Immunohistochemical detection of increased levels of protein-associated nitrotyrosine has become widely used as a surrogate marker of in situ inflammation. However, the potential consequences of protein-associated nitrotyrosine formation in terms of cellular immune recognition has received surprisingly little attention. Using a well-defined I-EK-restricted epitope of pigeon cytochrome c, we previously demonstrated that conversion of a single tyrosine residue to nitrotyrosine can have a profound effect on recognition by CD4 T cells. In this study, we used the MHC class I-restricted epitope of lymphocytic choriomeningitis virus glycoprotein (gp33) to demonstrate that conversion of tyrosine to nitrotyrosine can also profoundly affect recognition of MHC class I-restricted epitopes. Conversion of the Y4 residue of the gp33 epitope to nitrotyrosine completely abrogated recognition by gp33-specific T cells from P14 TCR-transgenic mice. In contrast, CD8+ T cells specific for “nitrated gp33” (NY-gp33) can be readily elicited in C57BL/6 mice after immunization with NY-gp33 peptide. Interestingly, T-T hybridomas specific for NY-gp33 peptide were found to fall into two distinct subsets, being specific for NY-gp33 presented in the context of either H-2Db or H-2Kb. This latter result is surprising in light of previous structural studies showing that Y4 comprises a critical TCR-contact residue when presented by H-2Db but that the same residue points downward into the peptide-binding groove of the MHC when presented by H-2Kb. Together, these results indicate that nitrotyrosine formation can impact T cell recognition both directly, through alteration of TCR-contact residues, or indirectly, through alterations in MHC-contact positions.
Immunohistochemical detection of the amino acid analog 3′-nitrotyrosine has recently become widely used as a hallmark of in situ inflammation (1). Although several putative pathways for the conversion of tyrosine to 3′-nitrotyrosine have been reported (2), up-regulation of NO synthase (NOS)4 at sites of inflammation followed by formation of the nitrating agent peroxynitrite (ONOO−) is thought to be one of the key primary pathways leading to production of 3′-nitrotyrosine (3). Whereas NOS up-regulation is usually transient and thus sometimes difficult to capture using conventional immunohistological approaches, 3′-nitrotyrosine is relatively stable once formed and thus constitutes a useful surrogate marker or “footprint” of inflammatory activity (4). The presence of 3′-nitrotyrosine and 3′-nitrotyrosine-modified proteins (protein-associated nitrotyrosine (NYP)) has been reported in a wide spectrum of diseases with an inflammatory component including atherosclerosis (5, 6), respiratory disease (7), transplant rejection (8), multiple sclerosis (9), Alzheimer’s disease (10), Parkinson’s disease (11), celiac disease (12), arthritis (13, 14), ischemia-reperfusion injury (15), autoimmune diabetes (16), autoimmune uveitis (17) as well as various cancer and infectious disease settings (18). Despite the fact that the formation of NYP is widely recognized as a surrogate marker of inflammatory processes, the immunological consequence of NYP accumulation has received surprisingly little attention. Recently, however, there has been increasing evidence regarding the potential immunogenicity of proteins containing a tyrosine-to-nitrotyrosine conversion (19). For example, anti-nitrotyrosine Abs have been identified in the synovial fluid of patients with arthritis (20) and in the serum of patients with systemic lupus erythematosus (20, 21) or acute lung injury (22). Previously, we have used an MHC class II-restricted model epitope system (the IEK-restricted epitope of pigeon cytochrome c (PCC)) to investigate whether conversion of tyrosine to 3′-nitrotyrosine can have an impact upon epitope recognition by CD4+ T cells (23). We observed that T cells specific for the conventional (unmodified) PCC epitope were completely unresponsive to stimulation with a synthetic peptide containing a tyrosine-to-3′-nitrotyrosine conversion. Conversely, T cells specific for nitrated PCC could be elicited after immunization with a synthetic PCC peptide containing a tyrosine-to-3′-nitrotyrosine conversion. T-T hybridomas specific for nitrated PCC peptide showed exquisite specificity as they were completely unresponsive to unmodified peptide. Most importantly, we observed that PCC-transgenic mice, which are normally tolerant toward immunization with PCC (24), exhibited a robust immune response after immunization with nitrated-PCC peptide (23). These results imply that T cells bearing receptors capable of “recognizing” nitrated “self” peptides are not subject to the constraints of negative selection in the thymus (central tolerance) and that NYP formed during periods of inflammation may therefore constitute an important class of “neoautoantigens” that may play a role in inflammation and or autoimmune disease. Similar findings were recently reported for another MHC class II-restricted model Ag, hen egg lysozyme, that was treated to contain the oxidative modifications nitro-tryptophan or nitrotyrosine (25).
In the present report, we demonstrate that specific recognition of nitrated epitopes by T cells is not restricted to MHC class II epitopes, but that conversion of tyrosine to nitrotyrosine can also profoundly affect recognition of MHC class I-restricted epitopes. To demonstrate this we used a well-characterized MHC class I-restricted epitope derived from the glycoprotein (gp33–41, referred to hereafter as gp33) of lymphocytic choriomeningitis virus (26). The gp33 epitope (KAVYNFATC) contains a single tyrosine residue (Y4) that has been shown to comprise an important TCR-contact residue when presented in the context of H-2Db (27, 28, 29). In this study, we demonstrate that conversion of the Y4 residue of the gp33 epitope to 3-nitrotyrosine completely abrogates recognition by H-2Db-restricted, gp33-specific T cells from P14 TCR-transgenic mice. Moreover, we demonstrate that T cells specific for “nitrated gp33” can be readily raised after immunization with nitrated gp33 peptide. In addition, we developed a panel of T-T hybridomas against the nitrated gp33 epitope and found that whereas some hybridomas were specific for nitrated gp33 presented in the context of H-2Db, others were specific for nitrated gp33 presented in the context of H-2Kb. This latter result was particularly surprising in light of previous structural studies showing that Y4 comprises a critical TCR-contact residue when presented by H-2Db but that the same residue points downward into the peptide-binding groove of the MHC when presented by H-2Kb and indeed comprises a secondary anchor residue in this context (27). Together, these results indicate that conversion of tyrosine to 3′-nitrotyrosine can have an impact on T cell recognition either directly, through a TCR contact, or indirectly through an altered MHC contact.
Materials and Methods
Wild-type C57BL/6 mice were obtained from The Jackson Laboratory. P14 TCR-transgenic mice (30) were a gift of Dr. B. Nelson (British Columbia Cancer Agency, Victoria, British Columbia, Canada). All animals were used at 8–20 wk of age. All animal studies were reviewed and approved by the Animal Care Committee at the University of Victoria.
Human IL-2 was provided by Dr. C. Reynolds (National Cancer Institute, Bethesda, MD). IL-2 production was assessed by either IL-2 capture ELISA (BD Pharmingen) or by bioassay using the IL-2-dependent cell line CTLL-2 (31). Abs for FACS (PE-CD8, FITC-H-2Db, FITC-H-2Kb, FITC-TCR Vβ panel) were purchased from BD Pharmingen. RMAS cells were a gift of Dr. S. Sad (National Research Council, Ottawa, Canada). The CD8-expressing T-T cell hybridoma partner BWLyt2-4 has been described previously (32) and was a gift of Dr. P. Marrack (National Jewish Medical Center, Denver, CO). HEK-293 cells expressing H-2Db or H-2Kb were a gift of Dr. J. Yang (National Cancer Institute, Bethesda, MD). Peptides gp33 (KAVYNFATM/C), short gp33 (AVYNFATC), NY-gp33 (KAV(nY)NFATM/C), and short NY-gp33 (AV(nY)NFATC) were synthesized by Sigma-Genosys. All synthetic peptides were shown by mass spectrometry analysis to be >80% pure and any contaminants were comprised predominantly of truncated peptides arising during synthesis. Where indicated, gp33 peptide was chemically modified by diluting peptide (1 mg/ml in H2O) 1:1 with 100 mM KPO4 (pH 7.8) plus 10 mM ONOO− or degraded ONOO− (Upstate Biotechnology) before use in assay.
IFN-γ ELISPOT analyses
C57BL/6 mice were immunized s.c. with 100 μg of peptide (gp33 or NY-gp33) mixed with IFA. Seven to 10 days after immunization, mice were euthanized and spleens were excised. Single-cell splenocyte suspensions were prepared in 10 ml of cRPMI (RPMI 1640, 10% FCS, 2 mM l-glutamine, 50 μM 2-ME, 10 mM HEPES, 10 mM MEM nonessential amino acids, 10 mM sodium pyruvate, and 50 μg/ml gentamicin) by mashing spleens through a 70-μM filter using the plunger from a 5-ml syringe followed by RBC depletion by ACK lysis. Splenocytes (3 × 105/well) were plated onto ELISPOT plates (Millipore MSIP; precoated overnight with 10 μg/ml anti-IFN-γ capture Ab (AN18; Mabtech) and then blocked for 2 h at 37°C with cRPMI) in triplicate in the absence of any stimulus, or in the presence of 10 μg/ml gp33, NY-gp33, or control SIINFEKL peptide. After overnight incubation at 37°C, ELISPOT plates were washed and incubated for 2 h at 37°C with 1 μg/ml biotinylated anti-mouse IFN-γ (mAb R4-6A2; Mabtech) followed by development with the Vectastain ABC Elite kit and Vectastain AEC substrate reagent according to the manufacturer’s instructions (Vector Laboratories). Spots were quantitated using a Zeiss automated ELISPOT reader and are reported as the number of spot-forming cells (SFC) per 106 splenocytes. The same protocol was used for ELISPOT analysis of short-term T cell lines (stimulated in vitro for 1 wk with the immunizing or corresponding opposite nitrated or non-nitrated peptide), except that responding cells (1 × 104 cells/well) were added to naive splenocytes (3 × 105 cells/well).
P14 T cell assays
Single-cell suspensions of P14 mouse splenocytes were prepared and RBC were removed by ACK lysis as described above. P14 splenocytes were incubated in triplicate in 96-well plates (1 × 105 cells/well) in the absence of any stimulus, or in the presence of varying amounts of the indicated peptide. After 72 h of incubation at 37°C, 100 μl of supernatant was harvested and levels of secreted IL-2 were determined by CTLL-2 proliferation assay.
Cell lines and T-T hybridomas
T cell lines were generated using splenocytes from mice immunized with gp33 or NY-gp33 peptides as described above. Briefly, splenocytes from immunized mice were incubated with peptide (10 μg/ml) plus IL-2 (10 U/ml) for 7 days. Cells were subsequently restimulated on a weekly basis with irradiated (3500 rad) syngeneic splenocytes that had been pulsed with gp33 or NY-gp33 peptide, as indicated, and subsequently washed to remove excess free peptide. After four sequential rounds of restimulation, cells were >95% CD8+ and were fused to the CD8-expressing T hybridoma fusion partner, BwLyt2-4, as previously described (32). T-T hybridomas were cloned by limiting dilution and were selected for the ability to secrete IL-2 in response to growth overnight in the presence of NY-gp33 peptide (10 μg/ml). TCR Vβ usage by T-T hybridomas was assessed by flow cytometry using a TCR Vβ screening panel (BD Pharmingen). MHC restriction of T-T hybridomas was assessed by stimulation in the presence of H-2Db- or H-2Kb-expressing HEK-293 cells that had been pulsed with gp33 or NY-gp33 peptides as indicated.
To test whether synthetic gp33 peptides containing a tyrosine-to-nitrotyrosine conversion at position Y4 maintained their ability to bind MHC class I molecules, we used an RMAS class I stabilization assay (33). As can be seen in Fig. 1, both gp33 peptide and modified gp33 peptide containing a tyrosine-to-nitrotyrosine conversion at position Y4 (NY-gp33) rescued the expression of H-2Db on the surface of RMAS cells. In addition, the gp33 epitope is unique in that it can bind to both H-2Db and H-2Kb molecules (26). Because previous structural studies have shown that amino acid Y4 comprises a secondary MHC-contact residue when presented in the context of H-2Kb (27), we anticipated that substitution of Y4 with a nitrotyrosine might disrupt the interaction between gp33 peptide and MHC. However, we observed that NY-gp33 peptide was also able to rescue the surface expression of H-2Kb on RMAS cells, albeit with what appeared to be a lower affinity.
Next, we determined whether conversion of amino acid Y4 to a nitrotyrosine might have any impact on recognition by gp33-specific T cells. For this we used splenocytes from P14 TCR-transgenic mice (expressing a transgenic TCR specific for the gp33 presented in the context of H-2Db). Splenocytes obtained from P14 TCR-transgenic mice produced IL-2 in a dose-dependent manner when stimulated with non-modified gp33 peptide, but were completely unresponsive to stimulation with the NY-gp33 peptide (Fig. 2 A).
Because lack of recognition of NY-gp33 by P14 T cells constitutes a negative result and to confirm that T cells were indeed capable of “seeing” NY-gp33 peptide, we immunized C57BL/6 mice with either gp33 or NY-gp33 peptide and then 7 days later assessed the immune response against each peptide by IFN-γ ELISPOT. As can be seen in Fig. 2 B, mice that had been immunized with gp33 or NY-gp33 peptides mounted a T cell response that was highly biased toward recognition of the immunizing peptide. This finding confirms that 1) T cells are capable of recognizing the nitrated gp33 peptide and 2) suggests that the majority of the Ag-specific T cells elicited by immunization recognize non-nitrated and nitrated gp33 peptides in a mutually exclusive manner. Interestingly, however, the mutually exclusive pattern of peptide recognition was not absolute as NY-gp33 peptide elicited a small number of IFN-γ-secreting cells from gp33-immunized mice and likewise gp33 peptide elicited a small number of IFN-γ-secreting cells from NY-gp33 immunized mice. Neither peptide elicited spot formation from splenocytes of naive (unimmunized) mice.
The ELISPOT results described above suggested that T cells capable of specifically recognizing nitrated gp33 peptide vs non-nitrated gp33 peptide could be readily raised after immunization with NY-gp33 peptide. To confirm this finding, we established short-term T cell lines from mice immunized with NY-gp33 peptide and used these lines to generate a panel of T-T hybridomas by fusing with the CD8-expressing fusion partner BWLyt2-4 (32). Two representative hybridomas derived from these fusions (4C8 and 24H1) were chosen for further analysis, based upon their specific recognition of NY-gp33 and their expression of distinct TCR Vβ chains (see below). As can be seen in Fig. 3, A, D, and E, T-T hybridomas 4C8 and 24H1 responded specifically to NY-gp33 peptide by producing IL-2 in a dose-dependent manner whereas neither hybridoma responded to standard (unmodified) gp33 peptide at any of the concentrations tested.
To ensure that T-T hybridomas were clonal in nature, we then determined the TCR Vβ expression pattern of 4C8 and 24H1 by FACS using a panel of TCR Vβ Abs. Although each hybridoma was found to express a single TCR Vβ chain (indicative of clonality), 4C8 and 24H1 each expressed a different TCR Vβ chain suggesting that recognition of NY-gp33 was not restricted to a specific TCR clonotype (see Table I). To further characterize these NY-gp33-specific T-T hybridomas, we used H-2Db and H-2Kb-expressing HEK-293 cell lines as APC to determine the pattern of H-2 restriction. We found that T-T hybridomas fell into two distinct subsets that recognized NY-gp33 presented in the context of either H-2Db or H-2Kb, but not both. Hybridomas expressing TCR Vβ 5.1 (4C8 is representative of this class) recognize NY-gp33 in the context of H-2Db whereas hybridomas expressing TCR Vβ 8.3 (24H1 is representative of this class) recognize NY-gp33 in the context of H-2Kb (see Fig. 3, B and C). Specific recognition of NY-gp33 in the context of H-2Db was not particularly surprising because amino acid Y4 has previously been shown to protrude up and out of the peptide-binding cleft when presented in the context of H-2Db and is thus expected to comprise an important TCR-contact residue (27, 29). However, the specific recognition of NY-gp33 in the context of H-2Kb was highly surprising considering that previous structural studies have shown that Y4 is oriented down into the peptide-binding cleft and constitutes a secondary MHC-contact residue when presented in the context of H-2Kb (27). Thus, to confirm our findings we used a second approach, taking advantage of a previous study showing that the full-length gp33 peptide (KAVYNFATC) can bind both H-2Db and H-2Kb, whereas the truncated peptide (AVYNFATC) can bind only to H-2Kb (26). The T-T hybridoma 4C8 responded to stimulation with the KAV(nY)NFATC but not the AV(nY)NFATC peptide confirming that it is restricted to H-2Db (Fig. 3,D). In contrast, the 24H1 hybridoma responded to stimulation with both the KAV(nY)NFATC and the AV(nY)NFATC peptides, confirming that it is restricted to H-2Kb (Fig. 3,E). Lastly, both the 4C8 and 24H1 hybridomas responded to stimulation with gp33 peptide that had been treated with the nitrotyrosine-promoting compound peroxynitrite, indicating that the nitrotyrosine modification present in synthetic NY-gp33 peptide was the same as the modification that is biologically produced after treatment with peroxynitrite (Fig. 3 F).
|T-T Hybridoma .||Restriction .||TCR Vβ .|
|T-T Hybridoma .||Restriction .||TCR Vβ .|
Restriction and TCR Vβ usage of the gp33-specific P14 T cell is as previously reported (27) and is shown for comparison only.
The 4C8 and 24H1 T-T hybridomas were absolutely specific in terms of their recognition of NY-gp33 vs gp33 peptides and, likewise, ELISPOT analysis of splenocytes from mice that had been immunized with either NY-gp33 or gp33 peptides revealed that the bulk of the response was against the immunizing peptide. However, during ELISPOT analysis of splenocytes from peptide-immunized mice, we repeatedly observed a weak response (low number of spots) in the presence of the opposite (non-immunizing) peptide (i.e., weak response elicited by gp33 peptide in splenocytes from NY-gp33-immunized mice and vice versa). We hypothesized that this observation could be explained by one of two different phenomenon: 1) cells elicited against the immunizing NY-gp33 or gp33 peptide could respond to the opposite peptide but with low affinity, resulting in a weaker response at the bulk population level or 2) there were two distinct populations in the responding repertoire of T cells, one that is absolutely specific for the immunizing peptide (major population) and a second population (minor) that can recognize both the immunizing peptide and corresponding opposite (modified or non-modified) peptide. To address these two possibilities, we immunized mice with either NY-gp33 or gp33 peptides and then expanded the cells in vitro in the presence of either the immunizing or corresponding opposite (modified or nonmodified) peptide. T cell cultures derived from mice that had been immunized with NY-gp33 and then expanded with NY-gp33 were predominantly specific for the NY-gp33 peptide but contained a minor population of cells responsive to non-modified gp33 (Fig. 4). Conversely, T cell cultures derived from mice that had been immunized with NY-gp33 and then expanded with non-modified gp33 were equally responsive to both NY-gp33 and non-modified gp33 (Fig. 4). The opposite pattern was observed in mice that had been immunized with non-modified gp33 and then stimulated in vitro with either NY-gp33 or gp33 peptide. Together, these findings imply that there is a unique, but minor, population of T cells within the bulk repertoire that can accommodate both the modified (NY-gp33) and non-modified (gp33) epitope.
In the present study, we provide evidence that, as previously observed with CD4 T cells (23, 25), CD8 T cells can specifically recognize epitopes containing a tyrosine-to-nitrotyrosine conversion. Y4 of gp33 has previously been shown to comprise an important TCR-contact point for T cells recognizing NY-gp33 in the context of H-2Db (27, 29). Indeed, the distal hydroxyl group of Y4 is itself a critical feature of TCR recognition because loss of this hydroxyl group in the tyrosine-to-phenylalanine mutation (Y4F) is known to reduce the affinity of the P14 TCR by at least 100-fold (29). Thus, it is not overly surprising that addition of a NO2 group at the 3′ position (next to the hydroxyl group) could also have a critical effect on recognition by P14 TCR. Nonetheless, specific recognition of the nitrated version of this well-known model epitope (or lack thereof in the case of P14) provides compelling evidence that nitration of any epitope containing a suitable tyrosine residue in a TCR-contact position could potentially result in formation of a unique epitope, perhaps with profound implications for the well-being of the host organism. Furthermore, we were quite surprised to find that T cells capable of recognizing NY-gp33 in the context of H-2Kb could also be readily generated. Previous structural studies have shown that when gp33 is presented by H-2Kb, the Y4 residue is positioned in a downward orientation where it actually contributes to MHC binding as a secondary anchor residue (27). It is unclear at this point how the 24H1 T-T hybridoma specifically recognizes NY-gp33 in the context of H-2Kb because in this orientation it is unlikely that the modified Y4 can be contacted directly by the TCR. Thus, we speculate that conversion of Y4 to nitrotyrosine introduces a shift in the way that gp33 is sitting in the H-2Kb peptide-binding cleft, resulting in some alteration in the face of the peptide that is presented to the TCR. Alternatively, the presence of nitrotyrosine could affect the conformation of the H-2Kb molecule itself thereby altering the interaction between the α-helix of the H-2Kb molecule and the TCR. This latter possibility is supported by previous structural analysis of the Y4F mutation of gp33 bound to H-2Kb which appears to induce a subtle alteration in that part of the α-helix of the H-2Kb molecule that interacts with the TCR (28). Although the precise molecular effect that 3-nitrotyrosine is conferring in terms of altering the presentation of NY-gp33 to T cells by H-2Kb would require confirmation by structural studies, it implies that conversion of tyrosine to nitrotyrosine may have an even broader immunological impact than we anticipated. Initially, we hypothesized that modification of only those tyrosines that were directly involved in TCR recognition might have an impact on T cell recognition. However, the results presented herein show that, in addition, nitration of tyrosines in non-TCR-contact positions can also have a profound impact on TCR recognition by indirectly affecting the face of the peptide/MHC molecule that is interacting with the TCR. Likewise, tyrosine nitration is only one of many amino acid modifications known to occur as a result of oxidative stress (34) and we assume that many other amino acid modifications could potentially have a similar impact on T cell recognition. Moreover, conversion of tyrosine to nitrotyrosine is not limited to the Ag side of the TCR peptide/MHC interface and a recent study has revealed that the TCR itself can become modified in the face of oxidative stress introduced by myeloid-derived suppressor cells (35). As described herein for epitope modification, these authors report that nitration of specific tyrosine residues within the TCR can have a profound immunological consequence, potentially resulting in the induction of Ag-specific tolerance by disrupting the interaction between TCR and peptide/MHC.
Recently, there has been growing interest in the presence of nitrotyrosine in many inflammatory conditions including a plethora of autoimmune disease, infectious disease, and cancer settings. Although the identity of the modified proteins in many cases is largely unknown, there are a growing number of publications describing the identification of nitrotyrosine-modified proteins using predominantly mass spectrometry-based approaches (4, 36, 37). In many instances, the modified proteins that have been identified comprise common and abundant self-proteins. These nitrotyrosine-containing self-proteins could potentially be processed through normal proteolytic and TAP-dependent processes and could end up being presented by MHC class I molecules on the surface of the cell as part of the repertoire of self-peptides. Previously, we demonstrated that NY-PCC-specific T cells can be readily raised in PCC-transgenic mice (23), implying that CD4 T cells specific for “nitrated-self” are not subject to the constraints of central tolerance. Together with the current study these findings suggest that nitrotyrosine-containing “self” peptides could easily be misinterpreted by the cellular immune system as being “foreign” and, given the right circumstances, could become targets of cellular immunity. Thus, we propose that nitrotyrosine-containing self-peptides (or other oxidatively modified peptides) could represent an important new class of neoautoantigens that warrant further investigation. Conversely, one of the major challenges faced by tumor immunologists is the identification of appropriate tumor Ags that are immunogenic because the vast majority of cancer Ags are self and are thus subject to the constraints of tolerance. Recently, nitrotyrosine has been observed in a number of human cancer settings including bladder carcinoma (38), colorectal carcinoma (39), breast cancer (40), esophageal squamous cell carcinoma (41), non-Hodgkin’s lymphoma (42), lung cancer (43), and melanoma (44) as well as in experimental tumor models (45). If our previous findings regarding the lack of central tolerance against nitrated-PCC in PCC-transgenic mice are a globally applicable phenomenon, then targeting of nitrotyrosine-containing “self” peptides present on tumor cells may be a rational, alternative approach for tumor immunotherapy.
Lastly, in the current study, we describe the development of T-T hybridomas that may prove to be valuable tools for the study of the immunological importance and consequences of nitrosative stress. The gp33 epitope is widely used in various immunological models and we anticipate that a number of systems may be useful avenues for providing the inflammatory conditions necessary for up-regulation of inducible NOS and the consequential formation of nitrated gp33. These include infection by lymphocytic choriomeningitis virus as well as other model systems in which gp33 is ectopically expressed including rat insulin promoter-gp33-transgenic mice (46), constitutive gp33-transgenic mice (47), and gp33-expressing tumor systems (48). Using TCR cDNAs obtained from the 4C8 and 24H1 T-T hybridomas described herein, we are planning to generate TCR-transgenic mice specific for NY-gp33. Such mice would comprise the first example of a transgenic mouse expressing a TCR specific for a posttranslationally modified Ag, and in the case of gp33-transgenic mice, a modified self-Ag that might be expected to form spontaneously during periods of inflammatory activity.
We thank Dr. James Yang (National Institutes of Health) for providing the H-2Db- and H-2Kb-expressing HEK-293 cell lines and Dr. Phillipa Marrack (National Jewish Medical Center) for providing the CD8-expressing BWLyt2-4 hybridoma fusion partner.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by a grant from the Canadian Institutes of Health Research (to J.R.W.).
Abbreviations used in this paper: NOS, NO synthase; NYP, protein-associated nitrotyrosine; PCC, pigeon cytochrome c; SFC, spot-forming cell.