Autistic effector T cells in mice with a point mutation in the LAT adaptor fail to respond to Listeria monocytogenes infection
Immo Prinz1,2,
Mischo Kursar1,2,
Hans-Willi Mittrücker2,
Enrique Aguado2,3,
Ulrich Steinhoff2,
Stefan H. E. Kaufmann2 and
Bernard Malissen1
1 Centre d'Immunologie de Marseille-Luminy, INSERM-CNRS-Université de la Méditerranée, Parc Scientifique de Luminy, Case 906, 13288 Marseille Cédex 9, France
2 Max Planck Institute for Infection Biology, Department of Immunology, Schumannstraße 21-22, D-10117 Berlin, Germany
3 Departamento de Bioquímica B e Inmunología, Facultad de Medicina, Universidad de Murcia, 30100 Murcia, Spain
Correspondence to: I. Prinz; E-mail: prinz{at}ciml.univ-mrs.fr
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Abstract
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The adaptor protein linker for activation of T cells (LAT) is an important transducer of extracellular T cell stimuli. In mice with a point mutation in LAT (LatY136F), TCR signaling is substantially compromised and LatY136F T cells are unresponsive to CD3 cross-linking in vitro. Nevertheless, LatY136F mice develop a polyclonal lymphoproliferation of CD4+ T cells, which display a Th2-polarized effector phenotype. In this study, LatY136F mice were infected with the intracellular bacterium Listeria monocytogenes and the antigen-specific responses of T cells were determined. Both CD4+ and CD8+ LatY136F T cells were unresponsive to L. monocytogenes infection. In contrast, when CD4+ T cells from wild-type mice were adoptively transferred into LatY136F hosts, they responded normally to L. monocytogenes, indicating that the LatY136F milieu permits Th1 responses. Furthermore, we analyzed whether the infection would influence the capacity of LatY136F CD4+ T cells to produce IL-4 and IFN-
. While L. monocytogenes infection results in Th1-type T cell responses in wild-type animals, we found that it did not shift the strong Th2 polarization of LatY136F T cells towards a Th1 pattern. In conclusion, our results suggest that the activation and Th2 polarization of the LatY136F CD4+ T cells is not influenced by infection with an intracellular pathogen known to induce robust Th1 responses, and is thus likely driven by T cell intrinsic mechanisms.
Keywords: LatY136F mutation, linker for activation of T cells (LAT), Listeria monocytogenes, signal transduction, T cell activation, Th1/Th2 cells
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Introduction
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The transmembrane adaptor molecule linker for activation of T cells (LAT) plays an essential role in the transduction and integration of the signals that finally result in T cell activation (1, 2). LAT contains nine conserved tyrosine residues that coordinate the assembly of T cell-signaling complexes within the cytoplasm. Most of the signaling activity of LAT is funneled through the four C-terminal tyrosine residues found at positions 136, 175, 195 and 235. Upon TCR-induced phosphorylation, these tyrosines manifest some specificity for the SH2-domain containing proteins they recruit. For instance, mutation of tyrosine (Y) 136 selectively eliminates binding of phospholipase C-
1 (PLC-
1), whereas the simultaneous mutation of Y175, Y195 and Y235 results in loss of binding of the Grb2/Grap adaptor molecules (3, 4).
In LAT null mutants, T cell development is completely blocked at the CD4 CD8 double-negative stage (5) A knock-in mutation that replaced tyrosine 136 of LAT with a phenylalanine (LatY136F) causes a partial block in
ß T cell development (6, 7). Over time, these mutant mice develop a fatal lymphoproliferative disorder featuring an over-abundance of polyclonal CD4+ T cells that chronically produce type-2 cytokines. This exaggerated Th2 differentiation causes massive lymphocyte infiltration in the lungs, tissue eosinophilia and maturation of plasma cells secreting IgE and IgG1 and is thus reminiscent of human allergic inflammation. These studies suggest that, although the LATY136 residue has a positive function during
ß T cell development, it exerts an additional inhibitory function that is critical for terminal differentiation and homeostasis of CD4+ T cells. The molecular mechanisms underlying this phenotype are as yet unclear, but they may involve defects in the recruitment of negative regulatory molecules to LAT, resulting in lymphoproliferation and autoimmunity. The intra-thymic selection of the CD4+ T cells that expand in LatY136F mice is dependent on MHC class II expression (6), suggesting that residual TCR-signaling activity in LatY136F mice is also responsible for the observed lymphoproliferation. However, in vitro cross-linking of the TCRCD3 complexes expressed at the surface of CD4+ T cells freshly isolated from LatY136F mice fails to induce PLC-
1 activation, Ca2+ mobilization and activation of the calcineurin-dependent transcription factors NF-ATc1 and NF-ATc2. Therefore, mutation of tyrosine 136 of LAT uncouples the TCR from Ca2+ flux-dependent signals. In contrast, the ability to induce RasErk activation in response to anti-CD3 antibody is still preserved in LatY136F T cells (7). Stimulation with a Ca2+ ionophore such as ionomycin was sufficient to induce IL-4 production, whereas treatment with phorbol 12-myristate 13-acetate (PMA) alone induced only a marginal response (I.P., unpublished results). To determine the in vivo relevance of these uncoordinated TCR signals and to determine whether they prevented antigen-specific responses, we infected LatY136F mice with the intracellular bacterium Listeria monocytogenes. In normal mice, L. monocytogenes infection induces at the same time robust innate and acquired immune responses, which are characterized by high frequencies of Listeria-specific CD4+ Th1 and CD8+ T cells (8, 9).
In order to facilitate the analysis of both CD4+ and CD8+ T cell responses specific for L. monocytogenes, we used a recombinant strain of Listeria monocytogenes-expressing ovalbumin (LmOVA) (10). In the C57BL/6 genetic background (H-2b), LmOVA infection induces the response to well-defined immunodominant MHC class I and class II epitopes. This system constitutes therefore an extremely sensitive model to follow antigen-specific T cell responses in vivo. In our study, we demonstrate that LatY136F mice completely ignored the T cell stimuli arising from LmOVA infection and failed to generate Listeria-specific T cells. While LmOVA induces a Th1-type T cell response in wild-type animals, no change was observed in the strong Th2 polarization of LatY136F T cells upon LmOVA infection.
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Methods
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Mice
LatY136F mice were generated as previously described (6) and back-crossed six times to a C57BL/6 background in the specific pathogen-free animal facility of Centre d'Immunologie de Marseille-Luminy. Infection studies were performed at the Max Planck Institute for Infection Biology, Berlin. In all experiments, mice were 56 weeks old. All experiments were performed in accordance with protocols approved by French and German law and European directives.
Bacteria and bacterial infection of mice
Mice were infected with the L. monocytogenes strain LmOVA and analyzed at 8 days post-infection. Bacteria were grown overnight in tryptic soy broth (TSB), washed twice in PBS, aliquoted in PBS, 10% glycerol, frozen and stored at 80°C. Aliquots were thawed and bacterial titers were determined by plating serial dilutions on TSB agar plates. For infections, aliquots were thawed and appropriately diluted in PBS. A total of 5 x 103 bacteria in a volume of 200 µl PBS or 200 µl PBS alone (mock infection) were injected into the lateral tail vein of mice. Dilutions of the inoculum were plated on TSB agar to control the bacterial dose. For determination of bacterial burdens in organs, mice were killed, livers and spleens were homogenized in PBS and serial dilutions of homogenates were plated on TSB agar plates. Colonies were counted after incubation at 37°C overnight.
Flow cytometric analyses
Rat IgG antibodies and mAbs specific for CD16/CD32 (clone 2.4G2), IFN-
(XMG1.2), CD8
(YTS169), CD4 (YTS191.1) and CD62L (Mel-14) were purified from rat serum or from hybridoma supernatants using protein G-sepharose. Purified antibodies were Cy5 or FITC conjugated according to standard protocols. PE-conjugated anti-CD4 (L3T4), APC- and PE-conjugated anti-IL-4 mAb (11B11) and allophycocyanin (APC)-conjugated anti-IFN-
(XMG1.2) were purchased from BD PharMingen, San Diego, CA, USA.
In vitro re-stimulation of cells and flow cytometric determination of cytokine expression
Spleens were removed and single-cell suspensions were prepared using an iron mesh sieve. RBCs were lysed and spleen cells were washed twice with RPMI 1640 medium supplemented with glutamine, Na-pyruvate, ß-mercaptoethanol, penicillin, streptomycin and 10% heat-inactivated FCS (complete RPMI medium). For the determination of cytokine expression, 4 x 106 cells were cultured in 1 ml of complete RPMI medium. T cells were stimulated for 4 h with 106 M of a listeriolysin-derived peptide Listeriolysin O (LLO190201) spanning amino acids 190201 (NEKYAQAYPNVS) or with the ovalbumin-derived petide OVA323339 (ISQAVHAAHAEINEAGR). During the final 3 h of culture, 10 µg ml1 brefeldin A was added. Cultured cells were then washed and incubated for 10 min with rat IgG antibodies and anti-CD16/CD32 mAb to block non-specific antibody binding. Subsequently, cells were stained with Cy5-conjugated anti-CD4 mAb, and after 30 min on ice, cells were washed with PBS and fixed for 20 min at room temperature with 4% PFA in PBS. Cells were washed with PBS, 0.1% BSA, permeabilized with PBS, 0.1% BSA, 0.5% saponin and incubated in this buffer with rat IgG antibodies and anti-CD16/CD32 mAb. After 5 min, conjugated anti-IFN-
mAb and conjugated anti-IL-4 mAb were added. After 20 min at room temperature, cells were washed with PBS and fixed with PBS, 1% PFA. Cells were analyzed using a FACSCalibur and the CellQuest software (BD Biosciences, San Jose, CA, USA) or FlowJo (Tree Star Inc., Ashland, OR, USA).
Generation of MHC class I tetramers and staining of cells with tetramers
Modified forms of the full-length cDNA of H-2Kb and human ß2-microglobulin were kindly provided by Dirk Busch (Institute for Immunology, Ludwig-Maximilians University, Munich, Germany). H-2KbOVA257264 tetramers were produced as described (11). For flow cytometry analysis, 2 x 106 cells were incubated for 15 min at 4°C with rat IgG antibodies, anti-CD16/CD32 mAb and streptavidin (Molecular Probes Inc.) in PBS, 0.5% BSA, 0.01% sodium azide. Cells were then stained for 60 min at 4°C with Cy5-conjugated anti-CD8
mAb, FITC-conjugated anti-CD62L mAb and PE-conjugated MHC class IOVA257264 tetramers. Subsequently, cells were washed with PBS, 0.5% BSA, 0.01% sodium azide and re-suspended in PBS. Propidium iodide was added prior to four-color flow cytometry analysis.
Carboxyfluorescein diacetate succinimidyl ester labeling and transfer of OT-II cells
TCR transgenic mice specific for the peptide OVA323339 (OT-II) were a kind gift from the Bundesamt für gesundheitlichen Verbraucherschutz und Veterinärmedizin, Berlin. OT-II CD4+ T cells were prepared from spleens and lymph nodes, RBCs were lysed and CD4+ T cells were purified using CD4 (L3T4) microbeads and an AutoMACS column (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Cells were washed twice in PBS, re-suspended at 1 x 107 cells ml1 and labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE;Molecular Probes Inc.) at a final concentration of 2 µM for 4 min at room temperature. The reaction was terminated with an excess of medium containing FCS. Effective CFSE labeling and purity of the OT-II CD4+ T cells was confirmed by FACS staining. A total of 5 x 106 labeled OT-II CD4+ T cells in 200 µl of PBS were injected into the lateral tail vein of mice 1 day prior to LmOVA infection.
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Results
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Elevated bacterial loads in LatY136F mice after LmOVA infection
Young LatY136F mice display a defective T cell development, but accumulate in the periphery large amounts of polyclonal CD4+ Th2 effector cells starting at an age of 45 weeks. These activated T cells are unresponsive to TCR cross-linking in vitro. To assess whether LatY136F mice could generate antigen-specific T cell responses under physiological in vivo conditions and in a highly supportive inflammatory environment, 5-week-old wild-type and LatY136F mice were infected with LmOVA. Mice were sacrificed at 8 days post-infection and the bacterial loads in both spleen and liver were determined. At this time point, control mice had completely cleared the infection, whereas LatY136F mice showed considerable bacterial titers in spleen and liver (Fig. 1). This result suggests that LatY136F mice are not able to clear a L. monocytogenes infection, an immune property that is strictly dependent on Listeria-specific T cell responses.

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Fig. 1. LatY136F mice fail to control LmOVA infection. LatY136F or wild-type littermate control mice were intravenously injected with 5 x 103 LmOVA or PBS. Eight days post-infection, mice were sacrificed and bacterial titers were determined in spleens and livers of three individual mice per group. Titers in LatY136F mice (triangles) or in wild-type littermate controls (squares) are shown as the logarithm of colony-forming units (CFU) per tissue. The broken horizontal line indicates the detection limit. Infection studies were performed two times with similar results.
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Lack of CD4+ T cell responses in LatY136F mice after LmOVA infection
To determine the magnitude of antigen-specific CD4+ T cell responses in wild-type and LatY136F mice 8 days after LmOVA infection, spleen cell suspensions were challenged for 4 h with an MHC class IIb-restricted epitope of LLO190201, which is immunodominant in the C57BL/6 background (H-2b) (12). As expected, the percentage of CD4+ T cells producing IFN-
in response to LLO190201 approximated 0.7% of all CD4+ T cells in spleens of wild-type mice (Fig. 2). In contrast, CD4+ T cells isolated from LatY136F mice showed no response to LLO190201, as documented by the lack of CD4+ T cells producing either IFN-
or IL-4 (Fig. 2). In conclusion, LatY136F mice failed to mount antigen-specific CD4+ T cell responses against LmOVA infection.
Lack of CD8+ T cell responses in LatY136F mice after LmOVA infection
The periphery of LatY136F mice also contains CD8+ T cells, although their frequency is low compared with CD4+ T cells (6). Their activated phenotype resembles that of LatY136F CD4+ T cells; however, they do not contribute to the lymphoproliferative disease observed in LatY136F mice. To determine whether these CD8+ cells were able to respond against LmOVA infection, we monitored the frequencies of wild-type and LatY136F CD8+ T cells that were specific for the immunodominant MHC class I H-2Kbepitope OVA257264. As documented by staining with H-2KbOVA257264 tetramers,
8% of the CD8+ T cells found in infected wild-type littermates were specific for this epitope (Fig. 3). In contrast, the frequency of
CD8+ T cells was not increased in LatY136F mice following LmOVA infection (Fig. 3). In conclusion, LatY136F mice failed to mount antigen-specific CD8+ T cell responses against LmOVA infection.

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Fig. 3. CD8+ T cells from LatY136F mice do not respond to LmOVA infection. LatY136F or wild-type littermate controls were injected intravenously with 5 x 103 LmOVA or with PBS. Eight days post-infection, spleen cell suspensions were prepared and directly stained with anti-CD8 mAb and anti-CD62L mAb to gate on effector CD8+ T cells, and with H-2KbOVA257264 tetramers to identify individual cells that were specific for this immunodominant epitope. (A) Dot plots show CD62L versus H-2KbOVA257264 expression for CD8+-gated cells. (B) Bar diagrams represent the mean ± SD of percentages of tetramer+ CD62L CD8+ T cells among total CD8+ T cells from three individually analyzed mice per group. Analyses of two independent infections were performed and gave similar results.
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LatY136F CD4+ T cells maintain their Th2 phenotype during LmOVA infection
We have shown above that TCR stimulation fails to activate LatY136F T cells in vivo. However, the effector CD4+ T cells from LatY136F mice do not suffer from a general activation defect since they readily produced cytokines upon unspecific stimulation with PMA and ionomycin. We next determined whether LmOVA infection could affect the prevalent Th2 polarization of CD4+ T cells observed in uninfected LatY136 mice. Comparison of CD4+ T cells from mock-infected or LmOVA-infected LatY136F mice illustrates that there were no significant differences in the production of IL-4 and IFN-
4 h after re-stimulation with PMA and ionomycin (Fig. 4). Therefore, although LmOVA infection induces a strong Th1 immune response in wild-type mice (8, 9), it does not shift the strong Th2 polarization displayed by the LatY136F CD4+ T cells towards a Th1 pattern (Fig. 4). This result suggests that the activation program driving the lymphoproliferation of the LatY136F CD4+ T cells as well as the acquisition of a Th2 phenotype is uncoupled from the TCR and likely depends on intrinsic uncoordinated signal transduction.
Wild-type CD4+ T cells adoptively transferred into LatY136F hosts respond normally to LmOVA infection
At 5 weeks of age, LatY136F mice already contained expanding populations of CD4+ T cells that chronically produce an over-abundance of type-2 cytokines (6, 7). To determine whether such a unique environment hampers the induction of Th1-type immune responses, we adoptively transferred CD4+ T cells purified from OT-II TCR transgenic mice into wild-type and LatY136F mutant mice. Both types of adoptively transferred mice were subsequently infected with LmOVA. Because OT-II CD4+ T cells were labeled with CFSE prior to transfer, it allowed us to monitor both their proliferation and cytokine production in response to infection with LmOVA. Four days after infection, we observed a strong proliferation of OT-II T cells transferred into both wild-type and LatY136F hosts (Fig. 5). In contrast, in uninfected control mice no proliferation of OT-II T cells occurred. Importantly, after in vitro stimulation with the OVA323339 peptide, the responding OT-II CD4+ T cells found in both wild-type and LatY136F hosts produced IFN-
but not IL-4 (Fig. 5). Therefore, the environment found in young LatY136F mice is permissive for Th1 responses regardless of an over-abundance of CD4+ T cells that chronically produce type-2 cytokines. This finding supports the view that the Th2 polarization affecting LatY136F CD4+ T cells likely occurred in a cell intrinsic mode.
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Discussion
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In this study we have employed a very sensitive in vivo detection system to analyze the capacity of LatY136F mice to mount antigen-specific T cell responses. Due to a point mutation that replaced the tyrosine 136 of the LAT adaptor protein with phenylalanine, these mice develop a lymphoproliferative disease involving polyclonal CD4+ effector T cells that chronically produce Th2 cytokines. By analyzing their T cell responses to well-defined immunodominant MHC class I and class II epitopes, we show here that T cells in LatY136F mice completely fail to respond to LmOVA infection. Provided that the polyclonal T cell repertoire present in LatY136F mice (6) comprises T cell precursors specific for the analyzed peptides, the TCR-signaling defect previously documented ex vivo in LatY136F T cells also extends to in vivo function. Therefore, the mutation of a single tyrosine residue in the LAT-signaling adaptor completely prevented the induction of antigen-specific CD4+ and CD8+ T cell responses in vivo, even under conditions where intracellular bacterial infection provided coincidentally potent T cell stimuli and a highly supportive inflammatory environment.
Two main conclusions can be drawn from our results. First, the decision of the LatY136F CD4+ T cells to acquire an activated Th2 phenotype appears intrinsically determined. This is suggested by the finding that infection with the intracellular bacterium LmOVA, a strong stimulus for Th1 immune responses (8, 9), did not even minimally shift the Th2 phenotype of the LatY136F CD4+ T cells towards Th1. Such a shift may not have been expected for LatY136F CD4+ effector T cells that had already reached a terminal Th2-differentiation stage. However, the ones generated during the course of LmOVA infection might have acquired a Th1 profile, as occurred for naive CD4+ T cells present in wild-type mice or in adoptively transferred LatY136F mice. In this context, it has to be noted that
30%, but not all LatY136F CD4+ T cells adopted an IL-4-producing Th2 phenotype. Approximately 10% produced IFN-
upon PMA/ionomycin stimulation, and remarkably, roughly 50% of those were able to produce IL-4 simultaneously. However, the inflammation induced during LmOVA infection did not influence these frequencies. Thus, our results allow us to conclude that the conversion of naive CD4+ LatY136F T cells into Th2 effectors is probably not due to chronic stimulation of these cells in the mere absence of extrinsic inflammatory stimuli (e.g. IL-12 and IFN-
), as has been suggested for naive wild-type T cells (1315).
The second conclusion of this study is that the phosphorylation of LAT tyrosine 136 and subsequent recruitment of PLC-
1 is a crucial requirement for the transduction of an activating TCR stimulus in vivo. This is demonstrated by the finding that T cells in LatY136F mice, which lack tyrosine 136, totally failed to respond to immunodominant bacterial antigens during LmOVA infection, although they display an activated effector phenotype. Along this line, it should be emphasized that effector and memory T cells can use non-TCR to secrete cytokines (16, review in 17). Similar mechanisms of autistic T cell activation, characterized by an independence from antigen-derived stimuli, may be also involved in the pathogenesis of human allergic inflammation. Therefore, it will be important to determine the molecular nature of the factors that lead to the proliferation and activation of the pathogenic T cells in the LatY136F model.
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Acknowledgements
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We thank Charles A. Stewart for carefully reviewing the manuscript, Marie Malissen, Sabine Jörg, Anne Gillet, Stéphanie Balor, Thomas Henry and Jean-Pierre Gorvel for advice. This work was supported by CNRS, INSERM, DFG, and by the European Union (MUGEN). I.P. was supported by a fellowship from Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche and by an EIF Marie Curie fellowship from the European Community.
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Abbreviations
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APC | allophycocyanin |
CFSE | carboxyfluorescein diacetate, succinimidyl ester |
LAT | linker for activation of T cells |
LLO | Listeriolysin O |
LmOVA | Listeria monocytogenes-expressing ovalbumin |
PLC- 1 | phospholipase C- 1 |
PMA | phorbol myristate acetate |
TSB | tryptic soy broth |
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Notes
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Transmitting editor: T. Hünig
Received 11 January 2005,
accepted 27 April 2005.
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