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Keywords:

  • Ca2+-flux;
  • CCL4;
  • γδ intestinal intraepithelial lymphocytes;
  • γδ T cells;
  • IFN-g

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Intestinal intraepithelial lymphocytes carrying the γδ TCR (γδ iIEL) are involved in the maintenance of epithelial integrity. γδ 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 γδ iIEL express the CD8αα homodimer. However, our knowledge about cognate ligands for most γδ TCR remains fragmentary and recent advances show that γδ 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 γδ iIEL was functional beyond its role during thymic selection. Using TcrdH2BeGFP (Tcrd, T-cell receptor δ locus; H2B, histone 2B) reporter mice to identify γδ T cells, we measured their intracellular free calcium concentration in response to TCR-crosslinking. In contrast to systemic γδ T cells, CD8αα+ γδ 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-γ upon TCR triggering in vitro. Notably, in vivo blocking of the γδ TCR with specific mAb led to a decrease of basal calcium levels in CD8αα+ γδ iIEL. This suggests that the γδ TCR of CD8αα+ γδ iIEL is constantly being triggered and therefore functional in vivo.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Heterodimers of the γδ TCR are shared by diverse T-lymphocyte populations comprising motile γδ 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 γδ T-cell populations 1, 2. A prototype for the latter is the compartment of intestinal intraepithelial lymphocytes carrying the γδ TCR (γδ iIEL), composed of γδCD8αα and γδCD8CD4 double negative (DN) populations. There is increasing evidence that the primary role of γδ iIEL and other tissue-resident γδ T cells is immune surveillance of their habitat and the maintenance of epithelial integrity 3–8. It is assumed that γδ 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 γδ TCR still remain unknown. Moreover, there have been convincing reports of alternative ways of γδ 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 γδ T cells can directly produce the effector cytokines IL-17A or IFN-γ in response to stimulation with IL-23 or IL-12/IL-18, respectively 21, 22. Therefore, it seems tempting to speculate that the γδ TCR may actually be dispensable for the in vivo function of γδ 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.

γδ iIEL as well as other iIEL carrying an αβ TCR (αβ 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, γδ iIEL and αβ iIEL can display on their surface T-cell activation markers such as CD69 and approximately 75% express the CD8αα 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 γδ TCR is involved in this process.

In this study, we investigated the functionality of γδ and αβ TCR expressed on freshly isolated systemic T lymphocytes and iIEL by measuring the increase of intracellular free calcium concentration ([Ca2+]i) levels after TCR stimulation on a single cell basis. Of note, we found that γδ and αβ iIEL had high levels of basal [Ca2+]i. Furthermore, we detected elevated basal [Ca2+]i levels in CD8αα+ when compared with [Ca2+]i in CD8αα γδ (DN) iIEL. These elevated basal [Ca2+]i levels correlated with lower responsiveness to TCR-specific stimulation. Furthermore, we were able to tune down basal [Ca2+]i levels of γδ CD8αα+ iIEL in vivo through the systemic administration of specific anti-γδ TCR mAb. Irrespective of the mechanism, this effect implied that diminished TCR signaling capacity resulted in lower basal [Ca2+]i levels and thus provided evidence that the γδ TCR was indeed functional and likely to be constantly triggered in vivo. Additional, albeit indirect support for a functional TCR in iIEL was offered by ex vivo stimulation assays demonstrating that TCR ligation of some γδ and αβ 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 γδ iIEL, must be functional in vivo.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Different basal intracellular equation image levels in systemic and intestinal γδ T cells

Monitoring of [Ca2+]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 γδ T cells, this was so far difficult, because the identification of bona fide γδ T cells depended on staining with mAb directed against the γδ TCR. In order to directly measure intracellular Ca2+ levels of γδ T cells in response to stimulation of their TCR, we thus made use of TcrdH2BeGFP (Tcrd, T-cell receptor δ locus; H2B, histone 2B) reporter mice 33. More precisely, we used F1 C57BL/6-Tcra−/−×TcrdH2BeGFP double heterozygous mice (γδ reporter mice) in which expression of the reporter H2BeGFP unambiguously identifies γδ T cells without touching their TCR. This system was chosen to avoid any false-positive GFP+ 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-CD8α, five populations of either systemic T cells or iIEL were defined (Fig. 1A). In the systemic T-cell compartment, CD8α expression identified αβCD8+ T cells (CD8+ p-αβ) while GFP expression identified γδDN T cells (CD8 p-γδ). In iIEL preparations, GFP+ γδ T cells were divided into CD8α (CD8 i-γδ, approximately 20% of all γδ T cells, corresponding to γδDN iIEL) or CD8α+ (CD8+ i-γδ, approximately 80% of all γδ T cells, corresponding to γδCD8αα+ iIEL). Finally, we gated GFPCD8α+ cells (CD8+ i-αβ), representing αβ CD8α+ iIEL. As a general observation, the iIEL compartment showed substantially higher basal [Ca2+]i levels than systemic T cells (Fig. 1B). The systemic populations had equal basal [Ca2+]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 Ca2+-fluxes (Fig. 1C). However, Ca2+ response amplitudes were higher in CD8+ p-αβ and CD8 p-γδ representing systemic T cells.

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Figure 1. γδ iIEL and systemic γδ T cells differ in basal intracellular [Ca2+] levels and Ca2+-flux responses to CD3/γδ TCR stimulation. (A) Representative FACS plots show the identification of specific T-cell subpopulations according to FSC/SSC (left column) and the surface marker CD8α versus γδ 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, CD8+ p-αβ (pink) and CD8 p-γδ (light blue) were designated. Within the iIEL population, CD8 i-γδ (green), CD8+ i-γδ (red) and CD8+ i-αβ (blue) were gated. (B) Histogram overlay showing basal [Ca2+]i levels of T-cell populations as gated in (A) over time (left panel). Summary plot of the basal [Ca2+]i level values of T-cell populations (right panel), columns show mean±SEM, n=3 independent experiments. (C) Overlay of representative ionomycin-induced Ca2+-flux responses of systemic and iIEL compartment T-cell subpopulations. Color coding of populations is as described in (A). (D) Representative Ca2+-flux kinetics of Indo-1AM-labeled CD8+ p-αβ (pink) and CD8 p-γδ (light blue) induced by the addition of anti-CD3 (clone 2C11, upper panel) or anti-γδ TCR (clone GL3, lower panel) monoclonal antibodies, followed by cross-linking with polyclonal anti-Hamster-Ab at the indicated time points. (E) Representative Ca2+-flux kinetics of CD8 i-γδ (green), CD8+ i-γδ (red) and CD8+ i-αβ (blue) subpopulations of Indo-1AM-labeled iIEL after addition of the same antibody combinations as described in (D). Data shown in panels (C–E) are representative for at least three independent experiments.

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Next, we studied the Ca2+-flux of isolated iIEL or systemic T cells from γδ reporter mice after TCR-clustering with antibodies. For this, we applied an anti-γδ TCR mAb clone (GL3) and an anti-CD3ε clone (145-2C11, here 2C11) and subsequently clustered them on the cell surface with secondary goat anti-hamster antibody. This procedure induced robust anti-CD3-induced Ca2+-fluxes in the systemic populations CD8+ p-αβ and CD8 p-γδ (Fig. 1D). Similarly, clustering with anti-γδ TCR mAb specifically induced Ca2+-flux of systemic CD8 p-γδ cells (Fig. 1D). However, in the iIEL compartment, we observed discrete Ca2+-fluxes in response to anti-CD3 or anti-γδ TCR mAb only in CD8 i-γδ but not in CD8+ i-γδ (Fig. 1E). This suggested that high basal [Ca2+]i levels in γδCD8αα+iIEL correlated with TCR-unresponsiveness. Taken together, we found that systemic αβ and γδ T cells showed comparable Ca2+-flux responses to TCR ligation, whereas CD8αα+ αβ and γδ iIEL were presumably pre-activated and thus refractory to further stimulation of the TCR complex and displayed high intrinsic [Ca2+]i levels. These results suggest a chronic stimulation of CD8α+ iIEL in vivo.

TCR stimulation and intracellular equation image-flux induce CCL4 and production by iIEL

Next, we sought to investigate the outcome of αβ- and γδ-specific TCR stimulation on isolated iIEL in ex vivo stimulation assays. Since systemic γδ T cells in lymph nodes, spleen and circulation 19, 21, 34 as well as intraepithelial γδ 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 γδ iIEL. We found that, irrespective of CD8α expression, γδ 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 γδ IEL are not ‘pre-wired’ toward a specific lineage 36. Therefore, we focused in this study on the well-established γδ IEL effector molecules CC chemokine ligand 4 (CCL4) and IFN-γ. Chemokine and cytokine production of αβ, γδ and total iIEL from WT mice was monitored by stimulation with plate-bound anti-γδ TCR (GL3 and GL4), anti-αβ TCR (H57-597, called H57) and anti-CD3 (2C11), respectively, followed by cytokine measurement in the supernatants. Here, αβ or γδ TCR triggering induced similar concentrations of CCL4 (Fig. 3A, upper panel), whereas higher amounts of IFN-γ were produced through anti-αβ TCR stimulation (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-αβ TCR (H57) and anti-γδ TCR (GL3) followed by intracellular staining. TCR engagement induced CCL4 production in both αβ and γδ iIEL populations (Fig. 3B, left panel), whereas more αβ iIEL than γδ iIEL produced IFN-γ (Fig. 3B, right panel). These results clearly showed that iIEL were not anergic in these assays and that the TCR in αβ and γδ 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 Ca2+ ionophore ionomycin showed that γδ iIEL populations had a better capacity to produce CCL4 (Fig. 3C, left panel) and αβ iIEL populations a better ability to produce IFN-γ in response to ionomycin-induced Ca2+-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-γ production in γδCD8αα+ iIEL (Fig. 3D). In contrast to γδ iIEL, αβ iIEL populations showed similar activation behavior either with PMA/ionomycin or TCR stimulation (Fig. 3E); however, αβ+CD4+ iIEL produced IFN-γ more efficiently after PMA/ionomycin stimulation 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 Ca2+ increase in the activation process. On the other hand, the importance of the γδ TCR, especially in γδCD8αα+ iIEL population, highlights a central role of this receptor for the function of γδ iIEL.

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Figure 2. TCR-ligation is a potent inducer of IFN-γ but not IL-17A production by CD8αα+ intestinal intraepithelial γδ T cells. Intracellular cytokine staining of intestinal intraepithelial γδ T cells gated as CD8βCD4TCR-βH2B-eGFP+ as detailed in Supporting Information Fig. 1. Isolated iIEL were cultured for 4 h either in medium (control), on plate-bound anti-γδ TCR mAb or stimulated by PMA/ionomycin before extra- and intracellular staining. Upper panels show intracellular IFN-γ versus CD8α surface staining. Lower panels show intracellular IL-17A versus CD8α surface staining. Numbers in quadrants indicate the percentage of cells in each. Data are representative of three independent experiments. Statistics for IFN-γ production are shown in Fig. 3.

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Figure 3. CCL4 and IFN-γ production by iIEL populations depends on TCR complex stimulation and correlates with ionomycin-induced intracellular [Ca2+] increase. Freshly isolated iIEL suspensions were stimulated in vitro. Subsequently, various T-cell populations were gated according to expression of TCR αβ or TCR γδ as well as co-receptors CD8α, CD8β, and CD4 and analyzed by intracellular cytokine staining as detailed in Supporting Information Fig. 2 and 3. (A) Representative quantification of CCL4 (upper panel) and IFN-γ (lower panel) in supernatants of iIEL stimulated by plate-bound anti-γδ TCR (clones GL3 and GL4), anti-αβ TCR (clone H57-597), anti-CD3 (clone 145-2C11) measured by cytokine bead array. N/S: no stimulation. (B) Intracellular FACS analysis of CCL4 (left panel) and IFN-γ (right panel) production in iIEL populations incubated on plates coated with anti-γδ TCR (clone GL3), anti-αβ TCR (clone H57), anti-CD3 (clone 2C11). Columns show mean±SEM, n=3 independent experiments. (C) Intracellular FACS analysis of CCL4 (left panel) and IFN-γ (right panel) production in the presence (black bars) or absence (white bars) of ionomycin (2 μg/mL) in the same iIEL populations. Columns show mean±SEM, n=3 independent experiments. (D) Direct comparison of CCL4 (left panel) and IFN-γ (right panel) production after stimulation with PMA/ionomycein ionomycin (black), anti-CD3 (red) or anti-γδ TCR (green) of γδ+CD8αα+ and γδ+DN iIEL populations by intracellular FACS analysis. N/S: no stimulation. Columns show mean±SEM, n=3 independent experiments. (E) Analysis as in (D) for αβ+CD4+, αβ+CD4+CD8α+, αβ+CD8αβ+ and αβ+CD8αα+ iIEL populations. All experiments were carried out with cells derived from WT C57BL/6 mice.

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In vivo anti-γδ TCR mAb treatment decreases equation image levels in iIEL

We hypothesized that the high basal [Ca2+]i levels observed in γδ iIEL (Fig. 1B) might be due to continuous TCR stimulation in situ. Taking into account that the anti-γδ TCR mAb clone GL3 could specifically activate γδ iIEL ex vivo and down-regulate surface γδ TCR complex levels in vivo39, we tested the effect of in vivo TCR modulation on basal [Ca2+]i levels of γδ iIEL. Therefore, reporter mice were treated with a regimen of three consecutive injections of 200 μg anti-γδ TCR mAb (GL3) at day −6, day −4 and day −2 before analysis. First, in vivo γδ TCR modulation induced down-modulation of CD3 and γδ TCR surface levels of γδ iIEL (Fig. 4A, upper panel), similar to what we showed previously 39. However, this protocol of repeated high-dose injection of anti-γδ TCR mAb did not alter the expression level of CD8α on the targeted γδ iIEL (Fig. 4A, upper panel) or the frequency of CD8α+ cells among all γδ iIEL (data not shown); neither did it significantly modulate the chronically activated phenotype of the γδ iIEL as assessed by surface activation markers (Fig. 4A, lower panel). Similarly, the activation status, as well as αβ TCR complex and CD8α expression on αβ iIEL (Fig. 4B), was not influenced by this regimen. Importantly, basal [Ca2+]i levels and amplitudes of ionomycin-induced Ca2+-fluxes were significantly decreased in CD8α+ iIEL derived from mice injected with GL3 compared with those from mock-treated animals (Fig. 4C, D). However, not only γδCD8αα+ iIEL but also αβCD8α+ iIEL cells showed a basal [Ca2+]i decrease. This was unlikely to be a direct effect of the GL3 mAb on αβ iIEL but may be due to changes in the composition of αβCD8α+ iIEL, e.g. through attraction of systemic αβ+CD8+ cells with lower basal [Ca2+]i levels into the gut epithelium 40. In contrast, basal [Ca2+]i levels of neither systemic CD8 p-γδ nor CD8 i-γδ were altered by GL3-treatment (Fig. 4C and D). These data suggest that the observed high basal [Ca2+]i levels of γδCD8αα+ iIEL reflect a constant TCR-specific activation in vivo, which could be partially blocked by anti-γδ TCR mAb treatment.

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Figure 4. Treatment with anti-γδ TCR mAb decreases ex vivo basal intracellular [Ca2+] levels in iIEL. (A and B) Representative histograms showing TCR, CD3, CD8α, CD69, CD44 and CD62L surface expression of γδ iIEL (A) and αβ iIEL (B), (gated as depicted in Supporting Information Fig. 1) from PBS (dotted line) and GL3 (black line) treated γδ reporter mice. Fluorescence minus one (FMO) control is shown as gray-filled histogram, all surface markers were similarly revealed with Streptavidin-PerCP. (C) Representative basal [Ca2+]i levels (left column) of iIEL in PBS (upper panel) or GL3-treated (lower panel) γδ reporter mice and corresponding ionomycin-Ca2+-flux induction kinetics (right column). The iIEL populations are shown as CD8 i-γδ (black line), CD8+ i-γδ (gray line) and CD8+ i-αβ (dotted line). (D) Comparison of the basal [Ca2+]i level values of PBS (black bars) or GL3 (white bars) treated γδ reporter mice. Columns show mean±SEM, n=3 independent experiments.

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In vivo anti-γδ TCR mAb treatment impairs the TCR responsiveness of γδ iIEL

Next, we investigated how γδ T cells from GL3-treated γδ reporter mice responded to TCR stimulation. As shown in Fig. 4A, the TCR complex was down-regulated but still present at residual levels on the cell surface of these γδ T cells. We found that anti-CD3 and anti-γδ TCR mAb clustering still elicited Ca2+-fluxes in CD8 p-γδ and CD8 i-γδ from mice injected with GL3, albeit with lower or almost flat amplitudes compared with those from mock-treated animals. The iIEL populations CD8+ i-γδ and CD8+ i-αβ only showed a decrease of basal [Ca2+]i, without evident mAb-induced Ca2+-flux neither in PBS nor in GL3 treated mice (Fig. 5A). The quantification of these changes, displayed as fold of basal [Ca2+]i levels after anti-CD3 and anti-γδ TCR mAb clustering, showed that CD8 p-γδ and CD8 i-γδ were affected by the GL3 treatment (Fig. 5B). In addition, iIEL from PBS- and GL3-treated γδ reporter mice were analyzed for responsiveness to ex vivo stimulation with GL3 and GL4, a different anti-γδ TCR mAb. In vivo treatment with GL3 reduced the TCR-dependent CCL4 and IFN-γ production of γδ iIEL (Fig. 5C). Surprisingly, the CCL4 and IFN-γ production capability of γβ iIEL from GL3-treated γδ reporter mice stimulated ex vivo with the anti-αβ TCR (H57) was increased (Fig. 5D). In conclusion, γδ 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-γδ TCR treatment. However, the down-modulation of their surface TCR in combination with the decoration of residual surface γδ 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 γδ T cells. However, it is at present not clear to what extent the responsiveness of γδ T cells to other stimuli, e.g. engagement of other receptors such as NKG2D or TLR, may be also altered by TCR modulation.

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Figure 5. Treatment with anti-γδTCR mAb impairs the TCR responses of γδ iIEL. (A) Ca2+-flux in PBS (gray line) or GL3 (black line) treated γδ reporter mice induced by the antibodies anti-CD3 (clone 2C11, left column) or anti-γδ TCR (clone GL3, right column). (B) Maximal fold increase of [Ca2+]i to average basal levels induced by anti-CD3 (clone 2C11, left panel) or by anti-γδ TCR (clone GL3, right panel) in the indicated T-cell populations of PBS (black bars) or GL3 (white bars) treated γδ reporter mice. Columns show mean±SEM, n=3 independent experiments. (C and D) CCL4 (left panel) and IFN-γ (right panel) measured by cytokine bead array in supernatants of iIEL from PBS (black bars) or GL3 (white bars) treated WT C57BL/6 mice. iIEL isolated from these two groups were either stimulated by plate-bound anti-γδ TCR clones GL3 and GL4 (C) or with anti-αβ TCR clone H57 (D). N/S: no stimulation. (C and D) One representative of three individual experiments is shown.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The question whether, after thymic selection, the TCR on γδ T cells had a physiological role at all was not unanticipated 19, 23. Our knowledge about cognate ligands of the γδ TCR remains limited and γδ T cells are equipped with a variety of receptors that can mediate T-cell activation and cytokine release. Here we provide evidence that the γδ TCR on γδ iIEL is functional in a normal mouse. We found that its down-modulation led to lower basal [Ca2+]i levels suggesting the γδ TCR on γδ iIEL to be constantly triggered in vivo.

The experiments carried out in the γδ reporter mice were an improvement to previous Ca2+-flux studies on γδ T cells 32, 41–44 because bona fide γδ T cells could be easily identified by their intrinsic fluorescence without the use of specific mAb directed against the γδ 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 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 Ca2+-fluxes in CD8α+ iIEL compartments were hardly detectable, possibly due to high basal [Ca2+]i levels in these cells. This was observed for both αβ iIEL and γδ iIEL. In contrast, CD8α γδ DN iIEL, which had lower basal [Ca2+]i levels, showed a sizeable Ca2+-flux. The reason for this dichotomy of CD8α+ and CD8α γδ iIEL is not clear. It is possible that the CD8αα homodimer directly modulates the iIEL's Ca2+ responses by direct interaction with the TCR. More likely, the interaction of CD8αα 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 CD8α cells are the precursors of CD8α+ γδ iIEL or whether CD8α+ and CD8α γδ iIEL represent largely unrelated populations that co-exist in the intestinal epithelium.

The observed intrinsically high basal [Ca2+]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 [Ca2+]i levels compared with autologous systemic blood lymphocytes 29. High basal [Ca2+]i levels were equally found in αβ and γδ iIEL thus raising the questioning whether both types of TCR experienced antigen-specific stimulation. Certainly, other factors may contribute to the activated phenotype of iIEL 46; however both αβ and γδ iIEL showed constitutive cytolytic activity in response to TCR engagement 46. In addition, it is likely that the TCR of αβCD8αα+ iIEL recognizes self-antigens 47, 48. Moreover, diminished Ca2+-fluxes in response to TCR stimulation were previously reported for memory CD4+ T cells compared with naïve T cells 49, 50. Collectively, it emerges that γδCD8αα+ iIEL, which had high basal [Ca2+]i levels, are chronically activated by their specific environment. Such continuous activation should at least in part be mediated by TCR triggering, because TCR modulation with anti-γδ TCR mAb reduced the high basal [Ca2+]i levels in CD8α+ γδ iIEL.

Administration of anti-γδ TCR was formerly used to ‘deplete’ γδ T cells in many experimental models for human disease. Several studies have reported profound effects of γδ TCR modulation in vivo thereby highlighting an important beneficial role for γδ 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 γδ T cells in TcrdH2BeGFP reporter mice we had previously reported that there is no depletion but that binding of anti-γδ TCR mAb rendered the target cells ‘invisible’ for further detection based on anti-γδ TCR mAb 39. However, at that time it was not further investigated what effect mAb treatment would have on γδ T-cell function in vivo. We favor a scenario where docking of the antibodies would presumably induce a limited initial activation of the γδ 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 [Ca2+]i levels in γδCD8αα+ iIEL from GL3-treated mice. This has technical implications for experimental in vivo administration of anti-γδ TCR antibody to block the biological functions of γδ iIEL. It appears that signaling through the TCR of γδ 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-γδ TCR-treated mice further highlight the physiological role of the TCR in γδ T cells 3, 51–56. Potential future therapeutic approaches to block γδ TCR signaling in humans may thus represent promising intervention strategies. In conclusion, the TcrdH2BeGFP reporter system enabled us to measure dynamic [Ca2+]i levels of γδ T cells in normal mice. Not ignoring the presence of NK-receptors or pattern recognition receptors expressed on γδ T cells we propose that the γδ TCR of CD8αα+ γδ iIEL is functional because it is constantly being triggered in vivo, most likely by ligands expressed on intestinal epithelial cells.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Mice

F1 C57BL/6-Tcra−/−×TcrdH2BeGFP reporter mice were obtained from crossbreeding Tcra/57 and TcrdH2BeGFP33. 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 Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit.

Antibodies

The mAb used for ex vivo iIEL stimulation directed against γδ TCR (clone GL3), CD3 (clone 145-2C11), αβ TCR (clone H57-597) (all Armenian hamster) were purified from hybridoma supernatants and γδ TCR (clone GL4) was a gift from Dr. Leo Lefrançois. For Ca2+-flux studies anti-γδTCR (clone GL3), CD3 (clone 145-2C11) and goat anti-Armenian hamster (anti-Hamster, Jackson ImmunoReasearch) were applied. For the analysis of T-cell populations by FACS the following mAb were used: γδTCR-FITC (clone GL3), γδTCR-biotin (clone GL3) and CD3-biotin (clone 145-2C11), CD8α-Cy5 or CD8α-biotin (clone Rm CD8), CD8β-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 αβ 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(ab′)2 Donkey anti-goat IgG-PE (Jackson ImmunoReasearch), ChromPure goat IgG (Jackson ImmunoReasearch) or anti-IL-17A-PE (clone ebio17B7, eBiosciences) and anti-IFN-γ-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 37°C, 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 γδ 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 γδ T-cell modulation

γδ reporter mice were treated with a regime of three consecutive intraperitoneal injections of purified anti-γδ TCR mAb at day −6, day −4 and day −2 before analysis (clone GL3, 200 μg/mouse). Control groups received mock injections with PBS.

equation image-flux measurements

iIEL and systemic T cells from γδ reporter mice were prepared for Ca2+-flux cytometry as described with minor modifications 58. In order to avoid a bias in the Ca2+-flux measurements, all the procedures were carried out at room temperature, without EDTA and with a final cell viability ≥92% determined by Trypan blue prior to Indo-1AM loading of the cells. Cells were incubated at a concentration of 0.5×107per mL with 5 μM Indo-1AM (Invitrogen, Molecular Probes) for 60 min at 37°C, stained with anti-CD8α-PE for 10 min and left at room temperature in the dark. The viability of cells after Indo-1AM loading was >90% as assessed by propidium iodide staining gated on the lymphocyte FSC/SSC population. Prior to data acquisition, the cell suspensions were warmed to 37°C in the dark for 10 min and then aliquoted in 200 μL, then CaCl2 was added to a final concentration of 1 mM and Ca2+-flux was measured with a LSRII (BD) cytometer equipped with a 355 nm UV laser at 37°C using a custom-built heating device adapted to cytometer tubes. After acquisition of the baseline levels for 60 s, anti-CD3 or anti-γδ TCR 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, 100 μg/mL of anti-CD3 (clone 145-2C11) with 180 μg/mL of anti-hamster and 100 μg/mL of anti-γδ TCR (clone GL3) with 180 μg/mL of anti-hamster final concentrations; iIEL compartment, 200 μg/mL of anti-CD3 with 180 μg/mL anti-hamster and 100 μg/mL of anti-γδ TCR (clone GL3) with 360 μg/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 Ca2+-flux (2 μg/mL). The kinetic Ca2+ 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 μg/mL of anti-γδ TCR (clone GL3 and GL4), anti-αβ 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-γ 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 μg/mL of anti-γδ TCR (clone GL3 or GL4), anti-αβ TCR (clone H57-597), anti-CD3 (clone 145-2C11) or in presence of PMA (10 ng/mL) and ionomycin (2 μg/mL), for 4 h. Brefeldin A (10 μg/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-γ. 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 mean±SEM 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 Ca2+-flux induction either with clones 145-2C11 or GL3) with the mean baseline levels (before antibody Ca2+-flux induction). These values obtained from iIEL or systemic T cells in PBS (control group) and anti-γδ TCR (GL3 group) treated mice conditions were compared using unpaired one-tailed t test. Values <0.05 were considered as significant (*).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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 Bakočević 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

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  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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