Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

The / T lymphocytes use clonally distributed TCR to recognize cell-bound antigens, usually in the form of peptides embedded in MHC molecules. The / TCR is an oligomeric complex containing variable, covalently bound  and  chains responsible for antigen recognition and four noncovalently associated monomorphic subunits, CD3, , , and  chain. The invariant subunits are crucial for efficient assembly of the TCR and, hence, for surface expression (1). In addition, they couple extracellular ligand binding into cytoplasmic signaling machinery and, therefore, form an essential and the most proximal component of TCR signal transduction (2).

Although some of the sequential biogenesis steps of the TCR complex are quite well-characterized, the final complex on the cell surface is surprisingly poorly defined: not only is the overall topology of the complex unknown, but so is even the basic stoichiometry of the TCR, the most commonly proposed structure being TCR₂CD3₂ (1).

Recently resolved three-dimensional structures of ectodomains of TCR  and / chains have now offered some potential insights into the puzzle of the TCR complex topology (3). The most striking feature of the structure of the C domain is the large 14-amino acid long FG loop that protrudes freely into the solvent on the external face of the C domain. It was soon proposed that this loop would interact with CD3 and, therefore, be part of the relay team in TCR signal transduction (3). Recent more detailed structural analyses and simple elegant antibody/epitope mapping of the TCR have added further details and suggested that the loop would form part of the interface between CD3 and the C domain (6, 7).

Here we report our finding that the TCR  chain lacking the complete 14-amino acid FG loop is able to support normal T cell development and function in transgenic mice.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

TCR- Mutagenesis.

Transfection of Cell Lines.

Mice.

Flow Cytometry and Antibodies.

T Cell Functional Assays.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Functional analysis of peripheral T cells from syngeneic control mice (WT), wild-type TCR- (), or mutant TCR- (-loop) transgenic mice. Proliferative response to SEB (A) or SEC 2 (B) superantigens after 48 h of culture. Helper T cell responses (C) assessed 14 d after immunization with NIP-OVA/CFA. Sera from the immunized mice were tested for the presence of anti-NIP IgGs by ELISA on plates coated with NIP-BSA. (D) Allogeneic killer assay. Splenocytes from H-2b control or transgenic mice were stimulated for 5 d with allogeneic H-2d BALB/c cells. Cytotoxic function was then tested on syngeneic H-2b (open symbols) or allogeneic H-2d (filled symbols) Na251CrO4-labeled target LPS blasts.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

Surface Expression of TCR Containing the Mutant, FG Loop- deleted  Chain.

To test whether the deletion of the complete 14-amino acid FG loop in the C domain would be deleterious for the TCR assembly and surface expression, we transfected TCR T cell thymoma 58 with retroviral vectors coding for either a control or a mutant  chain together with a wild-type  chain (10). To our initial surprise, the TCR surface expression was only slightly lower in the mutant case (Fig. 1 A). However, we must point out that the observed 30-50% reduction in the surface expression represents a handicap in the TCR assembly which, although small, is real since we have used a very efficient retroviral transfection system that allows us to create bulk transformants containing thousands of individual clones and which, therefore, provides us with a reliable statistical average. Functional analyses of these transfectomas consistently showed that the cells transfected with the mutant TCR  chain responded slightly less (about threefold) to antigenic stimulation as exemplified here by the dose-response curves to influenza hemagglutinin peptide HA 110-119 (Fig. 1 B) or SEC 3 superantigen (Fig. 1 C).

View larger version (21K): [in this window] [in a new window]   View larger version (11K): [in this window] [in a new window]   Fig. 1.   The mutant TCR  chain is functionally expressed on the cell surface. (A) Cells transfected with TCR  chain together with a control  () or mutant  chain (-loop) were stained with biotinylated anti-CD3 mAb followed by streptavidin-APC. Staining of cells transfected with mutant  chain only is shown as negative control. Numbers represent the mean of fluorescence intensity of CD3 staining. (B and C) Functional response of 105 TCR transfectants (, ; -loop, ) cultured with 105 irradiated antigen-presenting cells (B cell lymphomas A20) and the indicated concentrations of influenza hemagglutinin peptide HA 110-119 (B) or superantigen SEC 3 (C). After 20 h, the culture supernatant was collected and tested for the presence of lymphokines using the IL-2-dependent proliferation assay of HT2 cell lines.

Derivation of TCR  Chain Transgenic Mice.

To more rigorously assess the functional potential of the TCR containing the mutant  chain in normal physiological settings in vivo, we generated transgenic mice expressing either a wild-type or a loop-deleted version of the TCR  chain. The  transgenes, as in the above transfection studies, were derived from 14.3d T cell hybridoma expressing the TCR specific for influenza hemagglutinin peptide HA 110-119 in the context of I-E^d MHC class II molecules (16). In fact, it was the very same  chain (V8.2-J2.1) whose three-dimensional structure was first solved, thus providing us with the inspiration for the current study (3). Two characteristics of the transgenic lines used here were considered essential for straightforward interpretation of the data. First, the level of / TCR expression was identical in both lines (Fig. 2). Presumably the small handicap of the mutant  chain in the TCR assembly could be compensated by higher intracellular expression. Second, both transgenes were bred to TCR-^/ background to avoid any contribution of endogenous  chains for the observed / T cell behavior (11).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   FACScanTM analysis of thymocytes or lymph node cells from mutant TCR  chain transgenic mice. Thymocytes from littermate control (A and B), wild-type TCR- (C and D), or mutant TCR- (E and F) transgenic mice were triple-stained with anti-CD8-Cy5, anti-CD4- PE, and anti-V8 (F23.1-FLUOS) antibodies. Numbers in all dot plots are percentages of total cells. Histograms represent V8 expression of gated single positive CD4+ thymocytes, and numbers show the mean of fluorescence intensity of V8 staining. Lymph node cells from wild-type TCR- (G) or mutant TCR- (H) transgenic mice were stained with anti-C-FITC and anti-CD4-PE.

Mutant TCR  Chain Supports Normal / T Cell Development.

/ T cell development proceeds undisturbed and similarly in both TCR  chain transgenic lines as shown by flow cytometric analysis of thymic and lymph node cells (Fig. 2). Even the skewing into single positive CD4 thymocytes, as noted earlier for our wild-type TCR- transgenic mice (8), occurs to the same extent in both lines. As predicted, mAb H57-597 (anti-C [13]) does not bind to mutant TCR  chain (Fig. 2 H [6]). Interestingly, mAb F23.1 (anti-V8.1, 2, 3 [14]) binds equally well to both  chains, whereas mAb MR5-2 (anti-V8.1, 2 [17]) fails to react with the mutant, suggesting that the FG loop may form part of the MR5-2 epitope (not shown). Since the cellularity of thymi is normal in both cases, we assume that pre-TCR-mediated T cell expansion occurs normally in these mice.

Normal / T Cell Responses in Mutant  Chain Transgenic Mice.

Peripheral T cell responses were measured in several types of assays, and none of them, to our disappointment, showed any significant differences between mice of the different transgenic lines. The in vitro responses to anti-TCR antibodies (not shown) and to SEC 2 and SEB superantigens were repeatedly similar in all mice tested (Fig. 3, A and B). In addition, the in vivo CD4⁺ T cell responses measured by T cell help for hapten-specific IgG production were basically indistinguishable between control and mutant mice (Fig. 3 C). Finally, / T cells from mutant TCR  chain transgenic mice made as vigorous cytotoxic T cell responses against allogeneic targets as their control counterparts (Fig. 3 D). We also monitored the representation of four different V families by flow cytometry in peripheral T cells in order to reveal any subtle in vivo biases, but none were found (Table I). In addition, limited DNA sequence analyses of V 2 and 8 families from single / T cells revealed no obvious "mutant"-specific features (data not shown).

Thus far, we have found only a quantitative role in the TCR assembly process for the large solvent-exposed FG loop on the C domain. In transfectants, the TCR will assemble in the absence of the loop in the  chain but slightly less efficiently compared with the wild-type structure. Of course, the reduced surface expression leads to somewhat impaired function. However, we were able to show in vivo that TCRs are functionally expressed at the same level with or without the FG loop, and we did not find any qualitative or quantitative differences in their activity. This finding seems to rule out the models where the FG loop has an absolute role in TCR signaling. However, the apparent absence of any effect in vivo could also be due to the fact that some subtle compensatory mechanisms have been turned on in vivo (but not in cell lines), e.g., TCR affinities could be modulated, or new carbohydrate structures on the C domain could partially replace the FG loop functionally. Interestingly, all nonmammalian species studied to date, including birds, amphibians, reptiles, and fish, do not have the FG loop on their C domain (18); hence, our in vivo findings may not be that surprising.