrancois, L., T cell receptor-triggered activation of intraepithelial

lymphocytes in vitro. Int. Immunol. 1993. 5: 145–153.

38 Yamamoto, S., Russ, F., Teixeira, H. C., Conradt, P. and Kaufmann, S. H.,

Listeria monocytogenes-induced gamma interferon secretion by intest-

inal intraepithelial gamma/delta T lymphocytes. Infect. Immun. 1993. 61:

2154–2161.

39 Koenecke, C., Chennupati, V., Schmitz, S., Malissen, B., Forster, R. and

Prinz, I., In vivo application of mAb directed against the gammadelta TCR

does not deplete but generates ‘‘invisible’’ gammadelta T cells. Eur. J.

Immunol. 2009. 39: 372–379.

40 Monk, T., Spencer, J., Cerf-Bensussan, N. and MacDonald, T. T.,

Stimulation of mucosal T cells in situ with anti-CD3 antibody: location

of the activated T cells and their distribution within the mucosal micro-

environment. Clin. Exp. Immunol. 1988. 74: 216–222.

41 Ebert, E. C., Human intestinal intraepithelial lymphocytes have potent

chemotactic activity. Gastroenterology 1995. 109: 1154–1159.

Eur. J. Immunol. 2010. 40: 3378–3388 Cellular immune response 3387

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

42 Ebert, E. C., Interleukin 15 is a potent stimulant of intraepithelial

lymphocytes. Gastroenterology 1998. 115: 1439–1445.

43 Ebert, E. C., Activation of human intraepithelial lymphocytes reduces

CD3 expression. Clin. Exp. Immunol. 2003. 132: 424–429.

44 Solomon, K. R., Krangel, M. S., McLean, J., Brenner, M. B. and Band, H.,

Human T cell receptor-gamma and -delta chain pairing analyzed by

transfection of a T cell receptor-delta negative mutant cell line.

J. Immunol. 1990. 144: 1120–1126.

45 Cheroutre, H. and Lambolez, F., Doubting the TCR coreceptor function of

CD8alphaalpha. Immunity 2008. 28: 149–159.

46 Goodman, T. and Lefrancois, L., Intraepithelial lymphocytes. Anatomical

site, not T cell receptor form, dictates phenotype and function. J. Exp. Med.

1989. 170: 1569–1581.

47 Rocha, B., von Boehmer, H. and Guy-Grand, D., Selection of intraepithe-

lial lymphocytes with CD8 alpha/alpha co-receptors by self-antigen in the

murine gut. Proc. Natl. Acad. Sci. USA 1992. 89: 5336–5340.

48 Yamagata, T., Mathis, D. and Benoist, C., Self-reactivity in thymic

double-positive cells commits cells to a CD8 alpha alpha lineage

with characteristics of innate immune cells. Nat. Immunol. 2004. 5:

597–605.

49 Nagelkerken, L. and Hertogh-Huijbregts, A., The acquisition of a memory

phenotype by murine CD41 T cells is accompanied by a loss in their

capacity to increase intracellular calcium. Dev. Immunol. 1992. 3: 25–34.

50 Roederer, M., Bigos, M., Nozaki, T., Stovel, R. T., Parks, D. R. and

Herzenberg, L. A., Heterogeneous calcium flux in peripheral T cell

subsets revealed by five-color flow cytometry using log-ratio circuitry.

Cytometry 1995. 21: 187–196.

51 Hoffmann, J. C., Peters, K., Henschke, S., Herrmann, B., Pfister, K.,

Westermann, J. and Zeitz, M., Role of T lymphocytes in rat 2,4,6-

trinitrobenzene sulphonic acid (TNBS) induced colitis: increased mortal-

ity after gammadelta T cell depletion and no effect of alphabeta T cell

depletion. Gut 2001. 48: 489–495.

52 Dalton, J. E., Cruickshank, S. M., Egan, C. E., Mears, R., Newton, D. J.,

Andrew, E. M., Lawrence, B. et al., Intraepithelial gammadelta1

lymphocytes maintain the integrity of intestinal epithelial tight junctions

in response to infection. Gastroenterology 2006. 131: 818–829.

53 Inagaki-Ohara, K., Chinen, T., Matsuzaki, G., Sasaki, A., Sakamoto, Y.,

Hiromatsu, K., Nakamura-Uchiyama, F. et al., Mucosal T cells bearing

TCRgammadelta play a protective role in intestinal inflammation.

J. Immunol. 2004. 173: 1390–1398.

54 Kuhl, A. A., Pawlowski, N. N., Grollich, K., Loddenkemper, C., Zeitz, M.

and Hoffmann, J. C., Aggravation of intestinal inflammation by depletion/

deficiency of gammadelta T cells in different types of IBD animal models.

J. Leukoc. Biol. 2007. 81: 168–175.

55 Tsuchiya, T., Fukuda, S., Hamada, H., Nakamura, A., Kohama, Y.,

Ishikawa, H., Tsujikawa, K. and Yamamoto, H., Role of gamma delta T

cells in the inflammatory response of experimental colitis mice.

J. Immunol. 2003. 171: 5507–5513.

56 Shichita, T., Sugiyama, Y., Ooboshi, H., Sugimori, H., Nakagawa, R.,

Takada, I., Iwaki, T. et al., Pivotal role of cerebral interleukin-17-

producing gammadeltaT cells in the delayed phase of ischemic brain

injury. Nat. Med. 2009. 15: 946–950.

57 Mombaerts, P., Clarke, A. R., Rudnicki, M. A., Iacomini, J., Itohara, S.,

Lafaille, J. J., Wang, L. et al., Mutations in T-cell antigen receptor genes

alpha and beta block thymocyte development at different stages. Nature

1992. 360: 225–231.

58 Prinz, I., Gregoire, C., Mollenkopf, H., Aguado, E., Wang, Y., Malissen, M.,

Kaufmann, S. H. and Malissen, B., The type 1 cysteinyl leukotriene

receptor triggers calcium influx and chemotaxis in mouse alpha beta-

and gamma delta effector T cells. J Immunol. 2005. 175: 713–719.

Abbreviations: ab iIEL: iIEL carrying an ab TCR � [Ca21]i: intracellular

free calcium concentration � CCL4: CC chemokine ligand 4 � DN:

double negative � cd iIEL: intestinal intraepithelial lymphocytes

carrying the gd TCR � H2B: histone 2B � Tcrd: T cell receptor d locus

Full correspondence: Dr. Immo Prinz, Hannover Medical School,

Institute for Immunology Carl-Neuberg-Str. 1, 30625 Hannover,

Germany

Fax: 149-5115329722

e-mail: Prinz.Immo@mh-hannover.de

Received: 10/6/2010

Revised: 28/7/2010

Accepted: 22/9/2010

Accepcted article online: 5/10/2010

Eur. J. Immunol. 2010. 40: 3378–3388Frano H. Malinarich et al.3388

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu