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Response of Murine T Cells to the Synthetic Polypeptide Poly-Glu⁵⁰Tyr^{50 1}

Carol T. Cady^*, Michael Lahn^*, Michaelann Vollmer^*, Moriya Tsuji, Seong Jun Seo, Christopher L. Reardon^§, Rebecca L. O’Brien^* and Willi K. Born^2,*

^* Department of Immunology, National Jewish Medical and Research Center, Denver, CO 80206; Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, NY 10010; Department of Dermatology, Chung-Ang University Pil-dong, Chung-ku, Seoul, South Korea; and ^§ Department of Dermatology, University of Colorado Health Sciences Center, Denver, CO 80262

	Abstract

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Random heterocopolymers of glutamic acid and tyrosine (pEY) evoke strong, genetically controlled immune responses in certain mouse strains. We found that pE⁵⁰Y⁵⁰ also stimulated polyclonal proliferation of normal , but not ß, T cells. Proliferation of T cells did not require prior immunization with this Ag nor the presence of ß T cells, but was enhanced by IL-2. The T cell response proceeded in the absence of accessory cells, MHC class II, ß₂-microglobulin, or TAP-1, suggesting that Ag presentation by MHC class I/II molecules and peptide processing are not required. Among normal splenocytes, as with T cell hybridomas, the response was strongest with V1⁺ T cells, and in comparison with related polypeptides, pE⁵⁰Y⁵⁰ provided the strongest stimulus for these cells. TCR gene transfer into a TCR-deficient ß T cell showed that besides the TCR, no other components unique to T cells are needed. Furthermore, interactions between only the T cells and pE⁵⁰Y⁵⁰ were sufficient to bring about the response. Thus, pE⁵⁰Y⁵⁰ elicited a response distinct from those of T cells to processed/presented peptides or superantigens, consistent with a mechanism of Ig-like ligand recognition of T cells. Direct stimulation by ligands resembling pE⁵⁰Y⁵⁰ may thus selectively evoke contributions of T cells to the host response.

	Introduction

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The T cells respond in the course of many diseases and display various functional activities in experimental models of diseases, but the conditions that lead to their activation remain poorly understood. In most of the examples studied, the involvement of the TCR- in these responses has not been established, and even in cases where TCR dependence has been shown, uncertainty about the nature of the putative ligands remains (1).

What has become clear is that these ligands differ from those stimulating the ß T cells. Infections with pathogens or experimental immunization with Ags that readily elicit Ag-specific, MHC-restricted ß T cells do not provoke equally Ag-specific, MHC-restricted T cell responses. This is so despite observations that some T cells recognize MHC gene-encoded class I and II cell surface molecules (2) as well as structurally related molecules such as CD1c (3) and MICA/B (4). Some data indicate that these molecules are recognized by themselves without regard for bound ligands (2, 5), whereas others suggest that, to the contrary, MHC and MHC-like molecules are used merely as anchors for the ligands that the T cells recognize (6, 7). Recognition of the MIC molecules is further complicated by an involvement of NK cell receptors (8).

Although numerous soluble nonpeptidic molecules as well as certain proteins and peptides have been found to stimulate T cells (9, 10, 11, 12, 13, 14, 15, 16), the mechanisms of recognition have largely remained unresolved. In contrast, the response of a murine T cell hybridoma, DGT3, to a soluble peptide Ag composed exclusively of glutamic acid and tyrosine appeared to be based on a mechanism resembling that of TCR-ß ligand recognition (17). Poly-Glu⁵⁰ Tyr⁵⁰ (pE⁵⁰Y⁵⁰;³ formerly often termed polyGT) is a randomly synthesized heterocopolymeric polypeptide composed of glutamic acid and tyrosine, with an average length of 100 aa and a capacity to elicit strong immune responses in certain mouse strains (18, 19). However, hybridoma DGT3 was derived from a low responder DBA/2 mouse immunized with pE⁵⁰Y⁵⁰, and responded specifically to this Ag in a manner restricted by the nonclassical MHC class I molecule Qa-1^b (20). It was later shown that pE⁵⁰Y⁵⁰ bound to Qa-1^b but not to H2-K^k (21, 22), consistent with the proposed mechanism of MHC class I-restricted peptide recognition.

We have now examined the response of murine T cells to pE⁵⁰Y⁵⁰ in more detail. The experiments described here and in a separate study of epidermal T cells⁴ both demonstrate TCR-dependent polyclonal responses of normal murine T cells, but not ß T cells, to this polypeptide. These responses appear to be subset biased but independent of clonal selection, peptide processing and MHC class I/II expression. Our reports therefore describe a distinct type of TCR-dependent response more akin to polyclonal B cell receptor (BCR)-dependent responses and pattern recognition (23, 24) than to conventional Ag responses of T cells.

	Materials and Methods

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Mice

C57BL/10, AKR/J, TCR-ß^-/- (25), and TCR-^-/- mice (26) (both backcrossed onto the C57BL/6 genetic background) were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Mice deficient in ß₂m (27) and MHC class II (28) were purchased from Taconic Farms (Germantown, NY), and TAP-1^-/- mice (29) were obtained from The Jackson Laboratory. Mice deficient in both ß₂m and MHC class II were gifts from Dr. P. Marrack. These mice are of mixed genetic background (mostly C57BL), are also deficient in the invariant chain, and were derived in sequential crosses of mice lacking I-Aß^b (28) and invariant chain (30), and mice deficient in ß₂m (31). All mice used were 6–13 wk old.

Abs and reagents

Abs with specificities for murine CD3 (KT3) (32), TCR-ß (H57-597.2) (33), TCR- (GL3) (34) or (403A10) (35), TCR-V4 (UC3-10A6) (36), and TCR-V1 (2-11) (37) were purified from ascites or cell supernatants and conjugated with N-hydroxysuccinimido-biotin (H-1759, Sigma, St. Louis, MO), allophycocyanin (APC) using the APC conjugation kit, (Prozyme, San Leandro, CA), or FITC isomer on Celite (Sigma F-1628). Streptavidin-R-PE (PE) was purchased from Tago Immunologicals BioSource International (Camarillo, CA). GL3-PE, H57-597-PE, anti-CD44 (mAb IM7)-PE, anti-CD45RB (mAb 23G2)-PE, CD62L (mAb Mel-14)-PE, and streptavidin-CyChrome were purchased from PharMingen (San Diego, CA). The following reagents were purchased from Sigma: Con A (C-5275), poly-Glu-Tyr 1:1 (pE⁵⁰Y⁵⁰; P-0151), poly-Gly-Tyr 4:1 (P-0275), poly-Glu-Phe 4:1 (P-0687), poly-Glu-Leu 4:1 (P-0812), poly-Glu-Ala 6:4 (pEA; P-1650), poly-Glu-Ala-Tyr 6:3:1 (P-1650), and poly(A)sp-Glu 1:1 (P-1408). The polypeptides were dissolved either in dH₂O as 2 mg/ml stock solutions or in dH₂O containing 4% DMSO (see Discussion).

T cell purification

Murine splenocytes were isolated by mechanical dissociation of spleens, lysing the RBC using buffered ammonium chloride, and passing the washed remaining cells over sterile nylon wool coated with 5% FBS (38). The resulting cells were generally >80% CD3⁺ in immunocompetent and TCR-^-/- mice and >70% CD3⁺ in TCR-ß^-/- mice.

Culture medium

Culture medium was made from powdered IMDM dissolved in sterile water according to the instructions of the manufacturer (I-7633, Sigma). This medium was supplemented with sodium pyruvate, sodium bicarbonate, 2-ME, L-glutamine, nonessential amino acids, glucose, penicillin, streptomycin, gentamicin, and 10% FBS.

[³H]Thymidine proliferation assay

T cell-enriched splenocytes were cultured in triplicate at 1 x 10⁵ or 5 x 10⁵ cells/well in 96-well flat-bottom tissue culture plates (Falcon, Becton Dickinson, Franklin Lakes, NJ). The following stimuli were used as noted in the figure legends: medium alone, recombinant murine IL-2 (3.5 U/well), plate-bound anti-CD3 (KT3), Con A at 5 µg/ml, pEY and pEA dissolved in dH₂O in concentrations ranging from 2.5–40 µg/ml, or in dH₂O/4% DMSO, in concentrations ranging from 0.625–10 µg/ml. Cells were incubated for 42, 48, or 50 h at 37° in 10% CO₂. The cells were pulsed for the last 12 h with 0.5 µCi/well [³H]thymidine (NET027, NEN/Life Science, Boston, MA). The incorporated radioactivity was measured using a MicroBeta 1450 counter (Wallac Oy, Turku, Finland).

Flow cytometric analysis

Nylon wool-purified splenocytes were incubated for 15 min at 4°C with the anti-Fc receptor Ab 2.4G2, then stained with the derivatized Abs listed above and in the figure legends. Cells were analyzed cytofluorometrically on either an XL2 (Coulter, Miami, FL) or FACSCalibur (Becton Dickinson Immunocytometry Systems, Franklin Lakes, NJ) cytofluorometer. Forward and side scatter gates were used to exclude dead cells and to focus on cells with a scatter profile typical for lymphocytes.

For cell sorting, NAD splenocytes were stained with the FITC-derivatized anti-CD3 mAb KT3 and sorted on an EPICS Elite cell sorter (Coulter). Based on reanalysis, sorted cell populations were >97% pure.

CFSE proliferation assay

Nylon wool-purified splenocytes were resuspended at a concentration of 1 x 10⁷ cells/ml in balanced salts solution (BSS) containing 0.2 µM CFSE (39). CFSE was stored as a 10-mM stock solution in DMSO at -20°C. The cells were incubated with CFSE for 15 min at 37°C. Unincorporated CFSE was then removed by washing the cells three times with BSS. The washed cells were resuspended at a concentration of 5 x 10⁶ cells/ml in tissue culture medium, plated in 24-well flat-bottom tissue culture plates (5 x 10⁶ cells/well), and incubated for 50 h in medium alone or with the stimuli described above. At the end of the culture, cells were washed with staining buffer (BSS containing 2% FBS and 0.1% sodium azide). The cells were then preincubated with mAb 2.4G2 (Fc blocker) for 15 min at 4°C, washed, and stained with Abs. Unless otherwise stated, either 2-11-biotin or UC3-biotin were added first (20 min at 4°C), and then, after washing the cells, streptavidin-CyChrome and GL3-PE were added (20 min at 4°C). Cells were washed again and analyzed. All CFSE experiments were analyzed on the FACScalibur, gating first for live and TCR-⁺ cells and then on the V1⁺ and V4⁺ subsets. Percentages of cells proliferating were estimated by gating on populations with reduced CFSE fluorescence as indicated in Fig. 3.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Splenic T cells, but not ß T cells, proliferate in response to pE50Y50. A, Nylon wool-purified splenocytes from C57BL/10 mice were labeled with CFSE and cultured for 50 h with IL-2 at 5 x 106 cells/ml of medium alone, with pE50Y50 (40 µg/ml), or with Con A (5 µg/ml). Cells were stained with and gated on anti-TCR- (GL3-biotin/streptavidin-CyChrome) and anti-TCR-ß (H57-PE) Abs. Shown are distributions of CFSE-labeled cells within the gated T cell populations. Cells that have divided exhibit decreased CFSE fluorescence. B, Nylon wool-purified splenocytes from TCR-ß-/- mice were labeled with CFSE and cultured with IL-2 and pE50Y50 as described in A. Cells were stained with anti-TCR- (GL3-PE) and anti-V1 (2-11 biotin/streptavidin-CyChrome) Abs. The CFSE profiles shown are from cells coexpressing TCR- and V1.

Mice were immunized with pE⁵⁰Y⁵⁰, essentially as previously described by others (40). TCR-ß^-/- mice were injected i.p. with 200 µl of saline, 20 µg of pE⁵⁰Y⁵⁰ in saline, 20 µg of pEY in CFA (F-5881, Sigma), or CFA alone. Splenocytes were harvested 10 days after priming, enriched splenic T cells were restimulated in vitro, and their proliferative responses were measured in a [³H]thymidine proliferation assay as described above.

HT-2 stimulation assay

Hybridomas and transfectomas were tested for IL-2 secretion using the HT-2/MTT bioassay (41), as previously described in detail (42). For each cell, wells coated with 10 µg/ml anti-TCR- (mAbs 403-A10 or GL3) were used to confirm the cell’s ability to respond through the TCR. pEY and the other polypeptides were dissolved in dH₂O or dH₂O/4% DMSO at a concentration of 2 mg/ml and used in the assay at the indicated concentrations.

TCR genes and origins of hybridomas, the transfectoma, and the normal cell clones

Origins and expressed TCR genes of most of the hybridomas and T cell clones used in this study have been previously described (43, 44, 45, 46). Transfectoma 58.11 has also been previously described (42). This cell was generated by transfecting the TCR- genes of hybridoma 74BAS-86 (V1/V612) into a TCR gene loss variant of the ß T cell hybridoma DO-11.10. Hybridoma DGT3 was provided by Drs. Z. Korepa and J. Forman (University of Texas Health Science Center, Dallas, TX) with the permission of Dr. D. Vidovic (La Roche, NJ). We further characterized this cell using standard methods as previously described (43) and found that DGT3 expresses V2 and V6.1, with the following junctional amino acid sequences: V2, CAVW ME YSSG; V6.1, CALWE PNIGGIRT T. These sequences have not previously been reported. Throughout this study, V genes are designated according to the nomenclature introduced by Maeda et al. (47).

	Results

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Normal murine T cells proliferate in response to pE⁵⁰Y⁵⁰, with and without ß T cells

To determine whether normal T cells can respond to pE⁵⁰Y⁵⁰, we incubated nylon wool-nonadherent (NAD) splenocytes derived from mice genetically deficient in ß T cells (TCR-ß^-/-) with this polypeptide and measured incorporation of [³H]thymidine in vitro. For comparison, background-matched mice genetically deficient in T cells (TCR-^-/-) and T cell-sufficient mice (C57BL/6) were also tested (Fig. 1A). At 5 x 10⁵ cells/well, TCR-ß^-/- cells proliferated in response to pE⁵⁰Y⁵⁰, whereas TCR-^-/- cells did not. C57BL/6 cells showed a higher background response, but no increase due to pE⁵⁰Y⁵⁰. These data suggested that normal T cells responded to the polypeptide, without requirement for ß T cells. In addition, a repeat of this experiment with cytofluorometrically sorted, CD3⁺ NAD splenocytes indicated that the T cell response was accessory cell independent (Fig. 1B), a finding consistent with our experiments using cloned T cells and hybridomas (shown below).

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Murine splenic T cells proliferate in response to pE50Y50. A, Nylon wool-purified splenocytes (5 x 105 cells/well) prepared from C57BL/6 mice or from congenic mice deficient in ß T cells (TCR-ß-/-) or T cells (TCR--/-) were cultured for 42 h with and without pE50Y50 (40 µg/ml). During the final 12 h, the cells were pulsed with 0.5 µCi of [3H]thymidine. B, Nylon wool-nonadherent, cytofluorometrically sorted CD3+ splenocytes (105 cells/well) from TCR-ß-/- ( T cells) and TCR--/- mice (ß T cells) were cultured with pE50Y50 and IL-2 and pulsed with [3H]thymidine. Shown are the means and SDs of triplicate determinations in representative experiments.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. In vivo priming of TCR-ß-/- mice with pE50Y50 or CFA does not augment the in vitro response to pE50Y50. Mice were primed with pE50Y50 10 days before harvesting the spleen. Nylon wool-purified splenocytes (105 cells/well) were incubated with and without IL-2 and pE50Y50 or pEA (40 µg/ml each) for 48 h and pulsed as described in Fig. 1. Shown are the means and SDs of triplicate determinations.

To test for a possible subset bias in the response of splenic T cells to pE⁵⁰Y⁵⁰, we used the CFSE labeling technique in combination with V-specific mAbs. Large portions of splenic T cells express either V1 or V4 (37), and a small number of cells coexpress V1 and V4. We compared withtal TCR-⁺ splenocytes with those coexpressing either V1 or V4, both V1 and V4, or neither of the two V’s. An experiment analyzing the pE⁵⁰Y⁵⁰-induced proliferative response of V1⁺ TCR-⁺ splenocytes is shown in Fig. 3B. Of all TCR-⁺ V1⁺ cells, approximately one-third underwent at least one cell division upon stimulation with pE⁵⁰Y⁵⁰, 5 times the number that proliferated in the absence of the polypeptide. Fig. 4 summarizes these experiments, including a comparison between C57BL/10 and C57BL/6 mice, both of which have been known to be low responder strains to pE⁵⁰Y⁵⁰ (18). However, in a single experiment with an AKR/J mouse (high responder strain), a similar pattern of T cell proliferation was seen (not shown). Typically, the strongest responses were seen with the V1⁺ splenocytes, including both V1⁺V4^- and V1⁺V4⁺ cells. V4⁺V1^- cells showed variable and usually weaker responses (p < 0.06). TCR-⁺ splenocytes expressing neither V1 nor V4 showed no significant response to pE⁵⁰Y⁵⁰. In experiments with T cell hybridomas, we found among pE⁵⁰Y⁵⁰ responders a similar bias toward V1 expression. However, clones of normal T cells expressing other TCR- (46) also strongly responded to pE⁵⁰Y⁵⁰ (shown below). Differences between normal splenic T cell subsets in responsiveness to pE⁵⁰Y⁵⁰ could potentially reflect different states of prior activation, rather than differences in TCR structure. Therefore, we also compared freshly isolated splenic V1⁺ and V4⁺ cells for their expression of the activation/memory cell surface markers CD44, CD45RB, and CD62L, but there was no indication that V1⁺ populations were more stimulated or contained more memory cells than V4⁺ populations (not shown).

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Among splenic T cells, V1+ cells proliferate most strongly in response to pE50Y50. Nylon wool-purified C57BL/10 and C57BL/6 splenocytes were labeled with CFSE and cultured with IL-2 and pE50Y50 as described in Fig. 3. Cells were then stained with anti-TCR- (GL3-PE) together with either anti-V1 (2-11 biotin/streptavidin-CyChrome) or anti-V4 (UC3 biotin/streptavidin-CyChrome) Abs. In some of these experiments we examined cells that expressed both V1 and V4 by staining with 2-11 APC and UC3 biotin/streptavidin-CyChrome, respectively. The summarized data shown in Fig. 4 are based on CFSE profiles of gated T cell subsets. Mean percent values ± SDs of the fractions of proliferating cells within each subset are shown whenever at least three independent experiments were conducted. Data shown without SDs are based on at least three mice, but fewer than three independent experiments.

Because the subset-selective response to pE⁵⁰Y⁵⁰ was reminiscent of SAg responses of ß T cells, we first tested mice lacking MHC class II expression (I-Aß^b-/-; Fig. 5). In these mice, which also lack I-E expression due to their genetic background (C57BL/6), the response to pE⁵⁰Y⁵⁰ was maintained, indicating that MHC class II expression is not required for the development of pE⁵⁰Y⁵⁰-reactive T cells or for their response to pE⁵⁰Y⁵⁰ itself. Next, we tested mice lacking ß₂m. Here, the response to pE⁵⁰Y⁵⁰ was also maintained, indicating that ß₂m and ß₂m-dependent MHC class I expression are not required for the development of pE⁵⁰Y⁵⁰-reactive T cells. Moreover, T cells from ß₂m-deficient mice were also stimulated by pE⁵⁰Y⁵⁰ when cultured in medium containing serum only from ß₂m-deficient mice, thereby ruling out that serum ß₂m reconstituted the response (not shown). Mice lacking both MHC class II and ß₂m also maintained T cell reactivity with pE⁵⁰Y⁵⁰. Finally, mice deficient in TAP-1, and therefore lacking a major processing pathway for MHC class I-presented peptides (48), showed normal T cell responses to pE⁵⁰Y⁵⁰.

View larger version (26K): [in this window] [in a new window]   FIGURE 5. Genetic deficiencies in I-Aßb resulting in the absence of MHC class II expression, ß2m, or TAP-1 do not abrogate the T cell response to pE50Y50. Nylon wool-purified splenocytes were labeled with CFSE and cultured with IL-2 and pE50Y50 as described in Fig. 4. Cells were stained with anti-TCR- (GL3-PE) and either anti-V1 (2-11 biotin/streptavidin-CyChrome) or anti-V4 (UC3 biotin/streptavidin-CyChrome). The summarized data shown are based on CFSE profiles of gated T cell subsets. Mean percent values ± SDs of the fractions of proliferating cells within each subset are shown, derived as described in Fig. 4.

Responses of T cell hybridomas and clones

The subset bias in the response of normal splenocytes to pE⁵⁰Y⁵⁰ suggested involvement of the TCR-. We therefore examined hybridomas and cloned normal cell lines bearing known TCRs. All were originally derived from antigenically naive mice. Consistent with our finding that prior immunization was not required for the response of freshly isolated cells, certain hybridomas and clones nevertheless responded to pE⁵⁰Y⁵⁰.

The hybridomas were tested for cytokine responses in the absence of accessory cells, using the HT-2/MTT bioassay for IL-2 production. HT-2 cells, the indicator cells in this cytokine assay, by themselves did not respond to pE⁵⁰Y⁵⁰ (Fig. 6). In stimulation cultures with the polypeptide, the dose-response curves obtained with hybridomas was similar to those of the freshly isolated cells (not shown). This supported our initial assessment that accessory cells are not required for the response of T cells to the polypeptide (Fig. 1B) and indicated instead direct interactions between the responders and the polypeptide. Table I shows a survey of hybridoma responses, correlating the tissue origins of these cells, their expressed TCRs, their background cytokine production, their responses to pE⁵⁰Y⁵⁰, and their responses to the T cell mitogen Con A. Overall, the response pattern obtained was similar to that of the normal cells, although differences between V1⁺ and V4⁺ cells were more pronounced. Whereas V1⁺ hybridomas strongly responded to the peptide, V4⁺ hybridomas showed little or no reactivity, and other T cell hybridomas and ß T cell hybridomas were also nonreactive. In contrast to the response pattern observed with pE⁵⁰Y⁵⁰, all T cell hybridomas responded strongly to stimulation with Con A. We also tested a small number of T cell clones (Table II), originally derived from Plasmodium yoelii-immunized mice and described previously in more detail (46). Here, in addition to V1⁺ cells, V7⁺ cells also responded to pE⁵⁰Y⁵⁰, suggesting that additional types of TCR- are compatible with the response to pE⁵⁰Y⁵⁰. Moreover, we observed a response of T cell clones expressing the epidermal TCR- (V5/V1) to this peptide (see Footnote 4) and confirmed the response of DGT3 (Fig. 6), the first-described pE⁵⁰Y⁵⁰-reactive T cell hybridoma (17). As shown above (see Materials and Methods), DGT3 expresses V2. Another hybridoma of our own collection, also expressing V2, did not respond. Thus, it appears that V1 is not absolutely required for the T cell response to pE⁵⁰Y⁵⁰, albeit it perhaps represents the best fit.

View larger version (34K): [in this window] [in a new window]   FIGURE 6. Polypeptide preferences of T cell hybridomas and a TCR- transfectoma, 1 x 105 hybridoma cells (see Table I, Materials and Methods, and references for descriptions of individual cells), as well as the transfectoma 58.11 were incubated without accessory cells in medium alone or with titrated amounts of the polypeptides. Cytokines released were measured and quantitated using the HT-2/MTT bioassay (see Materials and Methods).

View this table: [in this window] [in a new window]   Table I. Responses of T cell hybridomas to pE50Y50 and Con A1

View this table: [in this window] [in a new window]   Table II. Responses of cloned T cells to pE50Y50

To assess the polypeptide specificity of at least V1⁺ T cells, we compared the responses of three V1⁺ hybridomas, 74BAS-86, 69BAS-122, and BNT-19.8.12, all independently derived and expressing slightly different TCRs (44), to pE⁵⁰Y⁵⁰ and similar polypeptides (Fig. 6, A, D, and E). Polypeptides containing glutamic acid and tyrosine at ratios other than 1:1 or containing other amino acids, all elicited smaller responses if compared with pE⁵⁰Y⁵⁰ or no responses at all. A similar response pattern previously reported for hybridoma DGT3 (17) is confirmed here (Fig. 6F), and we also found a similar hierarchy of stimulation with epidermal T cell clones expressing V5 (see Footnote 4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although the synthetic pE⁵Y⁵⁰ peptide mixture may not have biological relevance, the response of T cells to it is of interest for several reasons. First, having found that a fully defined synthetic peptide with alternating glutamic acid and tyrosine residues stimulated the same cells that responded to the random heterocopolymer, we conducted a database search for proteins containing repetitive EY sequences (data not shown). The search revealed several natural proteins containing such repeats, including predicted and actual proteins in bacteria, viruses, mice, and humans. Some of these or their derivatives potentially could stimulate T cell responses. Second, the original study by Vidovic et al. (17) suggested that this response can serve as a simple experimental model for T cell reactivity, and our new experiments support this assessment. Third, the response to pE⁵⁰Y⁵⁰ emphasizes that soluble protein Ags must be considered in addition to nonpeptidic Ags as potential stimulants of polyclonal T cell responses. Moreover, our new data reveal features of the response to pE⁵⁰Y⁵⁰ that are unlike any of the known ß T cell responses to Ags. In fact, the T cell response to pE⁵⁰Y⁵⁰ appears to have more in common with certain oligo- or polyclonal responses of B1 B cells than with conventional Ag responses of T cells (23). We suspect that the reactivity stimulated by the synthetic pE⁵⁰Y⁵⁰ reflects distinct functional properties of T cells that might normally determine their involvement in immune responses in infections and other diseases.

The response to pE⁵⁰Y⁵⁰ was first described with a single T cell hybridoma derived from a pE⁵⁰Y⁵⁰-sensitized mouse (17), and a second hybridoma derived in a similar manner was reported later by the same group (50, 51). The peptide specificity of the cytokine response and a requirement for Qa-1^b expression suggested a clonal, MHC-restricted mechanism of Ag recognition not unlike that used by ß T cells (17, 22). In the current study we have confirmed and extended the original observation to show that normal T cells freshly isolated from the murine spleen proliferate when stimulated with pE⁵⁰Y⁵⁰. However, instead of a clonal response, we found a T cell-selective, polyclonal, subset-biased response, elicited as a primary response in antigenically naive mice and not significantly altered by prior exposure of the mice to pE⁵⁰Y⁵⁰, CFA, or both. Our experiments do not formally exclude possible effects of Ag priming or cross-priming with environmental Ags. Nevertheless, it is noteworthy that T cell hybridomas derived from newborn mouse thymus (e.g., BNT-19.8.12 in this study) responded to the polypeptide as well. Therefore, antigenic priming or cross-priming are probably not essential mechanisms in this response, which, rather, seems to be "hard-wired," i.e., not requiring Ag selection. Secondly, whether using hybridomas, clones, or normal cells, there was no requirement for accessory cells other than the responding cells themselves, again in contrast to conventional peptide responses of T cells. Nevertheless, our experiments indicate that the response to pE⁵⁰Y⁵⁰ is TCR dependent, and proliferation is enhanced by IL-2. Among known Ag responses of ß T cells, only the so-called SAg responses exhibit a similar pattern (52). However, a hallmark of the SAg responses of ß T cells is the necessary involvement of MHC class II molecules, which serve both as presenting molecules for the SAgs and as restriction elements for the responding cells. Therefore, our finding that MHC class II was not required shows that the T cell response to pE⁵⁰Y⁵⁰ differs mechanistically from SAg responses.

There also was no requirement for MHC class I molecules, at least with regard to the majority of pE⁵⁰Y⁵⁰-reactive T cells. Neither was ß₂m-dependent MHC class I expression required for the development of V1⁺ or V4⁺ polypeptide-reactive populations. Since Qa-1^b is expressed in association with ß₂m (20), this implies that cells belonging to these subsets need not learn Qa-1^b recognition to become pE⁵⁰Y⁵⁰ reactive. Moreover, after replacing FBS, a potential source of ß₂m (53), with serum from ß₂m-deficient mice in the stimulation cultures, both subsets still responded to pE⁵⁰Y⁵⁰, indicating that ß₂m-dependent MHC class I was also dispensable during the actual stimulation assay (not shown). In addition, the observation that TAP-1 was not required removes this processing pathway of MHC class I-associated peptides from further consideration. And last, because the response to pE⁵⁰Y⁵⁰ was maintained in the absence of both MHC class II and ß₂m, the possibility that either type of MHC molecule can substitute for the other (as restricting element or merely as anchor) is removed as well. However, these findings do not preclude the possibility that a small part of the response relies on more conventional mechanisms of peptide recognition, and the two earlier described pE⁵⁰Y⁵⁰-reactive hybridomas (DGT3 and CTG3) as well as clone 291-F6 in this study may well differ in this respect. Perhaps this is because, unlike the majority of pE⁵⁰Y⁵⁰-reactive T cells in the spleen, hybridoma DGT3 expresses a TCR composed of V6.1 and V2. We recently confirmed that DGT3 in fact responds more strongly to pE⁵⁰Y⁵⁰ in the presence of accessory cells expressing Qa-1^b, whereas two V1⁺ hybridomas did not exhibit this preference (not shown).

In the responses of normal splenic T cells to pE⁵⁰Y⁵⁰, TCR involvement was suggested by the differential reactivity of V1⁺ and V4⁺ subsets, a bias that could not be ascribed to differences in prior activation, based on predominance of the memory/activation markers CD44, CD45RB, and CD62L (M. Lahn, unpublished observations). We and others have previously reported that splenic ß and T cell populations differ in their states of activation (54, 55), but in this comparison as well, prior activation is not likely to account for the difference in the response to pE⁵⁰Y⁵⁰ because T cell hybridomas representing the two types of T cells behaved like the fresh cells, and the transfer of a TCR- into an ß T cell hybridoma was sufficient to confer the response. All these observations are consistent with a role for the TCR- in the response to pE⁵⁰Y⁵⁰.

This dependence could reflect a requirement for direct peptide recognition via the TCR or a more indirect mechanism. The observations that V1⁺ T cells/clones/hybridomas were stimulated polyclonally and that even T cells and clones with structurally very different TCRs responded to pE⁵⁰Y⁵⁰ would still be consistent with a role for germline-encoded and polyclonally expressed components of the TCR- that are absent in TCR-ß. Indeed, we recently found that pE⁵⁰Y⁵⁰ binds more strongly to TCR- than to TCR-ß (C. Cady, unpublished observations). On the other hand, the observation that T cells expressing many different TCRs all respond to pE⁵⁰Y⁵⁰ appears to reflect a lack of specificity. It must be remembered, however, that pE⁵⁰Y⁵⁰ is a complex synthetic mixture of many different peptides. Furthermore, although normal splenic T cell subsets do not differ substantially in surface levels of expressed TCRs, the epidermal clones express TCRs at much higher levels than the hybridomas (not shown), a circumstance that might explain their response while hybridoma 70BET-49, expressing the same invariant TCR (V5/V1) at much lower levels, was nonresponsive. Last, as with the normal cells, some of the clones potentially express two TCR-. All the V7⁺ clones tested in this study also contained transcripts of productively rearranged V1 genes. However, by staining with mAb 2.11 (specific for V1.1), we did not detect significant levels of V1 on these clones or on an epidermal pE⁵⁰Y⁵⁰-reactive T cell clone (not shown).

We have previously reported that V1⁺ T cell hybridomas respond to certain peptides derived from 60-kDa heat shock proteins (15, 42, 44) and here we show that some of the same cells respond to pE⁵⁰Y⁵⁰. The response to pE⁵⁰Y⁵⁰, however, seems to be stronger, and it includes cells that were not stimulated by the 60-kDa heat shock proteins. Whether the two responses are based on related mechanisms has not yet been resolved.

The polyclonal subset-biased response of murine T cells to pE⁵⁰Y⁵⁰ also resembles other previously described polyclonal T cell responses in mice and humans including those to the Burkitt’s lymphoma Daudi (56), to mycobacterial Ags (9, 14), and to alkylamines (57). All these responses appear to be nonadaptive and mechanistically different from conventional Ag responses. Apart from their TCR dependence, these responses could mostly be based on germline-encoded pattern recognition instead of conventional Ag recognition. The broader subset-biased reactivity appears to be characteristic of T cells and thus may be related to a distinctive functional role of these cells. It has been argued before that T cells may function within the frontlines of host defenses (58), and that they fill a gap between the very rapid reactions of the innate host defenses and the more slowly developing Ag-specific adaptive immune responses (59). The subset-biased reactivity described here and in earlier studies fits well with these proposed early functions, and ß T cells may be excluded from this type of reactivity because of their potential ability to break self tolerance.

Whether the synthetic pE⁵⁰Y⁵⁰ actually mimicks natural ligands for T cells remains to be seen. In the course of our study we also found that the relatively high polypeptide concentrations required to elicit maximal responses with pE⁵⁰Y⁵⁰ (40 µg/ml) could be reduced 8- to 10-fold by dissolving the polypeptide in dH₂O/4% DMSO instead of dH₂O only (not shown). Under these conditions, pE⁵⁰Y⁵⁰ began to stimulate T cell responses at 10-nM concentrations. We speculate that the stimulatory polypeptides mimick ligands naturally occurring at high concentrations, perhaps being part of polyvalent structures such as certain polyanions found in extracellular matrix, glycosaminoglycans, nucleic acids, bacterial cell walls, or even eukaryotic cell walls where polyanionic proteins (e.g., CD43) protrude beyond the glycocalyx (60). Polyclonal T cells responsive to such stimuli may be equivalent to B1 B cells and their natural Abs (23), and could be part of early defensive measures in infection or of immunoregulatory responses in inflammation.

	Acknowledgments

 
We thank Dr. Kemal Aydintug for his help and Anatole Konoval and Xiang Yin for expert technical assistance.

	Footnotes

 
¹ This work was supported by National Institutes of Health Research Grant RO1AI40611 (to W.B.), an Arthritis Foundation Fellowship (to M.L.), National Institutes of Health Clinical Investigator Award AR01890-01 (to C.R.), and National Institutes of Health Grant KO4AI01291 (to R.O.).

² Address correspondence and reprint requests to Dr. Willi Karel Heinrich Born, Department of Immunology, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206.

³ Abbreviations used in this paper: pE⁵⁰Y⁵⁰, poly-Glu⁵⁰Tyr⁵⁰; BCR, B cell receptor, ß₂M, ß₂-microglobulin; APC, allophycocyanin; MTT, (3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); NAD, nylon wool nonadherent; CFSE, 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester; BSS, balanced salts solution; SAg, superantigen.

⁴ S. J. Seo, M. Lahn, C. Cady, M. Vollmer, R. L. O’Brien, W. K. Born, and C. L. Reardon. Activation of murine epidermal V5/V1-TCR⁺ T lymphocytes by Glu-Tyr polypeptides requires co-stimulatory signals. Submitted for publication.

Received for publication December 10, 1999. Accepted for publication May 30, 2000.

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