Antigenic epitopes; CD8 T-cells

Insulin β Chain Peptide (15-23) is a highly diabetogenic CD8 T-cell clone, G9C8, in the nonobese diabetic (NOD) mouse, specific to low-avidity insulin peptide B15-23, and cells responsive to this antigen are among the earliest islet infiltrates. We aimed to study the selection, activation, and development of the diabetogenic capacity of these insulin-reactive T-cells.[3]

There was good selection of CD8 T-cells with a predominance of CD8 single-positive thymocytes, in spite of thymic insulin expression. Peripheral lymph node T-cells had a naïve phenotype (CD44lo, CD62Lhi) and proliferated to insulin B15-23 peptide and to insulin. These cells produced interferon-gamma and tumor necrosis factor-alpha in response to insulin peptide and were cytotoxic to insulin peptide-coated targets. In vivo, the TCR transgenic mice developed insulitis but not spontaneous diabetes. However, the mice developed diabetes on immunization, and the activated transgenic T-cells were able to transfer diabetes to immunodeficient NOD.scid mice. Conclusions: Autoimmune CD8 T-cells responding to a low-affinity insulin B-chain peptide escape from thymic negative selection and require activation in vivo to cause diabetes.[3]

Proliferation and cytotoxicity assays: proliferation.[3]
Insulin-reactive CD8 T-cells from 5- to 8-week-old G9Cα−/−.NOD mice were cocultured with bone marrow–derived dendritic cells and B15-23 peptide or influenza hemagglutinin peptide IYSTVASSL (control) for peptide responses in RPMI complete medium. Similarly, soluble insulin or keyhole limpet hemocyanin (control) was used as antigen for protein cross-presentation responses. CD8 T-cells were purified from splenocytes of G9Cα−/−.NOD mice using positive selection beads (Miltenyi Biotec) with >95% purity. After 3 days of incubation, cells were pulsed with [3H]thymidine for 14 h to determine proliferation.

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Cytotoxicity.[3]
CD8 T-cells purified from 5- to 8-week-old G9Cα−/−.NOD mice, used directly ex vivo or prestimulated for 24 h with 1 μg/ml insulin B15-23 peptide, were harvested and further cocultured with 104 51Cr-sodium chromate (Amersham)-labeled P815 cells together with B15-23 insulin peptide at an effector-to-target (E:T) ratio of 10:1 for 16 h. Specific lysis was calculated as [(cytotoxic release − min)/(max − min)] × 100%, where the minimal release (min) corresponded to the spontaneous lysis, and the maximal lysis corresponded to lysis induced by addition of Triton-X100 (max).

In this study, researchers generated a T-cell receptor (TCR) transgenic mouse expressing the cloned TCR Valpha18/Vbeta6 receptor of the G9C8 insulin-reactive CD8 T-cell clone. The mice were crossed to TCRCalpha-/- mice so that the majority of the T-cells expressed the clonotypic TCR, and the phenotype and function of the cells was investigated.[3]

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In vivo immunization.[3]
G9Cα−/−.NOD mice were immunized intraperitoneally with 50 μg B15-23 peptide together with different concentrations of CpG 1826 oligonucleotides (Coley pharmaceuticals). Nine to twenty-one days later, a further injection of the same dose was given. Controls were injected with either 50 μg B15-23 peptide or the different doses of CpG alone.

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Diagnosis of diabetes.[3]
Diabetes was detected by initially testing for glycosuria and confirmed by blood glucose measurements >13.9 mmol/l/l.

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Adoptive transfer of diabetes.[3]
A total of 5–7 × 106 purified CD8 T-cells from G9Cα−/−.NOD mice, either taken directly ex vivo or preactivated by CpG-matured bone marrow–derived dendritic cells and insulin B15-23 peptide, were washed, resuspended in a volume of 200 μl of normal saline, and transferred intravenously to 6-week-old NOD.scid mice, 3-week-old NOD mice, and 6-week-old NOD mice. CD4 T-cells purified either from young 6-week-old mice (107) or from mice that had become diabetic (4–10 × 106) were transferred intravenously to 3- to 4-week-old G9Cα−/−.NOD mice.

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Generation of insulin-reactive TCR transgenic mice.[3]
Insulin-specific TCR transgenic mice were generated by isolating TCR genomic DNA from G9C8 cloned T-cells (23), which have previously been shown to have specific reactivity to amino acids 15–23 of the insulin B-chain. TCRα (Vα18S1 Jα18 Cα)- and β (Vβ6S1 Dβ1.1 Jβ2.3 Cβ1)-chain DNA was purified and cloned into pTαcass and pTβcass constructs. The cloned constructs were injected directly into NOD ova to generate independent TCRα and β founder lines, which were intercrossed to produce αβ TCR transgenic mice (G9NOD). Three independent founder lines each of TCRα (lines 17, 22, and 24) and β (lines 1, 2, and 3) were generated, and α lines 17, 22, and 24 were crossed with each of β lines 2 and 3. Results with each of these lines was similar in terms of selection and functional phenotype (data not shown), and, thus, TCRα line 22 and TCRβ line 2 were chosen for all subsequent studies.[3]
To generate repertoire-restricted αβ TCR transgenic mice, G9Cα−/−.NOD mice were generated by crossing the αβ TCR transgenic mice to NOD.Cα−/− mice. The NOD.Cα−/− mice were generated by backcrossing TCR.Cα−/− mice (B6/129 background) >20 generations to NOD/Caj mice in the laboratory of R.S. Sherwin (Yale University). G9RAG−/−.NOD mice were generated by crossing the αβ TCR transgenic mice to NOD.RAG2−/− mice, generated by backcrossing RAG2−/− mice to NOD/Caj mice >12 generations. To generate G9Cα−/−.RIP-B7.1 NOD mice, the RIP-B7.1 mice were 10 generations backcrossed to NOD.Cα−/− mice and intercrossed to generate NOD.Cα−/−RIP-B7.1 mice and then crossed with the G9Cα−/−.NOD mice to generate G9Cα−/−.RIP-B7.1 NOD mice. NOD.scid mice were were housed in microisolators or scantainers in barrier rooms.

[1]. Amrani A, et al. Progression of autoimmune diabetes driven by avidity maturation of a T-cell population. Nature. 2000;406(6797):739-742.
[2]. Takaki T, et al. Requirement for both H-2Db and H-2Kd for the induction of diabetes by the promiscuous CD8+ T cell clonotype AI4. J Immunol. 2004;173(4):2530-2541.

For unknown reasons, autoimmune diseases such as type 1 diabetes develop after prolonged periods of inflammation of mononuclear cells in target tissues. Here we show that progression of pancreatic islet inflammation to overt diabetes in nonobese diabetic (NOD) mice is driven by the 'avidity maturation' of a prevailing, pancreatic beta-cell-specific T-lymphocyte population carrying the CD8 antigen. This T-lymphocyte population recognizes two related peptides (NRP and NRP-A7) in the context of H-2Kd class I molecules of the major histocompatibility complex (MHC). As pre-diabetic NOD mice age, their islet-associated CD8+ T lymphocytes contain increasing numbers of NRP-A7-reactive cells, and these cells bind NRP-A7/H-2Kd tetramers with increased specificity, increased avidity and longer half-lives. Repeated treatment of pre-diabetic NOD mice with soluble NRP-A7 peptide blunts the avidity maturation of the NRP-A7-reactive CD8+ T-cell population by selectively deleting those clonotypes expressing T-cell receptors with the highest affinity and lowest dissociation rates for peptide-MHC binding. This inhibits the local production of T cells that are cytotoxic to beta cells, and halts the progression from severe insulitis to diabetes. We conclude that avidity maturation of pathogenic T-cell populations may be the key event in the progression of benign inflammation to overt disease in autoimmunity.[1]

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The NOD mouse is a model for autoimmune type 1 diabetes in humans. CD8(+) T cells are essential for the destruction of the insulin-producing pancreatic beta cells characterizing this disease. AI4 is a pathogenic CD8(+) T cell clone, isolated from the islets of a 5-wk-old female NOD mouse, which is capable of mediating overt diabetes in the absence of CD4(+) T cell help. Recent studies using MHC-congenic NOD mice revealed marked promiscuity of the AI4 TCR, as the selection of this clonotype can be influenced by multiple MHC molecules, including some class II variants. The present work was designed, in part, to determine whether similar promiscuity also characterizes the effector function of mature AI4 CTL. Using splenocyte and bone marrow disease transfer models and in vitro islet-killing assays, we report that efficient recognition and destruction of beta cells by AI4 requires the beta cells to simultaneously express both H-2D(b) and H-2K(d) class I MHC molecules. The ability of the AI4 TCR to interact with both H-2D(b) and H-2K(d) was confirmed using recombinant peptide libraries. This approach also allowed us to define a mimotope peptide recognized by AI4 in an H-2D(b)-restricted manner. Using ELISPOT and mimotope/H-2D(b) tetramer analyses, we demonstrate for the first time that AI4 represents a readily detectable T cell population in the islet infiltrates of prediabetic NOD mice. Our identification of a ligand for AI4-like T cells will facilitate further characterization and manipulation of this pathogenic and promiscuous T cell population.[2]

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In conclusion, we have shown that a CD8 T-cell responsive to a natural peptide of insulin, a major autoantigen in autoimmune diabetes, is selected in the thymus and can become highly pathogenic once activated in the periphery, causing diabetes. These T-cells are “ignorant” in a naïve state, but this can be readily overcome by stimuli that include direct activation or activation of antigen-presenting cells, either in pancreatic lymph nodes or in the islets. CD4 T-cells are likely to play a significant role, and this is currently under investigation. Further experiments aim to identify the endogenous stimulators that facilitate the “switch” of naïve and “ignorant” G9 cells into potent β-cell–destructive effector cells in the NOD mouse. Our study provides an important model not only for the study of immunopathogenic role of insulin-specific CD8 T-cells in autoimmune diabetes but also for better design of insulin-based immunotherapy.[3]

C44N12O13SH72

1009.18

1008.5062

247044-67-3

91808033

Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly; L-leucyl-L-tyrosyl-L-leucyl-L-valyl-L-cysteinyl-glycyl-L-alpha-glutamyl-L-arginyl-glycine; H-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-OH

LYLVCGERG; H-LYLVCGERG-OH

Typically exists as White to off-white solid at room temperature

-2.8

15

16

32

70

1810

7

[C@@H](NC(=O)[C@@H](N)CC(C)C)(C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CS)C(=O)NCC(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)O)CC1C=CC(O)=CC=1

CPSZWYPVFBXING-PVKJLKLCSA-N

InChI=1S/C44H72N12O13S/c1-22(2)16-27(45)37(63)53-31(18-25-9-11-26(57)12-10-25)41(67)54-30(17-23(3)4)42(68)56-36(24(5)6)43(69)55-32(21-70)39(65)49-19-33(58)51-29(13-14-34(59)60)40(66)52-28(8-7-15-48-44(46)47)38(64)50-20-35(61)62/h9-12,22-24,27-32,36,57,70H,7-8,13-21,45H2,1-6H3,(H,49,65)(H,50,64)(H,51,58)(H,52,66)(H,53,63)(H,54,67)(H,55,69)(H,56,68)(H,59,60)(H,61,62)(H4,46,47,48)/t27-,28-,29-,30-,31-,32-,36-/m0/s1

(4S)-4-[[2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-4-methylpentanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-methylpentanoyl]amino]-3-methylbutanoyl]amino]-3-sulfanylpropanoyl]amino]acetyl]amino]-5-[[(2S)-1-(carboxymethylamino)-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-5-oxopentanoic acid

Insulin beta Chain Peptide (15-23); 247044-67-3; Insulin ; A Chain Peptide (15-23); Insulin ?? Chain Peptide (15-23);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)