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*Diabetes
The Journal of Immunology, 2004, 173: 3103-3111.
Copyright © 2004 by The American Association of Immunologists

Diabetes Incidence Is Unaltered in Glutamate Decarboxylase 65-Specific TCR Retrogenic Nonobese Diabetic Mice: Generation by Retroviral-Mediated Stem Cell Gene Transfer1

Paula Y. Arnold, Amanda R. Burton and Dario A. A. Vignali2

Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TCR transgenic mice are valuable tools for dissecting the role of autoantigen-specific T cells in the pathogenesis of type 1 diabetes but are time-consuming to generate and backcross onto congenic strains. To circumvent these limitations, we developed a new approach to rapidly generate mice expressing TCR using retroviral-mediated stem cell gene transfer and a novel picornavirus-like 2A peptide to link the TCR {alpha}- and {beta}-chains in a single retroviral vector. We refer to these as retrogenic (Rg) mice to avoid confusion with conventional transgenic mice. Our approach was validated by demonstrating that Rg nonobese diabetic (NOD)-scid mice expressing the diabetogenic TCRs, BDC2.5 and 4.1, generate clonotype-positive T cells and develop diabetes. We then expressed three TCR specific for either glutamate decarboxylase (GAD) 206–220 or GAD 524–538 or for hen egg lysozyme 11–25 as a control in NOD, NOD-scid, and B6.H2g7 mice. Although T cells from these TCR Rg mice responded to their respective Ag in vitro, the GAD-specific T cells exhibited a naive, resting phenotype in vivo. However, T cells from Rg mice challenged with Ag in vivo became activated and developed into memory cells. Neither of the GAD-reactive TCR accelerated or protected mice from diabetes, nor did activated T cells transfer or protect against diabetes in NOD-scid recipients, suggesting that GAD may not be a primary target for diabetogenic T cells. Generation of autoantigen-specific TCR Rg mice represents a powerful approach for the analysis of a wide variety of autoantigens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The nonobese diabetic (NOD) 3 mouse is arguably the best-studied model of spontaneously arising organ-specific autoimmune disease (1, 2). Type 1 diabetes is characterized by autoreactive lymphocytes initially invading the pancreatic islets (insulitis) (3) and gradually destroying the insulin-producing {beta} cells (diabetes) (4). The autoantigens that are targeted by T cells and the course of disease in NOD mice appear to closely mimic type 1 diabetes in humans (5). The actions of both CD4+ and CD8+ T cells (1, 6), the balance of Th1/Th2 T cells and cytokines (2, 7, 8, 9, 10, 11, 12, 13, 14) as well as the function of regulatory T cells (15, 16, 17, 18, 19) all appear to be important factors in disease.

Over two dozen autoantigens have been implicated in the initiation or pathogenesis of diabetes (20), including glutamic acid decarboxylase (GAD) 65 (21, 22), IA-2 (23), IA-2{beta} autoantigen phogrin (24, 25, 26), insulin (27), and islet cell autoantigen ICA-69 (28). Numerous studies have indicated that GAD 65 is important in the initiation of diabetes, and various treatments with GAD protein, peptide or DNA by oral, nasal, i.v., intrathymic, or i.p. routes have inhibited disease (9, 10, 21, 29, 30, 31, 32, 33). Furthermore, one study suggested that antisense GAD expression in the pancreas inhibited diabetes (34).

The generation of autoantigen-specific CD4+ and CD8+ TCR transgenic (Tg) NOD mice has greatly increased our understanding of the mechanisms of initiation, progression, and protection from diabetes (5, 35, 36). Currently, only two diabetogenic CD4+ TCR Tg mice are available for study: 4.1 (37) and BDC2.5 (38), in which the TCR were derived from clones isolated from NOD islets and spleen, respectively. These Tg mice have made it possible to dissect mechanisms of {beta} cell destruction, contributions of the H-2Ag7 MHC class II molecule, and effects that other subsets of lymphocytes and particular molecules have on diabetes, mainly because the kinetics of the insulitis to diabetes transition is much faster compared with normal NOD mice. In addition, crossing these TCR Tg mice onto B6.H2g7, NOD-scid, or RAG–/– mice has been very useful in relating such factors as contributions of background genes, regulatory T cells, and alternative V{alpha} gene usage to the disease process. An important clue in dissecting disease in these models is missing however, as the 4.1 and BDC2.5 TCR recognize unknown islet cell Ags.

The only published CD4+ GAD-reactive TCR Tg mouse, specific for GAD 286–300, revealed that T cells in these mice were not diabetogenic but protective (39). This observation prompted speculation that GAD-reactive T cells may be protective and that immunization protocols act to amplify an existing protective response. A clear answer to this question requires the generation of many more GAD-specific TCR Tg mice specific for different T cell epitopes of GAD, yet making a large number of Tg mice in a reasonable amount of time is not feasible using current technology.

In this report, we created mice expressing TCR by combining two powerful technologies: a picornavirus-like ‘self-cleaving’ 2A peptide sequences to link TCR {alpha} and {beta} chains and by retroviral-mediated stem cell gene transfer to express the TCR in bone marrow of recipient mice. The 2A sequences have previously been used for the cotranslation of autologous proteins in several expression systems and host cells (40, 41, 42). The extremely rare 2A consensus motif (2A: Asp-Val/Ile-Glu-X-Asn-Pro-Gly; 2B: Pro) appears to impair normal peptide bond formation between the 2A Gly and the 2B Pro without affecting the translation of 2B (43). Through this ribosomal skip mechanism, highly efficient ‘cleavage’4 occurs between the first and second cistron. The TCR{alpha}-2A-TCR{beta} sequences (see Fig. 1) were cloned into a murine stem cell virus (MSCV)-based retroviral vector and expressed in mice using retroviral-mediated stem cell gene transfer. To avoid confusion with conventional Tg mice, we refer to mice that express TCR via retroviral-mediated gene transfer as ‘retrogenic’ (Rg) mice (i.e., ‘retro’ from retrovirus and ‘genic’ from transgenic).



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  		FIGURE 1. Construction of 2A-linked TCR vectors and method for generating TCR Rg mice. A, Schematic of the pMIG retroviral vector containing the 2A-linked TCR constructs, 5F2 TCR{alpha}-P2A-{beta}, 14B12 TCR{alpha}-P2A-{beta}, and 10E1 TCR{alpha}-T2A-{beta} produced by recombinant PCR. The constructs were cloned into an MSCV-based retroviral vector that contains an internal ribosomal entry site (IRES) and pMIG (GFP). The position and direction of primers used is shown. B, Amino acid sequence of the 2A regions of porcine teschovirus-1 (P2A) and Thosea asigna (T2A). Conserved residues are boxed. The cleavage point between the 2A and 2B peptides, and thus the NH2- and COOH-terminal cistrons, is indicated by the arrow. C, The oligonucleotide sequences used for PCR are shown. For the C{beta} primer, Y = equal amounts of primer with either A or G in that position and R = equal amounts of primer with C or T in that position. All constructs were verified by sequence analysis. D, Method for expression of TCR constructs in mice via retroviral-mediated stem cell gene transfer is shown.

 
We generated TCR Rg mice by reconstituting recipients with bone marrow transduced with retrovirus-containing genes encoding two diabetogenic TCR, BDC2.5 and 4.1, as positive controls and a TCR specific for hen egg lysozyme (HEL) 11–25 as a negative control. We then examined mice expressing TCR specific for GAD 206–220 and GAD 524–538. First, we asked whether these TCR could be expressed and function in three different congenic mouse strains, NOD, B6.H2g7, and NOD-scid. Second, we asked what effect these TCR had on diabetes progression. Generation of TCR Rg mice is a powerful means to generate NOD mice expressing TCR specific for various epitopes of countless autoantigens and to analyze the effects of these TCR in important congenic strains.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female NOD/LtJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). NOD-scid mice were obtained from The Jackson Laboratory and B6.H2g7 mice were kindly provided by C. Benoist (Joslin Diabetes Center, Boston, MA), and both were bred at Charles River Breeding Laboratories (Wilmington, MA) and St. Jude Animal Resources Center (Memphis, TN). Bone marrow recipient mice and adoptive transfer recipients were monitored weekly for diabetes by checking glucose in urine by Clinistix (Bayer, Elkhart, IN) and glucose in blood with glucometer (One Touch Profile; Lifescan, Milpitas, CA). Mice were considered diabetic following a positive reading by Clinistix and a blood glucose level above 200 mg/dL. All animal experiments were performed in an American Association for the Accreditation of Laboratory Animal Care-accredited, specific pathogen-free facility following national, state, and institutional guidelines. Animal protocols were approved by the St. Jude Institutional Animal Care and Use Committee.

Retroviral constructs and producer cell lines

TCR retroviral constructs were produced by recombinant PCR as previously described (44), using the primers and scheme (see Fig. 1, A and C) that are described elsewhere (45). BDC2.5 was cloned from plasmids provided by L. Teyton (The Scripps Research Institute, La Jolla, CA) and C. Benoist. Construct 4.1 was cloned from 4.1 TCR plasmids provided by P. Santamaria (University of Calgary, Alberta, Canada) GAD- and HEL-specific TCR were cloned from NOD-derived T cell hybridomas (46). Retroviral producer cell lines were generated as described with some modifications (47, 48, 49). Briefly, 293T cells were transiently transfected using Fugene (Roche Diagnostics, Indianapolis, IN). Retroviral producer cell lines were then generated by repeatedly transducing GP plus E86 cells six to eight times until a viral titer of >105/ml after 24 h was obtained.

Retroviral-mediated stem cell gene transfer

Retroviral transduction of murine bone marrow cells was performed as described (45, 48). Briefly, bone marrow was harvested from 8- to 10-wk-old donor mice 48 h after treatment with 150 mg/kg 5-fluoruracil (Pharmacia & UpJohn, Kalamazoo, MI). Bone marrow cells were cultured in complete DMEM with 20% FBS and the stem cells induced to proliferate with 20 ng/ml IL-3, 50 ng/ml IL-6, and 50 ng/ml stem cell factor (R&D Systems, Minneapolis, MN). Bone marrow cells were cocultured for 48 h with the retroviral producer cell lines described earlier. The nonadherent, transduced bone marrow cells were collected and washed. Sublethally irradiated mice (NOD = 750 rads, NOD-scid = 250 rad, B6.H2g7 = 1100 rads) were injected via the tail vein, with 4 x 106 bone marrow cells in PBS/2% FBS with 2 U/ml heparin. Mice were analyzed for TCR expression and function 8–10 wk posttransplant.

Histology

The pancreas was removed from each recipient mouse 8–10 wk posttransplant, put into 10% buffered Formalin, and embedded in paraffin, before 4-µm-thick sections, 100 µm apart (to insure counting of unique islets), were cut and stained with H&E at the St. Jude Histology Core Facility. Islets were scored blind for insulitis based on absence of any infiltrate (no insulitis), infiltrate in outer edges of islet (peri-insulitis), and infiltrate within islet (insulitis). An average of ~100 islets were scored per mouse.

Flow cytometric analysis of bone marrow recipient mice

Thymi, spleens, and pancreatic lymph nodes (pln) were removed from recipient mice, processed, RBC lysed, and the cells analyzed by flow cytometry using anti-CD3 PerCP-Cy5.5, anti-CD4-PE, and anti-CD8-allophycocyanin Abs (BD Pharmingen, San Diego, CA). Analysis of specific TCR and activation profiles of CD4+ T cells were performed using anti-CD4 PerCP-Cy5.5, anti-V{beta}8.1/8.2-biotin, or anti-V{beta}12-biotin along with PE-conjugated Abs to CD44, CD62 ligand (CD62L), CD25, or CD69, followed by streptavidin-allophycocyanin (BD Pharmingen). BDC2.5+ T cells were also analyzed with an anti-clonotypic mAb, aBDC (O. Kanagawa, Washington University School of Medicine, St. Louis, MO) (50). In some experiments, splenocytes from 4.1 TCR Tg mice were used to compare with those from the equivalent Rg mice (kindly provided by P. Santamaria).

Functional assays

Splenocytes (5 x 105/well) were cultured with a titration of peptide or protein in AIM-V serum-free medium (Invitrogen Life Technologies, Grand Island, NY) (NOD cells) or complete supplemented Eagle’s minimum essential medium (cSMEM) (NOD-scid and B6.H2g7 cells) in a 96-well flat-well plate. After 48 h, 75 µl of supernatant was removed for Multiplex cytokine analysis (51). Wells were pulsed with [3H]thymidine (1 µCi/well) (DuPont, Wilmington, DE) for the last 24 h of the 72-h assay. The following peptides were used: GAD 206–220 (TYEIAPVFVLLEYVT), GAD 524–538 (SRLSKVAPVIKARMM), and HEL 11–25 (AMKRHGLDNYRGYSL). Peptides used in functional assays were synthesized on a Rainin Symphony using either standard F-moc chemistry or Chiron pin technology. All peptides were produced by the Hartwell Center, St. Jude Children’s Research Hospital (Memphis, TN). Purity and quality were verified by HPLC and MALDI-TOF mass spectrometry. HEL protein was purchased from Sigma-Aldrich (St. Louis, MO). GAD 65 protein was purchased from Diamyd Diagnostics (Stockholm, Sweden).

Ag injection and adoptive transfer experiments

For Ag injection, NOD recipients of 14B12 (GAD 206–220) TCR-transduced bone marrow were injected i.p. with either 150 µM GAD 206–220 or 200 µg/ml recombinant GAD-65 in 0.5 ml PBS. After 3–4 days, splenocytes were analyzed for memory and activation markers as described earlier. For adoptive transfers, splenocytes (2.5 x 106 cells/ml cSMEM) from NOD recipients of TCR-transduced bone marrow (pooled from three mice per group) were stimulated with 5 µM Ag in a bulk culture. After 3 days the cells were washed and passed (2 x 105/ml) into cSMEM containing ~2.5 ng/ml IL-2 (C63 supernatant). After 3 additional days of expansion in IL-2, activated T cells were washed and transferred (10 x 106/mouse) via tail vein injection into 8- to 12-wk-old NOD-scid recipients alone or in combination with 15 x 106 splenocytes from prediabetic (8.5-wk-old) NOD donors. At the time of transfer, the activated 5F2 (HEL 11–25), 14B12 (GAD 206–220), and 10E1 (GAD 524–538) CD4+ T cells were 92%, 97%, and 99% positive for GFP and specific V{beta} TCR expression, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BDC2.5 and 4.1 TCR Rg mice develop diabetes

To assess the validity of our approach, we asked whether Rg mice expressing two diabetogenic TCR, BDC2.5 and 4.1, developed diabetes with an incidence that is comparable to data published for the Tg mice. We cloned these TCR into a MSCV-based vector, using a picornavirus-like 2A sequence for bicistronic translation of the TCR {alpha} and {beta} chains, followed by an internal ribosomal entry site, and GFP, abbreviated pMIG (Fig. 1, A and B). Retroviral producer cells were generated and used to transduce bone marrow isolated from NOD or NOD-scid donor mice and the retrovirally transduced bone marrow injected into syngeneic recipients as outlined (Fig. 1D). Flow cytometric analysis of PBL from the TCR Rg mice as early as 3.5–4.5 wk post-bone marrow transplant showed a clear increase in the percentage of GFP+ T cells expressing the appropriate V{beta} (Fig. 2A). This was evident in both NOD and NOD-scid mice, although the staining of BDC2.5 TCR-V{beta}4 is low in NOD-scid recipients but must be sufficient for selection. Similar results were seen with splenocytes analyzed 6–8 wk posttransfer (data not shown). TCR expression in the 4.1 Rg and Tg mice was comparable (Fig. 2B). Furthermore, the expression of BDC2.5 TCR was equivalent in NOD and NOD-scid mice (Fig. 2C).



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  		FIGURE 2. Expression and diabetes incidence in 4.1 and BDC2.5 Rg mice. A, PBL were isolated by orbital bleed from NOD and NOD-scid mice 3.5–4.5 wk following transfer of syngeneic bone marrow transduced with 4.1, BDC2.5, or vector alone (MIG). PBL from age-matched, untreated NOD mice were used as controls. Cells were stained with the indicated mAbs and analyzed by flow cytometry. Plots of NOD and NOD-scid cells are gated on live and live plus CD4+ T cells, respectively. For mice expressing GFP, numbers represent percentage of GFP+ cells that are TCR+. Data are representative of 5–20 mice per TCR. B, CD4+ splenocytes from 4.1 Rg or Tg mice were analyzed for V{beta}11 expression. C, Splenocytes from BDC2.5 NOD and NOD/SCID Rg mice were stained with anti-CD4 and either anti-TCR-V{beta}4 or the aBDC clonotypic Ab. Cells were gated on CD4 and GFP. D and E, NOD/SCID (seven per group) recipients of GFP vector, 4.1 TCR-transduced (D) or BDC2.5 TCR-transduced (E) bone marrow were followed for diabetes. Glucose levels in urine and blood were determined by Clinistix and Lifetouch glucometer, respectively. Mice having a glucometer reading level higher than 200 mg/dl were considered diabetic. F, A CD4+, GFP+, V{beta}4+ BDC2.5 Rg T cell line (5 x 106), generated from a BDC2.5 NOD bone marrow recipient, was transferred after 8 days rest in vitro into NOD-scid mice (five per group), and diabetes was monitored.

 
We then assessed the incidence of diabetes in BDC2.5 and 4.1 NOD-scid TCR Rg mice. Over 60% of both groups of mice developed diabetes by 80 days posttransfer (Fig. 2, D and E). Lastly, a T cell line generated from BDC2.5 Rg NOD mice transferred diabetes to NOD-scid mice (100% incidence within 47 days) (Fig. 2F). Taken together, these data suggest that the use of Rg mice to study type 1 diabetes is valid.

Expression and function of HEL- and GAD-specific TCR in Rg mice

We then cloned TCR from three CD4+ T cell hybridomas that were generated by immunization of NOD mice with HEL 11–25 (5F2: V{alpha}13.1, V{beta}8.1), GAD 206–220 (14B12: V{alpha}4.7, V{beta}8.2), or GAD 524–538 (V{alpha}4.9, V{beta}12) in IFA. Retroviral producers were used to transduce bone marrow isolated from NOD, B6.H2g7, or NOD-scid donor mice and reconstitute irradiated syngeneic recipients as described earlier (Fig. 1C). Flow cytometry of PBL from TCR Rg mice 3.5–4.5 wk post-bone marrow transplant demonstrated that all three TCR were expressed on a high percentage of GFP+ CD4+ T cells in NOD, B6.H2g7, and NOD-scid recipients (Fig. 3). Total GFP expression in the thymi, spleens, and pln of vector control NOD mice and TCR Rg mice was comparable (Fig. 4A). GFP+ thymocyte populations were comparable in all three TCR Rg NOD mice with no obvious skewing toward CD4+ cells (Fig. 4, B and C). There are several transcriptional, posttranslational, and receptor assembly mechanisms that are used to regulate TCR expression in the thymus. The characteristic increase in TCR expression seen upon transition from double positive to single positive thymocytes in unmanipulated mice also occurred in Rg mice (Fig. 4D). This is an important observation as it infers that the use of retroviral long terminal repeat promoter rather than the endogenous TCR promoter has not compromised TCR surface expression and T cell development. However, some differences were observed. Although 14B12 TCR expression was comparable to the NOD and GFP+ MIG vector controls, 5F2 and 10E1 TCR expression on double positive thymocytes was significantly higher. Whereas intriguing, it is currently unclear what consequence this has for T cell development and function, and many more TCR would have to be examined to determine whether a specific correlation exists. However, it is conceivable that the pattern of TCR expression reflects differences in TCR affinity, availability of the self-selecting ligand(s), and/or the efficiency and outcome of selection.



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  		FIGURE 3. TCR Rg mice express TCR on GFP+ CD4+ T cells. PBL were isolated by orbital bleed from NOD, B6.H2g7 and NOD-scid mice 3.5–4.5 wk following transfer of syngeneic bone marrow transduced with 5F2 TCR{alpha}-P2A-{beta} (HEL 11–25), 14B12 TCR{alpha}-P2A-{beta} (GAD 206–220), 10E1 TCR{alpha}-T2A-{beta} (GAD 524–538), or vector alone (MIG). PBL from age-matched, untreated NOD and B6.H2g7 mice were used as controls. Cells were stained with the indicated mAbs and analyzed by flow cytometry. Plots of NOD and B6.H2g7 cells are gated on live, CD4+ T cells. Plots of NOD-scid cells are gated on live cells. For mice expressing GFP, numbers represent percentage of GFP+ cells that are TCR+. Data are representative of 5–20 mice per TCR.

 


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  		FIGURE 4. TCR expression and T cell development in Rg mice. A, Thymus, spleen, and pln from 5F2, 14B12, 10E1 TCR Rg, and vector-transduced NOD bone marrow recipients were analyzed for GFP expression on total cells (live gate). B, Thymocytes (live gate) and pln (live, CD3+ gate) from NOD recipients of 5F2, 14B12, 10E1 TCR, or vector (MIG)-transduced bone marrow were analyzed by flow cytometry for expression of CD4 and CD8 T cell markers on GFP– or GFP+ cells. C, Thymocytes were gated on live cells and analyzed for CD4 and CD8 expression. D, Thymocytes were gated on CD4/CD8 double positive (DP) and CD4 single positive (SP) cells and CD3 expression in GFP– NOD control and GFP+ vector bone marrow recipients, or specific V{beta} TCR expression in GFP+ 5F2, 14B12, and 10E1 TCR bone marrow recipients determined. Data represent five mice per group, 8–10 wk post–bone marrow transplant. E, Thymus, spleen, and pln from 5F2, 14B12, 10E1 TCR, and vector-transduced NOD bone marrow recipients were analyzed for percentage of CD4+ V{beta}8.1/8.2+ (left) or CD4+ V{beta}12+ (right) T cells that are either GFP– or GFP+ cells. Data represent five mice per group 8–10 wk posttransplant.

 
In the periphery, there was some degree of skewing to CD4+ T cells (and corresponding decrease in CD8+ T cells) in the spleen (data not shown) and pln of the GAD-specific TCR Rg NOD mice, especially 10E1, which was not seen in the GFP– population (Fig. 4B). In the HEL-specific 5F2 TCR Rg mice, there was a large increase of double negative T cells (and decrease of CD4+ T cells) within the GFP+ population in the periphery. It is unclear whether this increase is related to the reduced percentage of CD4+ single positive thymocytes in the 5F2 mice (Fig. 4C). It should be noted that we have also seen a significant population of double negative T cells in the periphery of some conventional TCR Tg mice (e.g., our unpublished observations with 3A9 TCR tg mice). Despite little skewing, specific TCR were consistently expressed on a high percentage of GFP+ cells in the thymus, spleen, and pln among many mice that received the same TCR (Fig. 4E).

All three TCR were functional in NOD, B6.H2g7, and NOD-scid bone marrow recipients, as demonstrated by peptide-specific proliferation of splenocytes from bone marrow recipient mice 8–10 wk post–bone marrow transplant (Fig. 5). T cell proliferation was accompanied by IL-2 secretion, but very little IL-10, and no detectable IL-4, IL-5, or IFN-{gamma} were produced (Fig. 5 and data not shown). HEL 11–25-specific 5F2 cells and GAD 206–220-specific 14B12 Rg T cells also responded to whole protein Ag in vitro, whereas GAD 524–538-specific 10E1 Rg T cells did not respond to GAD protein (data not shown). Together, these data demonstrate that all three TCR were expressed and respond to their cognate peptide in all backgrounds tested. The fact that we were able to express these TCR in NOD-scid mice, illustrates that coexpression of endogenous V{alpha} chains is not necessary for development and expression of these TCR.



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  		FIGURE 5. Function of T cells from Rg mice. Splenocytes (5 x 105/well) from NOD, B6.H2g7, and NOD-scid TCR or vector-transduced (MIG) bone marrow recipients were cultured in the presence of indicated concentrations of peptide. Supernatants were removed at 48 h and cytokine concentration was determined by multiplex analysis (only IL-2 data are presented). Cultures were pulsed with [3H]thymidine during the last 24 h of a 72-h assay. Data represent two to five mice per group 8–10 wk post-bone marrow transplant.

 
Expression of the GAD 206–220 or GAD 524–538-reactive TCR does not accelerate or protect mice from diabetes

We followed NOD, B6.H2g7, and NOD-scid TCR Rg mice for diabetes incidence. Several initial experiments indicated that NOD recipients of vector-transduced bone marrow exhibit similar incidence of diabetes as untreated NOD mice, albeit with delayed kinetics (data not presented). Analysis of islet infiltrates in pancreata of NOD TCR Rg mice 8–10 wk posttransplant revealed no difference in severity of insulitis among vector, HEL-specific 5F2 TCR, or GAD-specific 14B12 and 10E1 TCR recipients (Fig. 6A). Furthermore, diabetes incidence in NOD Rg mice did not appear to be accelerated or ameliorated as a result of expression of either the HEL-specific or GAD-specific TCR (Fig. 6B). In addition, neither the HEL- nor GAD-specific TCR induced diabetes when expressed in B6.H2g7 or NOD-scid mice, two strains that do not normally develop diabetes (Fig. 6C).



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  		FIGURE 6. Expression of GAD 206–220 or GAD 524–538-specific TCR does not accelerate or protect from diabetes in TCR Rg mice. A, Islets in H&E-stained pancreas sections from NOD recipients of vector or TCR-transduced bone marrow were scored for lymphocytic infiltrates (no insulitis, peri-insulitis, or insulitis). Data represent means of five to seven mice, 8–10 wk posttransplant. B and C, NOD, B6.H2g7, or NOD-SCID recipients of vector or TCR-transduced bone marrow were followed for diabetes.

 
HEL- and GAD-specific Rg T cells exhibit a naive, resting phenotype in vivo

The observation that none of the GAD-specific TCR affected diabetes progression in NOD mice prompted us to analyze activation and memory phenotypes on TCR+ cells directly ex vivo. Although T cells expressing specific TCR were capable of responding to Ag in vitro, GFP+, CD4+, V{beta}+ T cells from NOD TCR Rg mice 8–10 wk post–bone marrow transfer exhibited a naive, resting phenotype. Specifically, the percentages of CD44high, CD25+, and CD69+ T cells in spleen (data not shown) and pln (Fig. 7A) were significantly decreased in 5F2 (HEL 11–25), 14B12 (GAD 206–220), and 10E1 (524–538) TCR Rg mice compared with their GFP– counterparts and with GFP+ T cells from vector recipients. Although the percentage of CD62Llow T cells from 5F2, 14B12, and 10E1 TCR Rg mice was not reduced compared with controls, this percentage was significantly decreased 20–25 wk post-bone marrow transfer (Fig. 7B). It should be noted that the percentage of CD44high, CD62Llow, CD25+, and CD69+ T cells in the MIG vector control is comparable to NOD controls, however the percentage of 14B12 cells with these phenotypes is significantly lower (Fig. 7B). Thus, it appears that although the TCR we expressed respond to their Ag in vitro, none of the TCR encounter Ag in a stimulatory manner in vivo. There are, however, two potential caveats. First, we cannot rule out the possibility that the T cells in the islets are activated and can ‘see’ Ag. Second, the analysis of cell surface phenotype may not be a sufficiently sensitive method to assess activation state. An alternative approach might be to assess the proliferation of CSFE-labeled T cells infiltrating the islets of adoptive recipients, although this would be a technically challenging experiment.



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  		FIGURE 7. T cells from TCR-transduced bone marrow recipients exhibit a naive, resting phenotype in vivo, yet can be stimulated by Ag challenge. A, Splenocytes from NOD recipients of TCR or vector-transduced bone marrow were gated on CD4 and V{beta}8.1/8.2 (left) or V{beta}12 (right) and analyzed for expression of memory markers CD44 and CD62L, and activation markers CD25 and CD69 on GFP– and GFP+ cells. Data represent five mice per group, 8–10 wk posttransplant. B, NOD recipients of bone marrow transduced with vector (MIG) or 14B12 TCR or were i.p. injected with no Ag, 150 µM GAD 206–220 or 200 µg/ml recombinant GAD-65 in 0.5 ml PBS. After 3–4 days, splenocytes were analyzed by flow cytometry for expression of CD44, CD62L, CD25, and CD69. Plots are gated on GFP, CD4, and V{beta} 8.1/8.2+. Data represent two to four mice per group, 20–24 wk posttransplant.

 
To eliminate the possibility that these TCR+ cells were anergic or unresponsive in vivo, we challenged vector or 14B12 TCR Rg NOD mice 20–24 wk posttransfer with GAD 206–220 or GAD protein in PBS i.p. (Fig. 7B). Analysis of GFP+, CD4+, V{beta}8.1/8.2+ T cells 3–4 days postimmunization revealed that GAD 206–220 or recombinant GAD-65 challenge induced a dramatic increase in percentage of CD44high and CD62Llow TCR Rg T cells. In addition, injection with peptide resulted in an increased percentage of CD69+ activated cells and a slight increase in CD25+ T cells. Conversely, GAD protein challenge induced a small increase in CD69+ activated T cells, but a substantial increase in the percentage of CD25+ T cells. Therefore, T cells expressing GAD 206–220-specific TCR are not tolerant in vivo, despite exhibiting a quiescent phenotype, and are quite capable of being activated by peptide or protein challenge.

Adoptive transfer of activated GAD-specific TCR+ T cells does not induce or protect from diabetes in NOD-SCID mice

We reasoned that the GAD-specific 14B12 and 10E1 TCR may not induce diabetes as a result of the quiescent nature of the T cells, i.e., T cells might have some diabetogenic, or even protective, capacity if first activated with Ag. In addition, T cells from the GAD 286–300 Tg mouse were shown to be protective, in that they blocked diabetes induced by transfer of prediabetic NOD splenocytes into NOD-scid mice (39). However, in vitro Ag-activated T cells from our NOD TCR Rg mice did not induce diabetes when transferred into NOD-scid mice, nor did these activated T cells significantly protect NOD-scid mice from diabetes induced by transfer of prediabetic NOD splenocytes (Fig. 8). In summary, despite being fully functional, our GAD-specific TCR Rg T cells were unable to induce or protect mice from diabetes, even when previously activated.



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  		FIGURE 8. Adoptive transfer of activated HEL or GAD-specific TCR+ T cells does not induce or protect from diabetes in NOD-scid mice. In vitro-stimulated CD4+, GFP+, V{beta}8.1/8.2+ (5F2 and 14B12 TCR) or V{beta}12+ (10E1 TCR) T cells (10 x 106) from NOD TCR Rg mice were transferred alone or in combination with 15 x 106 prediabetic NOD splenocytes into NOD-scid mice. NOD-scid mice (five per group) were monitored for diabetes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The studies presented clearly demonstrate the power of TCR expression in mice via retroviral-mediated stem cell gene transfer. Importantly both BDC2.5 and 4.1 TCR Rg mice developed diabetes, validating the use of this approach to study type 1 diabetes. Furthermore, we were able to express three novel H-2g7-restricted TCR (two GAD-specific TCR and one HEL-specific control) on three different congenic strains, and analyze function, phenotype and diabetogenic potential of the Rg T cells that developed. A similar experiment using TCR Tg mice generated by conventional Tg technology and followed by backcrossing onto three congenic strains, would have involved a considerably larger investment of time and resources. Analyzing the effects of TCR transgenes on pure genetic backgrounds is extremely important in type 1 diabetes research. It has been clearly shown that the pathogenic potential of a particular TCR can change significantly on different backgrounds, as illustrated using the BDC2.5 Tg TCR. Counter intuitively, it was shown that these T cells became less diabetogenic as it was further crossed onto the NOD background (52). In addition, Tg mice may exhibit founder effects related to the integration site of the transgene, whereas each TCR Rg mouse is essentially a separate founder.

Previously, other groups have expressed tumor-specific TCR by retroviral transduction of peripheral T cells (53, 54, 55). In this study we have transduced bone marrow stem cells with TCR{alpha} and TCR{beta} genes, thereby insuring expression of specific TCR during T cell development. Like TCR Tg mice, Rg mice express a given TCR on a large percentage of peripheral T cells. Our mice, however, have a larger component of ‘residual’ or ‘normal’ T cells, which are either host-derived or derived from untransduced stem cells. These T cells do not express the specific TCR and are fully capable of inducing diabetes in these mice. However, as indicated with the BDC2.5 and 4.1 TCR Rg mice this difference did not appear to alter diabetes incidence when compared with Tg mice (37). Unlike TCR Tg mice, TCR Rg mice cannot be bred, but we believe the advantages of this technique far outweigh this one disadvantage.

All the TCR we expressed could respond to Ag in vitro and in vivo, yet neither of the GAD-reactive TCR were found to be diabetogenic. Although the GAD 206–220-specific 14B12 Rg T cells responded to recombinant GAD protein, the GAD 524–538-specific 10E1 Rg T cells did not. Therefore, 14B12 TCR are ‘type A’ T cells, a designation introduced by Unanue and colleagues (56) to describe T cells that recognize peptide generated by endogenous protein Ag presentation as well as peptide added exogenously. In contrast, 10E1 are ‘type B’ T cells because they fail to recognize GAD protein and only recognize Ag in the form of GAD 524–538 peptide. These authors have proposed that type B T cells escape thymic negative selection, in which protein Ag is presented by thymic APCs. In the periphery, these T cells only recognize peptide Ag released in sites of inflammation, such as in tissues targeted for autoimmune attack. Therefore, type B T cells may be important mediators of autoimmunity. Although our observation that the type B 10E1 TCR is not diabetogenic argues against this theory, many more TCR specific for a variety of autoantigenic peptides would need to be examined to resolve this issue conclusively.

Although early studies indicated that GAD 65 was an important initiating autoantigen, our results support recent findings that suggest that GAD may not be critical in the development of diabetes. For example, various treatments with GAD protein, peptide or DNA by various routes of administration were shown to inhibit disease, and antisense GAD, expressed only in pancreatic islets, protected mice from diabetes (9, 10, 21, 29, 30, 31, 33). Nonetheless, the incidence of diabetes was unchanged in GAD knockout mice (57), and expressing GAD as a transgene was not protective (in some lines disease was actually accelerated) (58). Recently, von Boehmer and colleagues (59) clearly demonstrated normal diabetes incidence in GAD Tg mice, despite complete tolerance to GAD. Furthermore, only one diabetogenic GAD 524–543-specific CD4+ T cell line, 5A, has been reported (60), and only one GAD-specific CD8+ clone causes insulitis, but not diabetes (61). In fact, a CD4+ Tg specific for GAD 286–300 was not diabetogenic and instead was able to protect mice from diabetes (39). It is curious that the GAD 524–543-specific T cell line 5A (60) caused diabetes following adoptive transfer in to NOD-SCID mice whereas our GAD 524–538-specific 10E1 line did not. This did not seem to be due to differences in peptide specificity as 10E1 responded comparably to both peptides (data not shown). This difference could be due to either the method in which the T cell lines were generated, and thus their functional profile, or some aspect of their TCR affinity or fine specificity (type A vs type B as previously discussed). Clearly, additional studies will be required to resolve this discrepancy.

We did not find NOD T cells expressing our GAD 206–220- or GAD 524–538-reactive TCR to be significantly protective, either in NOD mice or NOD-scid mice injected with prediabetic NOD splenocytes. It is possible the GAD 286–300 Tg TCR is particularly unique in its protective ability or that GAD 286–300 is the only GAD epitope that generates protective T cells. Answering these questions definitively would require the generation of several more GAD 286–300-specific Tg lines. In addition, analyzing the contributions of factors such as background genes, coexpression of endogenous V{alpha} chains, or involvement of certain cytokines would require backcrossing each TCR Tg onto different backgrounds, such as B6.H2g7, NOD-scid, or NOD.IL-10 knockout mice.

Clearly, answering these critical questions using conventional TCR Tg technology would be very difficult and time consuming. However, generating TCR Rg mice using a combination of 2A-linked multicistronic retroviral vectors and retroviral-mediated stem cell gene transfer could prove to be an excellent way to answer important questions concerning the importance of TCR specificity for particular autoantigens and diabetes initiation, progression, and pathogenesis or protection. This general approach may also be useful for the dissection of other autoimmune diseases or pathogen models.


   Acknowledgments
 
We are very grateful to members of the Vignali laboratory for assistance with bone marrow harvest, the Hartwell Center (St. Jude Children’s Research Hosptial) for sequencing oligonucleotids and peptides, Richard Cross and Jennifer Hoffrage for FACS, and Janet Gatewood for multiplex cytokine analyses. We also thank Pere Santamaria, Christophe Benoist, Osami Kanagawa, and Luc Teyton for generously providing reagents.


   Footnotes
 
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.

1 This work was supported by the Juvenile Diabetes Research Foundation (1-2001-543), a Cancer Center Support CORE Grant (CA-21765), and the American Lebanese Syrian Associated Charities. P.Y.A. was supported by a National Institutes of Health Immunology and Molecular Biology Virology Training Grant (AI-07372) and a National Institutes of Health National Research Service Award (AI-10596). Back

2 Address correspondence and reprint requests to Dr. Dario A. A. Vignali, Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794. E-mail address: dario.vignali{at}stjude.org Back

3 Abbreviations used in this paper: NOD, nonobese diabetic; HEL, hen egg lysozyme; GAD, glutamate decarboxylase; Tg, transgenic; Rg, retrogenic; pln, pancreatic lymph nodes; MSCV, murine stem cell virus; cSMEM, complete supplemented Eagle’s minimum essential medium. Back

4 It should be emphasized that there is no known active, enzyme-driven cleavage event, even though "cleavage" is often used in the literature to describe this process. For consistency, this report will refer to this process as "cleavage". Back

Received for publication December 5, 2003. Accepted for publication June 22, 2004.


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