Experimental myasthenia gravis in congenic mice. Sequence mapping and H‐2 restriction of T helper epitopes on the α subunits of <i>Torpedo californica</i> and murine acetylcholine receptors

Bellone, Matteo; Ostlie, Norma; Liu, Sijin; Conti‐Tronconi, Bianca M.

doi:10.1002/eji.1830211003

Cited by 54 publications

(64 citation statements)

References 56 publications

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“…We purified TAChR from electric tissue of Torpedo californica as alkalistripped TAChR-rich membrane fragments (24). The AChR structure is very conserved along evolution: TAChR is highly homologous to mammalian MAChR and suitable to induce EMG (4).…”

Section: Purification Of Torpedo Achr (Tachr)mentioning

confidence: 99%

“…The AChR structure is very conserved along evolution: TAChR is highly homologous to mammalian MAChR and suitable to induce EMG (4). We measured the protein concentration by the Lowry assay (25) and the TAChR concentration as ␣-bungarotoxin (␣BTX)-binding sites (24). The TAChR preparations we used contained 3.8 -5.8 nmol of sites/mg protein.…”

Section: Purification Of Torpedo Achr (Tachr)mentioning

confidence: 99%

“…The TAChR preparations we used contained 3.8 -5.8 nmol of sites/mg protein. SDS-PAGE analysis (24) showed that the TAChR preparations contained only the four TAChR subunits as the main protein bands. For cell cultures, we diluted the TAChRrich membrane fragments in RPMI 1640 as needed and sterilized them by UV irradiation.…”

Section: Purification Of Torpedo Achr (Tachr)mentioning

confidence: 99%

“…We determined the epitope repertoire on the AChR ␣ subunit, which dominates the sensitization of anti-TAChR CD4 ϩ cells in B6 mice (24,26), by using a panel of synthetic peptides ϳ20 residue long and overlapping by ϳ5 residues that spanned the sequence of the TAChR ␣ subunit. We reported their characterization previously (24).…”

Section: Peptidesmentioning

confidence: 99%

“…We reported their characterization previously (24). We used solutions of the individual peptides in PBS sterilized by UV irradiation and stored frozen.…”

Section: Peptidesmentioning

confidence: 99%

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Transgenic Expression of IL-10 in T Cells Facilitates Development of Experimental Myasthenia Gravis

Ostlie

Karachunski

Wang

et al. 2001

The Journal of Immunology

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Ab to the acetylcholine receptor (AChR) cause experimental myasthenia gravis (EMG). Th1 cytokines facilitate EMG, whereas Th2 cytokines might be protective. IL-10 inhibits Th1 responses but facilitates B cell proliferation and Ig production. We examined the role of IL-10 in EMG by using wild-type (WT) C57BL/6 mice and transgenic (TG) C57BL/6 mice that express IL-10 under control of the IL-2 promoter. We immunized the mice with doses of AChR that cause EMG in WT mice or with low doses ineffective at causing EMG in WT mice. After low-dose AChR immunization, WT mice did not develop EMG and had very little anti-AChR serum Ab, which were mainly IgG1, whereas TG mice developed EMG and had higher levels of anti-AChR serum Ab, which were mainly IgG2, in addition to IgG1. At the higher doses, TG mice developed EMG earlier and more frequently than WT mice and had more serum anti-AChR Ab. Both strains had similar relative serum concentrations of anti-AChR IgG subclasses and IgG and complement at the muscle synapses. CD8+-depleted splenocytes from all AChR-immunized mice proliferated in the presence of AChR and recognized a similar epitope repertoire. CD8+-depleted splenocytes from AChR-immunized TG mice stimulated in vitro with AChR secreted significantly more IL-10, but less of the prototypic Th1 cytokine IFN-γ, than those from WT mice. They secreted comparable amounts of IL-4 and slightly but not significantly reduced amounts of IL-2. This suggests that TG mice had reduced activation of anti-Torpedo AChR Th1 cells, but increased anti-AChR Ab synthesis, that likely resulted from IL-10-mediated stimulation of anti-AChR B cells. Thus, EMG development is not strictly dependent on Th1 cell activity.

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Section: Purification Of Torpedo Achr (Tachr)mentioning

confidence: 99%

Section: Purification Of Torpedo Achr (Tachr)mentioning

confidence: 99%

Section: Purification Of Torpedo Achr (Tachr)mentioning

confidence: 99%

Section: Peptidesmentioning

confidence: 99%

“…We reported their characterization previously (24). We used solutions of the individual peptides in PBS sterilized by UV irradiation and stored frozen.…”

Section: Peptidesmentioning

confidence: 99%

See 3 more Smart Citations

Transgenic Expression of IL-10 in T Cells Facilitates Development of Experimental Myasthenia Gravis

Ostlie

Karachunski

Wang

et al. 2001

The Journal of Immunology

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Preferential pairing of T and B cells for production of antibodies without covalent association of T and B epitopes

et al. 1994

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T cell from H-2b mice recognize at least 12 sequence regions on the Torpedo acetylcholine receptor (TAChR) alpha, gamma and delta subunits. Immunization of C57BL/6 mice with individual synthetic TAChR sequences known to contain CD4+ epitopes resulted in most cases (10 out of 12 peptides) in anti-peptide antibody (Ab) production, indicating that short TAChR sequences contain both CD4+ and B epitopes. Immunization of C57BL/6 mice with a mixture of a CD4+ epitope peptide, from the TAChR or from an unrelated protein, plus another TAChR sequence forming a "pure" B epitope (T alpha 63-80), induced in most cases anti-peptide Ab and CD4+ cell sensitization only against the peptide containing the CD4+ epitope. However, when the T epitope peptide T alpha 360-378 was co-injected with the B epitope, Ab were also produced against the B epitope peptide. Injection of the individual peptides T alpha 360-378 and T alpha 63-80 at different and distant sites along the back of mice elicited sensitization of CD4+ cells and Ab production only against peptide T alpha 360-378. Therefore, when optimal cooperation between T and B cells occurs, spatial proximity but not covalent association of the B and the CD4+ epitope is necessary for production of Ab against the B epitope.

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In vitro priming of cytotoxic T lymphocytes against poorly immunogenic epitopes by engineered antigen‐presenting cells

et al. 1994

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Cytotoxic T lymphocytes (CTL) recognize antigenic peptides presented by major histocompatibility complex class I (MHC-I) molecules on the surface of target cells. Optimal induction of CD8+ CTL depends on the amount of relevant peptide/MHC-I complexes and the presence of co-stimulatory molecules on antigen-presenting cells (APC). The antigen-processing defective mutant cell line RMA-S, when cultured at low temperature, expresses high amounts of MHC-I molecules that do not contain endogenously derived peptides. These "empty" MHC-I molecules can be stabilized by addition of MHC-binding peptides. RMA-S cultured at low temperatures with selected peptides have been used for in vitro CTL induction with conflicting results. RMA-S cells do not express detectable amounts of B7 co-stimulatory molecule. This could explain their unpredictable efficiency as APC. We have evaluated whether RMA-S cells, stably transfected with cDNA encoding for the human B7.1 molecule could provide effective co-stimulation for CD8+ T lymphocytes. RMA-S/B7 cells, loaded with different synthetic peptides, demonstrated a high and sometimes unique efficiency for in vitro primary CTL induction, even when "sub-optimal" antigen peptides were used. Most importantly, RMA-S/B7 cells pulsed with naturally processed peptides extracted from the poorly immunogenic B16 melanoma cells were able to prime CD8+ cells against B16 melanoma. We conclude that the use of RMA-S/B7 cells as APC represents an ideal strategy for in vitro CTL immunization without prior in vivo priming. This system may also help to address the issue of the different contributions of co-stimulation and relative occupancy of MHC-I by single peptide epitopes in CTL priming.

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Experimental myasthenia gravis in congenic mice. Sequence mapping and H‐2 restriction of T helper epitopes on the α subunits of Torpedo californica and murine acetylcholine receptors

Cited by 54 publications

References 56 publications

Transgenic Expression of IL-10 in T Cells Facilitates Development of Experimental Myasthenia Gravis

Transgenic Expression of IL-10 in T Cells Facilitates Development of Experimental Myasthenia Gravis

Preferential pairing of T and B cells for production of antibodies without covalent association of T and B epitopes

In vitro priming of cytotoxic T lymphocytes against poorly immunogenic epitopes by engineered antigen‐presenting cells

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