T cells in mice expressing a transgenic human TCRß chain get positively selected but cannot be activated in the periphery by signaling through TCR

Chandrashekhar Pasare, Paushali Mukherjee, Adrienne Verhoef1, Pratima Bansal, Sanjeev K. Mendiratta, Anna George, Jonathan R. Lamb2, Satyajit Rath and Vineeta Bal

National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110 067, India
1 Department of Biology, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK
2 Respiratory Medicine Unit, University of Edinburgh, Edinburgh EH8 9AG, UK

Correspondence to: V. Bal


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TCR–CD3 complex-mediated signaling is crucial for both developmental selection and antigenic activation of T cells. We report that mice expressing a recombined human TCRß chain (Tg), which have normal development of T cells, mounted very weak responses to immunization with protein antigens as well as the HA307–319 peptide recognized by the human T cell clone HA1.7 from which the transgene is derived. An anti-CD3{epsilon} mAb triggered equivalent proliferation from Tg and non-Tg T cells, but an anti-human TCRß mAb induced proliferation poorly in Tg T cells in contrast to human T cells or HA1.7. In Tg mice, T cells expressing endogenous TCR were CD44high, whereas most transgene-expressing T cells remained CD44low, suggesting that transgene-expressing cells are not activated in the periphery to participate in immune responses. However, anti-human TCRß could induce some activation markers on T cells and cross-linking of the Tg TCR by plate-coated anti-human TCRß efficiently induced T cell proliferation. Human TCRß-mediated Tg T cell activation could be rescued by exogenous IL-2, as well as by the calcium ionophore A23187, but not by phorbol esters. Thus, this human TCRß chain functions efficiently for positive selection of mouse T cells, but not for their peripheral activation, probably because of a lack of oligomerization leading to defects in signaling for calcium flux and IL-2 induction. The data thus suggest an early point of separation of signaling pathways between positive selection and peripheral activation of T cells.

Keywords: signal transduction, repertoire development


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The TCR mediates decision-making signals at many points during T cell development and response. In the thymus, successful TCRß rearrangement is `sensed' in association with pre-T{alpha} chains (13) and the CD3 complex (4) to permit further development. Successful TCR{alpha} chain rearrangement then allows the cells to express functional TCR on the surface, which mediates the positive and negative selection signals that mold the developing repertoire (5): positive selection rescuing from and negative selection inducing programmed cell death. Cells that survive these selective events are exported to the periphery as mature naive T cells.

When mature peripheral T cells respond to peptide–MHC-induced ligation of their TCR and associated co-stimulatory signals, a number of signaling events occur. These include activation of tyrosine kinases like ZAP-70, Lck and Fyn (6), phosphorylation of immunoreceptor-associated tyrosine activation motifs, especially on the CD3{varepsilon} and CD3{zeta} chains (7), and recruitment of phospholipase C{gamma} (8), leading to protein kinase C and Ras activation. At another level calcium influx induces calcineurin leading to the triggering of transcription factors c-Rel and NF-AT essential for IL-2 gene transcription (9). Other pathways triggered include those acting via adapter proteins like Grb2 and Sos, resulting in the sequential activation of Ras, Raf1 and MAP kinases, which ultimately induce transcription factors such as c-Fos and c-Jun (1012). The outcome of these events is proliferation and/or differentiation, as well as secretion of a variety of cytokines. Successful signaling through the TCR can result in activation and proliferation. There are also differences in the signal requirements both for production and maintenance of `naive' and `memory' T cells, suggesting that their states of differentiation are distinct (1316).

In mice transgenic (Tg) for recombined TCR{alpha} and TCRß chains, the majority of the T cells in the peripheral repertoire express only the Tg receptor chains, since the presence of a recombined TCR gene during development allelically excludes the corresponding endogenous chains (17,18). The resultant repertoire restriction is obviously far more severe in TCR{alpha}ß Tg than in mice Tg for TCRß alone, since the Tg TCRß chain usually combines with a large number of endogenous TCR{alpha} chains in the peripheral repertoire (19). In a variety of such TCRß Tg mice, variations in the peripheral repertoires have been reported ranging from severely restricted (20) to relatively normal for recognition of both nominal antigens and alloantigens (21). Repertoire restriction, when observed in such systems, has usually been attributed to the paucity of chain diversity (22).

We have investigated the peripheral T cell repertoire in mice expressing a TCRß transgene derived from a human T cell clone (23), since human TCRß transgenes have been reported to be able to substitute completely for mouse TCRß in thymic development as well as for peripheral alloresponsiveness (23,24). However, we report here that the apparent repertoire restriction seen in these mice is associated with the failure of efficient T cell activation via the human TCRß in the absence of extensive cross-linking, despite normal positive selection occurring in the thymus.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Mice expressing the recombined human TCRß gene from the T cell clone HA1.7 were a generous gift of Dr M. J. Owen (Imperial Cancer Research Fund, London, UK). The transgene was bred from the original H-2bxH-2k background (23) to the CBA/CaJ (H-2k) background over nine generations before use. These Tg mice were maintained as heterozygotes in the Small Animal Facility under conventional environmental conditions at the National Institute of Immunology. For screening of transgene expression in each litter, mice were bled, mononuclear cells were separated on Ficoll-Hypaque density gradients and then stained with anti-human TCRß for flow cytometry. Tg mice had >90% of their T cells staining positively with anti-human TCR-Cß antibody (data not shown). Either non-Tg littermates or ageand gender-matched CBA/CaJ mice were used as controls. Mice were used at 8–12 weeks of age. All mice were bred and used with the approval of the Institutional Animal Ethics Committee.

Reagents
The mouse anti-human TCRß mAb JOVI-1 used recognizes the Tg as well as 50–75% of human peripheral blood T cells (25) and was the generous gift of Dr M. J. Owen. The hamster anti-mouse CD3{varepsilon} (500-A2) mAb was a gift of Dr C. A. Janeway (Yale Medical School, New Haven, CT). Other mAb used were hamster anti-mouse TCRß (H57-597, HB218; ATCC, Rockville, MD) and mouse anti-human CD3{varepsilon} (OKT3, CRL8001; ATCC). All mAb were used as culture supernatants.

Immunization
Mice were immunized in their hind footpads either with 100 µg/mouse of ovalbumin (OVA; Sigma, St Louis, MO) or HA307–319 peptide emulsified in complete Freund's adjuvant (CFA; Difco, Detroit, MI). Seven days after immunization draining inguinal and popliteal lymph nodes were dissected and single-cell suspensions prepared for T cell proliferation assays.

T cell proliferation assays
Triplicate cultures of cells from lymph nodes (3x105/well) in Click's medium (Irvine Scientific, Irvine, CA) containing 10% FCS (Life Technologies, Grand Island, NY) in 96-well flat-bottomed plates (Nunc, Roskilde, Denmark) were stimulated with appropriate antigens in titrating doses. Cultures were incubated at 37°C in a 5% CO2 atmosphere for 96–120 h. [H3]thymidine (0.5 µCi/well; Amersham, Aylesbury, UK) was added for the last 12–16 h of the culture period. Cells were harvested and counted by scintillation spectroscopy (Betaplate; Wallac LKB Pharmacia, Turku, Finland). Results are expressed as c.p.m. ± SE.

When using mAb as stimuli, culture supernatants were added to the wells in titrating doses. Wherever mentioned, recombinant IL-2 (Boehringer Mannheim India, Mumbai, India), phorbol myristate acetate (PMA; Sigma) or the calcium ionophore A23187 (Sigma) were added in culture at indicated concentrations. Coating of mAb onto plates was done where appropriate by immobilizing affinity-purified goat anti-mouse Ig or Protein A (Sigma) at 10 µg/ml in 100 µl volume as a first layer, followed after washing by appropriate mAb in titrating dilutions. Plates were incubated at 37°C for 30 min and washed with sterile PBS before adding cells. Assays with mAb stimulation were of 48–60 h duration.

For experiments involving thymocytes, single-cell suspensions were prepared from thymi and 5x105 cells/well used. For generating secondary T cells, splenocytes (5x106/well in 24-well plates) were incubated with a 1:10 dilution of anti-mouse CD3{varepsilon} mAb culture supernatant for 48 h, washed and rested for 48 h, and were then re-stimulated with titrating doses of soluble or plate-bound anti-human TCRß.

For experiments using human peripheral blood mononuclear cells (PBMC), heparinized blood from healthy volunteer donors was layered onto Ficoll-Paque (Pharmacia, Uppsala, Sweden) for PBMC separation. PBMC were used at 1x105/well. For analyzing responses of T cell clone HA1.7 to anti-human TCRß mAb and anti-CD3{varepsilon} mAb, HA1.7 (1x104 cells/well) was stimulated with titrating amounts of the mAb in presence of irradiated antigen-presenting cells (APC) (1x105/well), cultures were pulsed with [H3]thymidine after 66–72 h, harvested and counted.

Flow cytometry
Tg mice were screened by staining blood mononuclear cells with the transgene-specific mAb JOVI-1 on ice for 45 min, followed after washing by goat anti-mouse Ig–FITC (Jackson ImmunoResearch, West Grove, PA). For some experiments, splenic cells from Tg mice and non-Tg littermates were subjected to preliminary enrichment for T cells by passing them over nylon wool columns and then used for staining. For two-color flow cytometric analysis, cells were stained with either anti-mouse TCRß mAb (followed by anti-hamster Ig–FITC; Jackson ImmunoResearch) or anti-human TCRß mAb (followed by anti-mouse Ig–FITC; Jackson ImmunoResearch) as well as with anti-CD44–phycoerythrin (PE). For examination of T cell activation markers, the cells were either left without any treatment or stimulated with anti-CD3 or anti-TCR mAb for 48 h, separated on Ficoll-Hypaque (Pharmacia) density gradients and stained for CD4 and CD8 versus CD25 or CD44 or CD95. For staining, FITC-labeled mAb against mouse or human CD25 and PE-labeled mAb against mouse and human CD95 and mouse CD44 (all from PharMingen, San Diego, CA) were used along with biotinylated anti-CD4 and anti-CD8 antibodies followed by streptavidin–FITC or streptavidin–PE (Jackson ImmunoResearch) as appropriate. Samples were run on a flow cytometer (Bryte; BioRad, Hemel Hampstead, UK) and analyzed using FlowJo (Treestar, San Jose, CA) software.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The human TCRß transgene expressed in the Tg mice uses Vß3 and is derived from the HA307–319-specific HLA-DR1-restricted human T cell clone, HA1.7 (26,27). The generation and characterization of Tg mice has been reported earlier (23). With increasing age, the proportion of T cells expressing endogenous TCRß goes up in these mice (23 and data not shown).

Human TCRß Tg mice respond poorly to nominal antigens
Lymph node cells from Tg mice or non-Tg littermates immunized with OVA in CFA were challenged with graded doses of OVA or protein extract from Mycobacterium tuberculosis, H37Rv (generously provided by Dr R. S. Kamat, Bai Jerbai Wadia Children's Hospital, Mumbai, India) in T cell proliferation assays in vitro. The results (Fig. 1A and BGo) show that non-Tg mice responded well to OVA as well as to mycobacterial proteins, while the Tg mice showed very poor responses to both. Mice were next immunized with the influenza hemagglutinin peptide, HA307–319 (kindly synthesized by Dr D. M. Salunke, National Institute of Immunology, New Delhi, India) in CFA. Figure 1Go(C) shows that Tg mice showed very poor peptide-specific responses in vitro, while non-Tg littermates responded well, suggesting that the human TCRß chain from HA1.7 was not providing the capability even for mounting an immune response against the HA307–319 peptide.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1. Peripheral T cells from Tg mice respond very poorly to a variety of antigens. T cell proliferative response to titrated doses of OVA (A), mycobacterial protein extract, (H37Rv; B) and HA307–319 peptide (C) from Tg (filled symbols) and non-Tg (open symbols) mice immunized with OVA in CFA (A and B) or with HA307–319 in CFA (C) are shown (mean ± SE). Data are representative of five independent experiments.

 
Signaling through TCRß is deficient in Tg T cells despite normal TCR–CD3 levels
Whether natural exposure to environmental and food antigens results in any activation of Tg T cells in the Tg mice was also analyzed by flow cytometry. Figure 2Go shows that splenic T-enriched cells from non-Tg and Tg mice show comparable levels of staining for TCRß expression (Fig. 2A–CGo) and for CD3 (data not shown). A large proportion of T cells from non-Tg mice show high levels of CD44 (Fig. 2DGo) and almost all cells expressing mouse TCRß in Tg mice also show high levels of CD44 (Fig. 2EGo). In contrast, only a small minority of the human TCRß-expressing Tg T cells show high levels of CD44 (Fig. 2FGo). Thus, it appears that Tg T cells are not easily activated by natural stimuli either.



View larger version (41K):
[in this window]
[in a new window]
 
Fig. 2. Tg-expressing T cells show low levels of CD44. Splenic T-enriched cells from non-Tg (A and D) and Tg (B, C, E and F) were stained for mouse TCRß or human TCRß versus CD44. Mouse TCRß staining is shown in (A) and (B), and human TCRß staining in (C). Appropriate TCR-expressing cells from (A)–(C) were gated and analyzed for CD44 levels as shown in (D–F) respectively. The thin line in each panel shows negative controls, while the thick lines represent stained samples. Data are representative of three independent experiments.

 
Whether Tg T cells can be at all activated by a signal transmitted through the TCR–CD3 complex was the next question analyzed. Figure 3Go(A) shows that anti-human TCRß mAb triggered a high proliferative response from human PBMC and only a background response from non-Tg spleen cells, as expected. However, Tg spleen cells also showed a very weak response despite majority of the peripheral T cells in Tg mice showing the expression of human TCRß (23 and Fig. 2Go). Addition of irradiated human PBMC (1x105/well) to transgenic splenocytes did not enhance the proliferative response (Fig. 3AGo). When T cells were stimulated through CD3 using an anti-CD3{varepsilon} mAb (Fig. 3BGo), the proliferative responses induced by anti-mouse CD3{varepsilon} were if anything somewhat higher in Tg than in non-Tg cells as reported earlier (23). The ability of the anti-human TCR mAb and anti-human CD3 to trigger normal human peripheral T cells and the T cell clone HA1.7 were compared and were found to be similar (Fig. 3CGo), ruling out the possibility that the TCRß chain of the HA1.7 TCR was specifically unable to transduce activating signals. This suggested that there may be a signaling defect specific to the TCR component of Tg T cells.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3. Tg T cells, but not human T cells, respond poorly to signaling by anti-human-TCR mAb. Responses of Tg splenocytes ({circ}), non-Tg splenocytes ({triangleup}), human PBMC ({square}) and Tg splenocytes with irradiated human PBMC ({blacksquare}, only A) to reciprocal log dilutions of anti-human TCR (A) and anti-mouse CD3 mAb (B) are shown. Data are representative of five independent experiments. Responses of either HA1.7 or human PBMC to anti-human TCR and anti-human CD3 mAb (C) are also shown. Data are representative of two independent experiments.

 
Induction of T cell activation markers on Tg T cells by anti-human TCRß
Since addition of anti-human TCRß mAb to Tg T cells brings about only minimal proliferation of T cells, we investigated if it at least induces early activation markers. Splenic cells from Tg or non-Tg mice and human PBMC were stimulated with anti-mouse CD3, anti-human CD3, anti-mouse TCRß and anti-human TCRß antibodies for 48 h, and cells were harvested and stained for early activation markers versus CD4 and CD8 together in a two-color flow cytometric analysis. Cells expressing CD4/CD8 were gated as T cells for analysis of activation markers. Figure 4Go shows that anti-mouse CD3 mAb can up-regulate CD44 expression on Tg T cells well but anti-human TCR mAb does so very weakly (Fig. 4BGo), whereas both anti-mouse CD3 and anti-mouse TCR mAb bring about up-regulation of CD44 (Fig. 4AGo) in non-Tg T cells. Similarly, only anti-mouse CD3 mAb and not anti-human TCR mAb can up-regulate CD95 and CD25 expression on Tg T cells (Fig. 4D and GGo respectively), while anti-mouse CD3 and anti-mouse TCR mAb induce CD95 and CD25 expression in almost all non-Tg T cells (Fig. 4C and FGo), and anti-human CD3 and anti-human TCR mAb on all human T cells (Fig. 4E and HGo). The forward scatter of T cells from the same experiment shows that the size of T cells shows a correlation with the expression of the activation markers. Thus, anti-human TCR mAb brings about only a minor increase in the cell size of Tg T cells but anti-mouse CD3 induces a large increase (Fig. 4JGo), as do the respective anti-CD3 and anti-TCR mAb in non-Tg T cells (Fig. 4IGo) and human T cells (Fig. 4KGo).



View larger version (58K):
[in this window]
[in a new window]
 
Fig. 4. Tg T cells show some marginal up-regulation of CD44, CD95 and CD25 and minimal increase in cell size after stimulation with anti-human TCR mAb. Gated non-Tg (A, C, F and I), Tg (B, D, G and J) and human (E, H and K) CD4+/CD8+ T cells are shown analyzed for levels of CD44 (A and B), CD95 (C–E), CD25 (F–H) and forward scatter (I–K). Activation marker levels on anti-mouse TCR-stimulated (thick line), anti-CD3-stimulated (broken line) and anti-human TCR-stimulated (thin line) T cells are shown. Shaded curves show staining levels on unstimulated T cells in each panel. Data are representative of two independent experiments.

 
Extensive cross-linking of anti-human TCRß rescues T cell proliferation in both primary and secondary Tg T cells
Since the Tg T cells could not be efficiently triggered to express even early activation markers with anti-TCRß mAb added in culture, we attempted to increase the TCR ligation density by using plate-coated antibody. As shown in Fig. 5Go, the anti-human TCRß antibody added in culture stimulated Tg splenocytes very poorly compared to human PBMC (Fig. 5AGo). However, when the same antibody was used in the plate-bound form, the Tg T cells responded very well (Fig. 5BGo). Neither Tg nor non-Tg T cells showed such an enhancement of response to plate-bound antibody if anti-CD3{varepsilon} was used (Fig. 5C and DGo). Since activation requirements of naive and memory T cells are different (1316), we examined the activation of naive and primed Tg T cells. Thymocytes were used as a source of predominantly naive T cells. Thymocytes from Tg or non-Tg mice were stimulated with anti-human TCRß antibody in culture or in the plate-coated form. There was no response to the antibody added in culture, but the Tg T cells responded well to the plate-bound anti-human TCRß (Fig. 6AGo). Non-Tg T cells did not respond, as expected, and there were no differences in the responses to anti-CD3 used either in culture or in plate-bound form (data not shown).



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 5. Anti-TCR mAb can activate Tg T cells after increased cross-linking. T cell responses to reciprocal log dilutions of anti-human TCRß mAb added in culture (A) or coated on plates (B) and to anti-mouse CD3 added in culture (C) or coated on plates (D) are shown for Tg spleen cells ({square}), non-Tg spleen cells ({triangleup}) or human PBMC ({circ}). Data are representative of four independent experiments.

 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6. Naive and primed T cells from Tg mice have similar activation requirements. (A) Response of thymocytes from Tg (filled symbols) and non-Tg (open symbols) thymocytes to anti-human TCRß mAb either added in culture (circles) or in the plate-coated form (squares). (B) Responses of anti-CD3 mAb-stimulated Tg T cells from spleen to anti-human TCRß mAb added in culture ({circ}) or in plate-coated form (•). Data are representative of three independent experiments.

 
Next, all peripheral T cells were converted to secondary cells by activating them through CD3 before analyzing their responses to TCR-mediated signaling. Tg or non-Tg splenocytes were activated by plate-bound anti-CD3 for 48 h, rested in medium for a further 48 h and were then stimulated with anti-human TCRß. Figure 6Go(B) shows that even secondary T cells from the Tg mice could not be activated by anti-human TCRß in culture, while they responded well to plate-coated anti-human TCRß. Thus, the signaling defect in the human TCRß-expressing Tg T cells is related to TCR cross-linking and is not related to the activation status of the T cells.

Exogenous IL-2 and calcium ionophore can overcome the signaling defect in Tg T cells
Non-Tg and Tg splenocytes were stimulated in the presence of anti-mouse and anti-human TCR mAb respectively in the presence or absence of IL-2. Figure 7Go(A) shows that the presence of exogenous IL-2 (10U/ml) can potentiate the anti-human TCR-mediated proliferative response of Tg T cells (3to 10-fold in different experiments), whereas the response of non-Tg splenocytes to anti-mouse TCR is only marginally enhanced. The calcium ionophore A23187 and the phorbol ester PMA were similarly used with appropriate anti-TCR mAb (Fig. 7B and CGo) to stimulate non-Tg or Tg splenocytes. While addition of PMA had no major effect on the human TCR-mediated proliferation of Tg T cells, addition of A23187 resulted in a significant increase in anti-human TCR-mediated response in Tg T cells (Fig. 7BGo), suggesting that TCR-mediated signaling may be defective in calcium flux induction in Tg T cells.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 7. Exogenous IL-2 and calcium ionophore rescue Tg T cells from the block in signaling through human TCRß. Responses of splenocytes from Tg or non-Tg mice to anti-human TCR or anti-mouse TCR mAb respectively with or without 10 U/ml IL-2 (A), 0.1 µM A23187 (B) or 0.1 µM PMA (C) are shown. Responses of splenocytes to IL-2/A23187/PMA alone without anti-TCR mAb (hollow bars), to the respective anti-TCR mAb (hatched bars) and to both together (stippled bars) are shown. Background counts in the absence of any stimulation were <10,000 c.p.m. Data are representative of three independent experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The selection, maturation and development of a diverse repertoire of T cells in the thymus is influenced by peptide-loaded MHC molecules, CD4 or CD8 co-receptors as well as the diversity of the TCR themselves. For mice expressing recombined TCR{alpha} and TCRß chains as transgene, most TCR in the periphery are of transgenic origin (19) and hence the repertoire is restricted to the specificity of the transgene. In contrast, T cells in mice transgenic for a single chain of the TCR have been shown to express the Tg chain with many different complementary endogenous chains. Mice transgenic for a single chain of TCR have been shown to mount alloresponses (24,28), an indication that the transgenic chains participate in positive selection in the thymus. In the mice used in the present study, >90% of peripheral T cells in young mice express the Tg human TCRß chain with a diverse TCR{alpha} usage (23). The numbers of CD4CD8, CD4+CD8+ and CD4+/CD8+ T cells in the Tg thymus are comparable with non-Tg thymus (data not shown and 23). This suggests that all the essential signals required for positive selection are adequately delivered by the species-hybrid TCR. It could therefore be expected that mature peripheral T cells would also respond well to TCR-mediated signals in these mice.

To explore the functional capability of peripheral T cells, mice were immunized with nominal antigens. Tg mice mounted very weak (~10,000-fold less as compared to their non-Tg counterparts) T cell responses (Fig. 1Go), possibly due to the restricted TCR repertoire in these mice because of the presence of a single Tg TCRß chain in spite of its being associated with a variety of TCR{alpha} chains. The Tg mice are on H-2k background and HA1.7, the original T cell clone from which the human TCRß chain has been derived, can recognize its nominal peptide HA307–319, although weakly, on I-Ek (29). It was therefore possible that Tg mice might mount efficient T cell responses to this peptide at the least, even if their T cell repertoire was otherwise limited. However, their response against HA307–319 is also negligible (Fig. 1CGo), suggesting that the T cell defect in these mice may not be due to a limited T cell repertoire. Alloreactivity appears to be an intrinsic feature of the positively selected T cells and can be mediated by the mature T cell repertoire even without immunization. Therefore, a relatively restricted TCR repertoire and a failure of effective immunization (Fig. 1Go) may not compromise alloreactivity. While T cells from these mice have previously been shown to be able to mount alloreactive responses (23), we find that the alloreactive responses mounted by Tg T cells are quantitatively weaker by ~100-fold (data not shown). This apparent discrepancy in the findings using alloresponses with these Tg mice may be due in part to the differences in genetic backgrounds, since earlier (23) the mice were in a mixed 129 (H-2b)xCBA/CaJ (H-2k) background, while we have bred them to homogeneity on the CBA/CaJ (H-2k) background before use. Also, the earlier study does not provide a dose–response titration of the alloresponse and quantitative differences may therefore have been missed. T cells from these Tg mice have been shown to respond well to staphylococcal enterotoxin B (23), known to activate human Vß3-expressing cells (30); the relative efficiency of that activation cannot be estimated, since no comparison can be made with human or non-Tg T cells on a per cell basis. Hence, we decided to look for signaling through CD3 and TCR on these cells using mAb. The original T cell clone, HA1.7 from which the TCRß transgene is derived, responded equally well to anti-human TCR and anti-human CD3 mAb (Fig. 3CGo), but surprisingly, in spite of most of the peripheral T cells expressing the transgene, Tg T cells could mount only a minimal response to anti-human TCR mAb (Fig. 3AGo) despite retaining their ability to respond to anti-CD3 mAb (Fig. 3BGo). This restricted response was not due to the inability of mouse APC to provide adequate accessory signals to the responding T cells because addition of irradiated human APC as bystander cells to these wells did not enhance the response (Fig. 3AGo). These data thus suggest that the presence of this human TCRß chain as part of the mouse TCR complex may be responsible for deficient signaling in peripheral T cells. While these data provide one explanation for the lack of immune responses in the Tg mice, they do not rule out the additional presence of a restricted repertoire as a result of the transgenic TCRß chain. It is also possible that the xenogenic origin of the TCRß chain may be related to this hypothetical repertoire restriction and may be overcome by the provision of a human MHC element. However, since signaling through the TCR appears to be compromised in the Tg T cells, there is no way of examining the repertoire-restricting role, if any, of the transgenic TCRß chain.

Although active immunization failed to elicit a strong immune response, the Tg mice were not found susceptible to infections under normal housing conditions. Hence, we looked for the evidence of environmental antigenic exposure and T cell activation status. Increased surface levels of CD25 and CD44 as well as increase in size have been used as early TCR-mediated T cell activation markers (3133). CD44 up-regulation is one of the features of activated or memory T cells (32,34). Unlike CD44, only recently activated T cells express the high-affinity IL-2 receptor (CD25), but neither naive nor memory T cells do (35). Up-regulation of CD25 is also seen in anergic T cells (36). It has also been shown that, following signaling through the CD3–TCR complex, the size of the responding T cell increases before cell division takes place and in some instances of abortive signaling such an increase in cell size is still seen (37). The induction of Fas (CD95) on T cells upon activation is also well known (38). The cumulative evidence of chronic exposure of T cells to various immunogens in Tg mice was analyzed using CD44 expression on TCRß-expressing cells. While the majority of the human TCRß-expressing Tg T cells continued to show low levels of CD44, most of the endogenous TCRß-expressing T cells in the transgenic mice showed up-regulation of CD44 (Fig. 2Go) suggesting that the Tg-expressing T cells may not be functionally active resulting in expansion and accumulation of the endogenous TCRß-expressing pool with age.

Since human TCRß ligation with mAb stimulated Tg T cells very weakly (Fig. 3Go), we next examined if the defect in signaling seen was restricted to T cell proliferation or also extended to the induction of early activation markers on T cells. Our data show that there is some low-level induction of each of these early activation markers by the anti-human TCRß mAb in Tg T cells (Fig. 4Go), but it is far weaker than that brought about by anti-CD3{varepsilon} mAb or seen in human T cells. So what sort of a signal is given to the Tg T cells by the anti-human TCRß mAb? T cells from Tg mice do not seem to become anergic since they respond well to anti-CD3 after priming with anti-human TCR in vitro (data not shown). Additionally, only endogenous TCR-expressing cells from Tg mice, but not transgene-expressing cells, show high levels of CD44 in vivo (Fig. 2Go) further suggesting that transgene-expressing cells hardly ever get activated in vivo, explaining increasing numbers of endogenous TCRß-expressing cells in Tg mice with increasing age.

Under normal circumstances, anti-TCR or anti-CD3 mAb induce aggregation of CD3–TCR complexes and this oligomerization is thought to be necessary for successful activation (39). Fluid-phase mAb appear to induce this oligomerization by being cross-linked via the Fc receptors on APC (40). Plate-coating of the anti-human TCRß mAb dramatically enhanced its stimulatory capacity for Tg T cells, while this is not true of either the anti-CD3{varepsilon} mAb or for human T cells (Fig. 5Go). This suggests that the defect in signaling via the Tg TCRß chain may be related to a relative failure of oligomerization and that the defect may be localized to the transmission of signal from the TCR{alpha}ß to the CD3 complex (41). Whether this occurs due to altered assembly of the CD3–TCR complex due to conformational constraints imposed by the xenogenic origin of the TCRß chain and whether the specific VDJ portion of this transgene or its species-specific human TCR-Cß region are responsible for such alteration remains to be examined. However, mice expressing a single mouse TCRß chain as a transgene have been reported to be capable of mounting an immune response against the antigenic peptide (21) as well as clearing infections such as lymphocytic choriomeningitis virus (42). Therefore, it is possible that the defect in these human TCRß Tg mice may be imposed by the presence of a xeno-TCR chain. If a relative lack of TCR{alpha}ß-driven oligomerization-related signals is at the root of the defect seen here, it is quite likely that the relatively few peptide–MHC complexes presented by APC during immunization (4345) are insufficient to activate these T cells, leading to a lack of immune responses in vivo (Fig. 1Go). This may help explain the fact that higher densities of activating ligands, such as superantigen–MHC complexes or allo-MHC molecules, are somewhat better at activating these Tg T cells (23 and data not shown). It is thus not surprising that, even though in old Tg mice exposed to environmental stimuli the proportions of T cells bearing endogenous TCRß go up (and is presumably responsible for their normal resistance to infections), practically all Tg-expressing T cells remain CD44low, as would be expected of naive T cells (Fig. 2Go).

Normally, murine splenic T cell populations are a mixture of naive and experienced secondary T cells, and naive T cells are known to require relatively stringent stimuli for complete activation (16). Hence, another question was whether the inability of these Tg T cells to respond to the anti-human TCRß mAb was because all the transgene-expressing peripheral T cells are naive and TCR cross-linking brought about by the antibody is not adequate to trigger them. A comparison between the anti-human TCRß mAb-driven responses of responding thymocytes, which can be considered predominantly naive T cells, versus secondary T cells generated by anti-CD3 stimulation in Fig. 6Go shows that the defect seen here persists in both naive and secondary T cells.

The precise nature of the defect of human TCRß-mediated signaling in Tg T cells is the obvious next issue. Since proliferation is severely compromised, failure of induction of IL-2 was a distinct possibility. Interestingly, IL-2 supplementation rescues the proliferative defect (Fig. 7AGo), suggesting that failure to induce IL-2 may be one of the defects of signaling Tg T cells through human TCRß. IL-2 induction is crucially dependent on TCR clustering (46), raising the possibility that the human TCRß may not allow efficient TCR oligomerization, causing failure of IL-2 induction.

Further analysis of the signals capable of rescuing Tg T cells from the human TCRß-mediated signaling defect showed that the calcium ionophore A23187 could induce such proliferation, while PMA could not (Fig. 7B and CGo). Sustained calcium flux induction in T cells is also thought to be dependent on TCR oligomerization (47,48) and is in turn essential for IL-2 induction (49). On the other hand, activation of the MAP kinase pathway (a major consequence of phorbol esters) is not sufficient by itself for IL-2 induction (50). CD28–CD80/CD86 interactions synergize MAP kinase activation (51) and supplementation with anti-CD28 could not restore proliferative potential to the anti-human TCRß-mediated signal in Tg T cells (data not shown). It is therefore plausible that a failure of TCR oligomerization is responsible for the lack of activation of Tg T cells due to absence of calcium flux and IL-2 induction.

Finally, it has been shown that positive selection is mediated by low-avidity MHC–TCR interactions (52,53), that low-avidity triggering of the TCR fails to generate TCR oligomerization (39,48,54) and that T cell positive selection can occur in the absence of TCR oligomerization (55). It is therefore possible that TCR oligomerization is not required for positive selection but is needed for activation and such a possibility would explain why the Tg human TCRß chain can mediate positive selection but not peripheral activation.

In conclusion, the results presented here demonstrate that Tg T cells expressing a recombined human TCRß chain receive activation signals through the Tg TCRß chain poorly. The defect appears to be related to the level of TCR oligomerization required for peripheral T cell activation to take place, probably lies in the conformation or molecular communication between TCR{alpha}ß and CD3, since CD3-mediated signaling is normal, and is probably at the level of calcium flux responses and IL-2 induction. These defective signals are adequate to mediate positive selection in the thymus but not for activation of peripheral mature T cells, suggesting that further analysis of these T cells may help identify points of divergence in signaling for the two processes.


    Acknowledgments
 
We would like to acknowledge the technical assistance of Mr Inderjit Singh, and the extensive advice of Drs R. K. Anand and R. K. Juyal in maintaining the Tg mice used. This project was partly supported by grants to V. B. from the British Council and the Wellcome Trust, and to J. R. L. from the Wellcome Trust and the Medical Research Council. The National Institute of Immunology is funded by the Department of Biotechnology, Government of India.


    Abbreviations
 
APC antigen-presenting cell
CFA complete Freund's adjuvant
OVA ovalbumin
PBMC peripheral blood mononuclear cell
PE phycoerythrin
PMA phorbol myristate acetate
Tg transgenic

    Notes
 
Transmitting editor: A. J. McMichael

Received 30 January 2000, accepted 28 September 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Groettrup, M., Ungewiss, K., Azogui, O., Palacios, R., Owen, M. J., Hayday, A. C. and von Boehmer, H. 1993. A novel disulfide-linked heterodimer on pre-T cells consists of the T cell receptor beta chain and a 33 kd glycoprotein. Cell 75:283.[ISI][Medline]
  2. Mombaerts, P., Clarke, A. R., Rudnicki, M. A., Iacomini, J., Itohara, S., Lafaille, J. J., Wang, L., Ichikawa, Y., Jaenisch, R. and Hooper, M. L. 1992. Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages. Nature 360:225.[ISI][Medline]
  3. Fehling, H. J., Krotkova, A., Saint-Ruf, C. and von Boehmer, H. 1995. Crucial role of the pre-T-cell receptor alpha gene in development of alpha beta but not gamma delta T cells. Nature 375:795.[ISI][Medline]
  4. Malissen, M., Gillet, A., Ardouin, L., Bouvier, G., Trucy, J., Ferrier, P., Vivier, E. and Malissen, B. 1995. Altered T cell development in mice with a targeted mutation of the CD3- epsilon gene. EMBO J. 14:4641.[Abstract]
  5. von Boehmer, H. 1994 Positive selection of lymphocytes. Cell 76:219.
  6. Weil, R., Cloutier, J. F., Fournel, M. and Veillette, A. 1995. Regulation of Zap-70 by Src family tyrosine protein kinases in an antigen-specific T-cell line. J. Biol. Chem. 270:2791.[Abstract/Free Full Text]
  7. Reth, M. 1989. Antigen receptor tail clue. Nature 338:383.[ISI][Medline]
  8. Imboden, J. B. and Stobo, J. D. 1985. Transmembrane signaling by the T cell antigen receptor. Perturbation of the T3–antigen receptor complex generates inositol phosphates and releases calcium ions from intracellular stores. J. Exp. Med. 161:446.[Abstract]
  9. Weiss, A. and Imboden, J. B. 1987. Cell surface molecules and early events involved in human T lymphocyte activation. Adv. Immunol. 41:1.[ISI][Medline]
  10. Wassarman, D. A., Therrien, M. and Rubin, G. M. 1995. The Ras signaling pathway in Drosophila. Curr. Opin. Genet. Dev. 5:44.[Medline]
  11. Olson M. F., Ashworth, A. and Hall, A. 1995. An essential role for Rho, Rac, and Cdc 42 GTPases in cell cycle progression through G1. Science 269:1270.[ISI][Medline]
  12. Alberola-Ila, J., Takaki, S., Kerner, J. D. and Perlmutter, R. M. 1997. Differential signaling by lymphocyte antigen receptors. Annu. Rev. Immunol. 15:125.[ISI][Medline]
  13. Van de Velde, H., Lorre, K., Bakkus, M., Thielemans, K., Ceuppens, J. L. and de Boer, M. 1993. CD45RO+ memory T cells but not CD45RA+ naive T cells can be efficiently activated by remote co-stimulation with B7. Int. Immunol. 5:1483.[Abstract]
  14. Bradley, L. M., Atkins, G. G. and Swain, S. L. 1992. Long-term CD4+ memory T cells from the spleen lack MEL-14, the lymph node homing receptor. J. Immunol. 148:324.[Abstract/Free Full Text]
  15. Akbar, A. N., Salmon, M. and Janossy, G. 1991. The synergy between naive and memory T cells during activation. Immunol. Today 12:184.[ISI][Medline]
  16. Luqman, M. and Bottomly, K. 1992. Activation requirements for CD4+ T cells differing in CD45R expression. J. Immunol. 149:2300.[Abstract/Free Full Text]
  17. Uematsu, Y., Ryser, S., Dembic, Z., Borgulya, P., Krempenfort, P., Berns, A., von Boehmer, H. and Steinmetz, M. 1988. In transgenic mice the introduced functional T cell receptor ß gene prevents further expression of endogenous ß genes. Cell 52:831.[ISI][Medline]
  18. Bluthmann, H., Kisielow, P., Uematsu, Y., Malissen, M., Krimpenfort, P., Berns, A., von Boehmer, H. and Steinmetz, M. 1988. T cell specific deletion of T cell receptor transgenes allows functional rearrangement of endogenous {alpha} and ß genes. Nature 334:156.[ISI][Medline]
  19. von Boehmer, H. 1990. Developmental biology of T cells in T cell receptor transgenic mice. Annu. Rev. Immunol. 8:531.[ISI][Medline]
  20. O'Brien D. P., Baecher-Allan, C. M., Jr, Burns, R. P., Shastri, N. and Barth, R. K. 1997. Elimination of T-cell-receptor beta chain diversity in transgenic mice restricts antigen-specific but not alloreactive responses. Immunology 91:375.[ISI][Medline]
  21. Perkins D. L., Wang, Y., Fruman, D., Seidman, J. G. and Rimm, I. J. 1991. Immunodominance is altered in T cell receptor (ß-chain) transgenic mice without the generation of a hole in the repertoire. J. Immunol. 146:2960.[Abstract/Free Full Text]
  22. Rimm I. J., Krenger, W., Beland, J. L., Geller, M. C., Di Savino, E., Yui, K., Katsumata, M. and Ferrara, J. L. 1996. TCR-beta transgenic mice fail to mediate a GVHR due to defects of allorecognition and subsequent IL-2 generation. Bone Marrow Transplant. 17:835.[ISI][Medline]
  23. Viney J. L., Prosser, H. M., Palmer, D. B., Lipoldova, M., Lamb, J. R. and Owen, M. J. 1993. Analysis of T cell repertoire and function in mice transgenic for the human Vß3 TCR. Int. Immunol. 5:1541.[Abstract]
  24. Rothe J., Ryser, S., Mueller, U., Steinmetz, M. and Bluethmann, H. 1993. Functional expression of a human TCRß gene in transgenic mice. Int. Immunol. 5:11.[Abstract]
  25. Viney. J. L., Prosser, H. M., Hewitt, C. R. A., Lamb, J. R. and Owen, M. J. 1992. Generation of monoclonal antibodies to a T cell receptor ß chain expressed in transgenic mice. Hybridoma 11:701.[ISI][Medline]
  26. Lamb, J. R., Eckels, D. D., Lake, P., Johnson, A. H., Hartzman, R. J. and Woody, J. N. 1982. Antigen-specific human T lymphocytes clones: induction, antigen specificity, and MHC restriction of influenza virus-immune clones. J. Immunol. 128:233.[Abstract/Free Full Text]
  27. Lamb, J. R., Eckels, D. D., Lake, P., Woody, J. N. and Green, N. 1982. Human T-cell clones recognize chemically synthesized peptides of influenza haemagglutinin. Nature 300:66.[ISI][Medline]
  28. Listman J. A., Rimm, I. J., Wang, Y., Geller, M. C., Tang, J. C., Ho, S., Finn, P. W. and Perrkins, D. L. 1996. Plasticity of the T cell receptor repertoire in TCR beta-chain transgenic mice. Cell. Immunol. 167:44.[ISI][Medline]
  29. Lechler, R. I., Bal, V., Rothbard, J. B., Germain, R. N., Sekaly, R., Long, E. O. and Lamb, J. 1988. Structural and functional studies of HLA-DR restricted antigen recognition by human helper T lymphocyte clones by using transfected murine cell lines. J. Immunol. 141:3003.[Abstract/Free Full Text]
  30. Herman, A., Kappler, J. W., Marrack, P. and Pullen, A. M. 1991. Superantigens: mechanisms of T cell stimulation and role in immune responses. Annu. Rev. Immunol. 9:745.[ISI][Medline]
  31. Schuh, K., Twardzik, T., Kneitz, B., Heyer, J., Schimpl, A. and Serfling, E. 1998. The interleukin 2 receptor alpha chain/CD25 promoter is a target for nuclear factor of activated T cells. J. Exp. Med. 188:1369.[Abstract/Free Full Text]
  32. Budd, R. C., Cerottini, J. C., Horvath, C., Bron, C., Pedrazzini, T., Howe, R. C. and MacDonald, H. R. 1987. Distinction of virgin and memory T lymphocytes. Stable acquisition of the Pgp-1 glycoprotein concomitant with antigenic stimulation. J. Immunol. 138:3120.[Abstract/Free Full Text]
  33. Ernst, D. N., Weigle, W. O., McQuitty, D. N., Rothermel, A. L. and Hobbs, M. V. 1989. Stimulation of murine T cell subsets with anti-CD3 antibody. Age-related defects in the expression of early activation molecules. J. Immunol. 142:1413.[Abstract/Free Full Text]
  34. MacDonald, H. R., Budd, R. C. and Cerottini, J. C. 1990. Pgp-1 (Ly 24) as a marker of murine memory T lymphocytes. Curr. Top. Microbiol. Immunol. 159:97.[ISI][Medline]
  35. Nelson, B. H. and Willerford, D. M. 1998. Biology of the interleukin-2 receptor. Adv. Immunol. 70:1.[ISI][Medline]
  36. Ramensee H. G., Kroschewski, R. and Frangoulis, B. 1989. Clonal anergy induced in mature V beta 6+ T lymphocytes on immunizing Mls-1b mice with Mls-1a expressing cells. Nature 339:541.[ISI][Medline]
  37. Chai, J. G. and Lechler, R. I. 1997. Immobilized anti-CD3 mAb induces anergy in murine naive and memory CD4+ T cells in vitro. Int. Immunol. 9:935.[Abstract]
  38. Nagata, S. 1994. Fas and Fas ligand: a death factor and its receptor. Adv. Immunol. 57:129.[ISI][Medline]
  39. Reich Z., Boniface, J. J., Lyons D. S., Borochov, N., Wachtel, E. J. and Davis, M. M. 1997. Ligand-specific oligomerization of T-cell receptor molecules. Nature 387:617.[ISI][Medline]
  40. Looney, R. J. and Abraham, G. N. 1984. The Fc portion of intact IgG blocks stimulation of human PBMC by anti-T3. J. Immunol. 133:154.[Abstract/Free Full Text]
  41. Samelson L. E., Patel, M. D., Weissman, A. M., Harford, J. B. and Klausner, R. D. 1986. Antigen activation of murine T cells induces tyrosine phosphorylation of a polypeptide associated with the T cell antigen receptor. Cell 46:1083.[ISI][Medline]
  42. Brandle, D., Brduscha-Riem, K., Hayday, A. C., Owen, M. J., Hengartner, H. and Pircher, H. 1995. T cell development and repertoire of mice expressing a single T cell receptor alpha chain. Eur. J. Immunol. 25:2650.[ISI][Medline]
  43. Germain, R. N. 1994. MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell 76:287.[ISI][Medline]
  44. Demotz, S., Grey, H. M. and Sette, A. 1990. The minimal number of class II MHC–antigen complexes needed for T cell activation. Science 249:1028.[ISI][Medline]
  45. Harding, C. V. and Unanue, E. R. 1990. Quantitation of antigen-presenting cell MHC class II/peptide complexes necessary for T-cell stimulation. Nature 346:574.[ISI][Medline]
  46. Holsinger, L. J., Graef, I. A., Swat, W., Chi, T., Bautista, D. M., Davidson, L., Lewis, R. S., Alt, F. W. and Crabtree, G. R. 1998. Defects in actin-cap formation in Vav-deficient mice implicate an actin requirement for lymphocyte signal transduction. Curr. Biol. 8:563.[ISI][Medline]
  47. Hashemi, B. B., Slattery, J. P., Holowka, D. and Baird, B. 1996. Sustained T cell receptor-mediated Ca2+ responses rely on dynamic engagement of receptors. J. Immunol. 156:3660.[Abstract]
  48. Boniface, J. J., Rabinowitz, J. D., Wulfing, C., Hampl, J., Reich, Z., Altman, J. D., Kantor, R. M., Beeson, C., McConnell, H. M. and Davis, M. M. 1998. Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands. Immunity 9:459.[ISI][Medline]
  49. Timmerman, L. A., Clipstone, N. A., Ho, S. N., Northrop, J. P. and Crabtree, G. R. 1996. Rapid shuttling of NF-AT in discrimination of Ca2+ signals and immunosuppression. Nature 383:837.[ISI][Medline]
  50. Utting, O., Teh, S. J. and Teh, H. S. 2000. A population of in vivo anergized T cells with a lower activation threshold for the induction of CD25 exhibit differential requirements in mobilization of intracellular calcium and mitogen-activated protein kinase activation. J. Immunol. 164:2881.[Abstract/Free Full Text]
  51. Zhang, J., Salojin, K. V., Gao, J.-X., Cameron, M. J., Bergerot, I. and Delovitch T. L. 1999. p38 mitogen-activated protein kinase mediates signal integration of TCR/CD28 costimulation in primary murine T cells. J. Immunol. 162:3819.[Abstract/Free Full Text]
  52. Suzuki, H., Guinter, T. I., Koyasu, S. and Singer, A. 1998. Positive selection of CD4+ T cells by TCR-specific antibodies requires low valency TCR cross-linking: implications for repertoire selection in the thymus. Eur. J. Immunol. 28:3252.[ISI][Medline]
  53. Liu, C. P., Crawford, F., Marrack, P. and Kappler, J. 1998. T cell positive selection by a high density, low affinity ligand. Proc. Natl Acad. Sci. USA 95:4522.[Abstract/Free Full Text]
  54. Germain, R. N. 1997. T-cell signaling: the importance of receptor clustering. Curr. Biol. 7:R640.[ISI][Medline]
  55. Takahama, Y., Suzuki, H., Katz, K. S., Grusby, M. J. and Singer, A. 1994. Positive selection of CD4+ T cells by TCR ligation without aggregation even in the absence of MHC. Nature 371:67.[ISI][Medline]




This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Request Permissions
Google Scholar
Articles by Pasare, C.
Articles by Bal, V.
PubMed
PubMed Citation
Articles by Pasare, C.
Articles by Bal, V.