Sulfated beef insulin treatment elicits CD8+ T cells that may abrogate immunologic insulin resistance in type I diabetes.

Naquet, Philippe; Ellis, Janet; Kenshole, Anne B.; Semple, John W.; Delovitch, Terry L.

doi:10.1172/jci114323

Cited by 14 publications

(13 citation statements)

References 26 publications

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“…1C Lower). Rather, some DC/M4 peptides consist of C-terminal A-chain residues A20 and A21 disulfide-linked to core B-chain residues, and such peptides are not immunogenic (4,22 2A). This peak was absent from precipitated DC/MO samples (Fig.…”

Section: Methodsmentioning

confidence: 99%

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Naturally processed heterodimeric disulfide-linked insulin peptides bind to major histocompatibility class II molecules on thymic epithelial cells.

Forquet

Hadžija

Semple

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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We determined whether disulfide-linked insulin peptides that are immunogenic in vitro for CD4+ T cells bind to major histocompatibility complex class II in vivo.Radiolabeled recombinant human insulin (rHI) was injected into BALB/c mice, and processed rHI peptides bound to I-Ad molecules on different thymic antigen-presenting cells were characterized. The A6-All/B7-B19 and A19-A21/B14-B21 disufide-linked I-Ad-bound MATERIALS AND METHODS Recombinant HI (rHI) purified from Escherichia coli K-12 strain CA7233 transformed with plasmid plac 9/4 PI (Fig. 1A) was radiolabeled (-3 mCi/mmol; 1 Ci = 37 GBq) at 19 distinct amino acids (Fig. 1B) (8). Labeled rHI was indistinguishable from commercially available HI (8) and was injected into 4-to 6-week-old female BALB/c (H-2d) mice.Cortical epithelial cells (thymic nurse cells; TNCs), corticomedullary DC rosettes (T-ROS), and a separate MHC class III medullary DC/macrophage (MO) population were isolated (9). The DC/M4 population was recovered as lowdensity cells (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Remaining tissue fragments were digested with collagenase type XI (0.5 mg/ml, from Clostridium histolytum; Sigma) to obtain T-ROS and with 0.02% trypsin (0.5 ml per thymus; Sigma) in Hanks' balanced salt solution (HBSS) containing DNase I (8 pug/ml; Sigma) to purify TNCs. Before assay, TNCs were allowed to reexpress surface MHC class II during culture (16 hr, 370C) in RPMI 1640 (GIBCO) containing 10 mM Hepes (pH 7.4), 2 mM L-glutamine, 50 AuM 2-mercaptoethanol, penicillin (100 units/ ml), streptomycin (100 pg/ml), and 5% (vol/vol) fetal calf serum (CRPMI) and supplemented with interleukin 2 (IL-2; 15 units/ml).APC yields (per mouse) were 4.2 x 104 for DCs/Mos, 1.4x 105 for T-ROS, and 3.5 x 104 for TNCs. Enrichment of APCs was assessed by immunofluorescence using the anti-I-Ad MK-D6 (10), J1lD and 33D1 (both detect thymic DCs) (11), anti-CD4 GK 1.5 (12), or anti-CD8 53-6.62 (13) monoclonal antibodies. The relative I-Ad surface densities were TNCs > T-ROS > DCs/Mos, with the density on TNCs being :2-fold greater than that of DCs/M4s. APCs represented =60%o of each preparation, and their relative purity was 85-90%, as reported (14).Noncovalently linked membrane-associated peptides were acid eluted (15 min, 4°C; HBSS at pH 3.6) from thymic APCs and resolved by reversed-phase C18 HPLC (15). Intracellular peptides were extracted from the acid-treated cell pellet, passed through Sephadex G-50 to obtain "insulin-sized peaks," and analyzed by C18 HPLC (15 Pharmacia). After cell lysis and washing of the Sepharose beads in PBS containing 0.2% digitonin, immune complexes were dissociated with 8 M urea (pH 4.0) and passed through Sephadex G-25 to fractionate "insulin-sized" peaks. Labeled

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Section: Methodsmentioning

confidence: 99%

“…We previously demonstrated that heterodimeric disulfidelinked insulin peptides are presented by B cells to CD4+ T cells in vitro (4)(5)(6). Since this suggested that a disulfide-linked peptide binds to MHC class II, we analyzed whether human insulin (HI) is processed into a disulfide-linked peptide that binds to MHC class II in vivo.…”

mentioning

confidence: 99%

Naturally processed heterodimeric disulfide-linked insulin peptides bind to major histocompatibility class II molecules on thymic epithelial cells.

Forquet

Hadžija

Semple

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…Insulin and proinsulin are targets of autoantibodies (13)(14)(15) and T cells (16)(17)(18)(19)(20)(21)(22)(23) in diabetic and prediabetic subjects. Anti-insulin antibodies are the first autoantibodies detected in children at risk for diabetes (15).…”

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confidence: 99%

Recognition of a subregion of human proinsulin by class I-restricted T cells in type 1 diabetic patients

Toma

Haddouk

Briand

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

117

171

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Proinsulin is a key autoantigen in type 1 diabetes. Evidence in the mouse has underscored the importance of the insulin B chain region in autoimmunity to pancreatic beta cells. In man, a majority of proteasome cleavage sites are predicted by proteasome cleavage algorithms within this region. To study CD8 ؉ T cell responses to the insulin B chain and adjacent C peptide, we selected 8-to 11-mer peptides according to proteasome cleavage patterns obtained by digestion of two peptides covering proinsulin residues 28 to 64. We studied their binding to purified HLA class I molecules and their recognition by T cells from diabetic patients. Peripheral blood mononuclear cells from 17 of 19 recent-onset and 12 of 13 long-standing type 1 diabetic patients produced IFN-␥ in response to proinsulin peptides as shown by using an ELISPOT assay. In most patients, the response was against several class I-restricted peptides. Nine peptides were recognized within the proinsulin region covering residues 34 to 61. Four yielded a high frequency of recognition in HLA-A1 and -B8 patients. Three peptides located in the proinsulin region 41-51 were shown to bind several HLA molecules and to be recognized in a high percentage of diabetic patients.autoimmunity ͉ beta cell ͉ CD8 T cells ͉ human autoantigens ͉ proteasome T ype 1 diabetes is characterized by the activation of lymphocytes against autoantigens expressed by pancreatic beta cells. T lymphocytes play a key role in the disease process. Diabetes has been reported in a patient deprived of B lymphocytes (1). In the nonobese diabetic (NOD) mouse, CD8 ϩ T cells play a pivotal role in the initiation of autoimmunity (2). Beta 2 microglobulindeficient NOD mice do not develop insulitis unless beta cell class I expression is restored (3). CD8 ϩ T cells are responsible for beta cell destruction in transgenic mice over expressing beta cell-specific CD8 ϩ T cells (2). In man, CD8 ϩ T cells and IFN-␥-positive cells are major components of insulitis (4-9). Recurrent diabetes in recipients of isografts from a discordant identical twin is accompanied by predominant CD8 ϩ T cell infiltration (10). However, few studies have characterized recognition of beta cell antigens by CD8 ϩ T cells (11,12).Proinsulin has been ascribed a key role in diabetes. Insulin and proinsulin are targets of autoantibodies (13-15) and T cells (16)(17)(18)(19)(20)(21)(22)(23) in diabetic and prediabetic subjects. Anti-insulin antibodies are the first autoantibodies detected in children at risk for diabetes (15). In the NOD mouse, transfer of insulin-specific T cells accelerates diabetes (24), and insulin exposure prevents diabetes (25). Diabetes development is altered in mice lacking the expression of proinsulin genes (26-28). Evidence in the mouse has underscored the importance of the proinsulin region encompassing the insulin B chain in diabetes autoimmunity (25,29). CD8 ϩ T cells that transfer diabetes in the NOD recognize an insulin B chain epitope (30). In man, a majority of proteasome cleavage sites are predicted within ...

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“…Because of this close homology, thymic IGF-2 would be a good candidate for inducing central immune self-tolerance of the whole insulin family although the tolerance to insulin per se would be weaker. This again might explain why B-and Tcell autoreactivity to insulin has been equally observed in diabetic and related non-diabetic individuals [33].…”

Section: Central Self-tolerance Of the Insulin Familymentioning

confidence: 93%

“…This is indirectly reflected by the frequency and titer of antibodies obtained after active immunization with the three peptides [32]. In the same line of reasoning, the high occurrence of anti-insulin autoantibodies in the normal population [33] could be linked to the low level of INS expression within the human thymus. The IGF-2 protein contains peptide sequences that have been highly conserved throughout evolution of the insulin family.…”

Section: Central Self-tolerance Of the Insulin Familymentioning

confidence: 95%

Presentation of Neuroendocrine Self in the Thymus: Toward a Novel Type of Vaccine / Immunotherapy

Geenen¹,

Brilot²,

Hansenne³

et al. 2004

DDRO

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Slightly after the emergence some 400 millions years ago of the first signs of adaptive immune response, tolerogenic pathways developed in order to preserve the integrity of self from potential autoimmune toxicity. Amongst those tolerogenic pathways, the thymus occupies a central place both by deleting self-reactive T cells that are produced in the thymus during random recombination of gene segments encoding the variable parts of the T-cell receptor for antigen (TCR) (negative selection), and by generating self-antigen specific regulatory T cells (Tr). A repertoire of neuroendocrine-related genes are transcribed by thymic stromal cells -epithelial and 'nurse' cells (TEC/TNC), dendritic cells (DC) and macrophages (MΦ) -in such a way that a dominant protein precursor is expressed in the thymus environment. Oxytocin (OT) and neurokinin A (NKA) are the dominant thymic precursors for the neurohypophysial hormone and tachykinin families, respectively. With regard to the insulin gene family, all members are transcribed following a precise cell topography and hierarchy in the profile of gene expression: IGF2 (TEC/TNC) > IGF1 (MΦ) >> INS (medullary TEC and/or DC). This hierarchy implies that IGF-2 is more tolerated than IGF-1, and much more than Insulin (Ins). The low level of INS transcription in the thymus also explains why Ins displays immunogenic properties, as well as the significant prevalence (±40%) of anti-Ins autoantibodies in the general population. Ins administration failed in providing tolerance or protection toward islet ß cells in type 1 diabetes (T1D). In contrast, the presentation of IGF-2 B11-25, the homologous sequence of Ins B9-23, to peripheral blood mononuclear cells (PBMC) isolated from DQ8+ T1D adolescents significantly increases IL-10 secretion and IL10 expression. Given the potent regulatory/suppressive properties of IL-10 on the autoimmune response toward islet ß cells, these data support that IGF-2 derived sequences constitute a strong basis for the development of an antigen-specific driven tolerogenic approach for T1D prevention and/or cure.

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Sulfated beef insulin treatment elicits CD8+ T cells that may abrogate immunologic insulin resistance in type I diabetes.

Abstract: The in vitro responses of T cells from 13

Cited by 14 publications

References 26 publications

Naturally processed heterodimeric disulfide-linked insulin peptides bind to major histocompatibility class II molecules on thymic epithelial cells.

Naturally processed heterodimeric disulfide-linked insulin peptides bind to major histocompatibility class II molecules on thymic epithelial cells.

Recognition of a subregion of human proinsulin by class I-restricted T cells in type 1 diabetic patients

Presentation of Neuroendocrine Self in the Thymus: Toward a Novel Type of Vaccine / Immunotherapy

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