T Cell Mediated Conversion of a Non-Anti-La Reactive B Cell to an Autoreactive Anti-La B Cell by Somatic Hypermutation †
Abstract
:1. Introduction
2. Results
2.1. Hybridoma Fusion from a Human La Transgenic Mouse after Adoptive Transfer of T Cells from Non-Transgenic Mice Immunized with Human La
2.2. The Hybridomas Originate from the La Transgenic Mouse
2.3. The Hybridomas Originate from Individual B Cells
2.4. Reactivity of Anti-La Mabs to Denatured and Native Human and Mouse La Protein
2.4.1. Analysis by SDS-PAGE and Immunoblotting Using La Proteins Recombinantly Expressed in E. coli and Total Extracts from Murine or Human Cell Lines
2.4.2. Coprecipitation Study of Native Human and Mouse La Protein from Total Cell Extracts of a Mouse 3T3 Cell Line Expressing the Human La Transgene
2.4.3. Immunofluorescence Analysis Using Human and Murine Cell Lines
2.5. Epitope Mapping
2.6. Lack of Anti-La Reactivity of the 312B Germline Sequence
3. Discussion
4. Materials and Methods
4.1. Recombinant Human La Protein Expression and Characterization
4.2. Mice
4.3. T Cell Isolation, Adoptive Transfer, and Hybridoma Fusion
4.4. Hybridoma Fusion
4.5. Sequence Analysis
4.6. Construction, Expression, and Purification of Recombinant Antibodies
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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mab | Isotype | Vl | Jl | Vh | Dh | Jh | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
E | %ID | E | %ID | E | %ID | E | %ID | E | %ID | ||
SW5 | IgG2b | 21–12 | 98% | JK1 | 86% | VH7183.a7.10 | 94% | DSP2.7 DSP2.5 | 91% | JH3 | 98% |
7B6 | IgG1 | 8–21 | 98% | JK1 | 100% | VHJ606.a6.127 | 88% | DSP2.8 DSP2.7 DSP2.5 | 100% | JH3 | 100% |
5B9 | IgG2a | 8–21 | 98% | JK5 | 100% | J558.33 | 91% | DSP2.2 | 100% | JH3 | 100% |
24BG7 | IgG1 | kk4 | 99% | JK2 | 100% | J558.22 | 94% | DST4.3 DST4.2 | 100% | JH3 | 100% |
22A | IgG1 | kk4 | 99% | JK5 | 94% | J558.80.186 | 89% | DSP2.9 | 89% | JH2 | 100% |
27E | IgG2b | aa4 | 97% | JK1 | 100% | J558.22 | 93% | DSP2.3 | 100% | JH2 | 100% |
312B | IgG1 | 12–44 | 98% | JK2 | 100% | J558.6.96 | 94% | DSP2.9 | 100% | JH2 | 96% |
2F9 | IgG1 | 21–5 | 99% | JK4 | 100% | 3609.21.174 | 96% | DST4.3 DST4.2 | 100% | JH2 | 100% |
32A | IgG1 | aa4 | 99% | JK1 | 100% | J558.84.190 | 93% | DSP2.x | 100% | JH2 | 100% |
16C | IgM | bt20 | 99% | JK2 | 100% | J558.16.106 | 96% | DFL16.3 | 100% | JH4 | 100% |
13C5B | IgM | aa4 | 98% | JK1 | 100% | J558.47 | 97% | DSP2.11 DSP2.10 | 100% | JH4 | 98% |
312G | IgM | kk4 | 98% | JK5 | 100% | VHQ52.a19.61 | 98% | DFL16.3 | 100% | JH4 | 100% |
mab | Number of Mutations | n-Nucleotides | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
FWR1 | CDR1 | FWR2 | CDR2 | FWR3 | CDR3 | FWR4 | |||||||||
N | A | N | A | N | A | N | A | N | A | N | A | N | A | Upstream Jl | |
SW5 | 1 | 1 | 2 | 2 | 1 | 1 | 1 | 1 | 4 | 3 | +3 | ||||
7B6 | 2 | 2 | 1 | 1 | +2 | ||||||||||
5B9 | 1 | 1 | 1 | 1 | +1 | ||||||||||
24BG7 | 1 | 1 | 1 | 1 | 1 | 1 | −1 | ||||||||
22A | 2 | 1 | 1 | 2 | 2 | +1 | |||||||||
27E | 2 | ||||||||||||||
312B | 1 | 1 | 1 | 1 | 1 | +1 | |||||||||
2F9 | 1 | +1 | |||||||||||||
32A | 1 | 1 | 1 | 1 | |||||||||||
16C | +2 | ||||||||||||||
13C5B | |||||||||||||||
312G | −1 |
mab | Number of Mutations | n-Nucleotides | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
FWR1 | CDR1 | FWR2 | CDR2 | FWR3 | CDR3 | |||||||||
N | A | N | A | N | A | N | A | N | A | N | A | Upstream Dh | Downstream Dh | |
SW5 | 1 | 1 | 8 | 5 | 8 | 3 | 2 | 2 | +2 | +1 | ||||
7B6 | 5 | 2 | 2 | 2 | 5 | 13 | 8 | 6 | 2 | +4 | ||||
5B9 | 3 | 1 | 6 | 4 | 4 | 1 | 6 | 5 | 3 | 1 | +6 | +6 | ||
24BG7 | 9 | 6 | 3 | 2 | 3 | 3 | 3 | 2 | +3 | +3 | ||||
22A | 3 | 3 | 3 | 2 | 2 | 12 | 7 | 8 | 4 | 1 | +12 | −3 | ||
27E | 8 | 6 | 4 | 3 | 3 | 3 | 5 | 2 | +5 | +6 | ||||
312B | 3 | 3 | 2 | 1 | 1 | 1 | 3 | 3 | 4 | 2 | 2 | 1 | +10 | −4 |
2F9 | 1 | 1 | 1 | 1 | −5 | +13 | ||||||||
32A | 2 | 3 | 3 | 6 | 6 | 5 | 2 | +6 | ||||||
16C | 3 | 2 | 2 | 2 | 1 | 1 | +7 | +6 | ||||||
13C5B | 1 | 1 | 3 | 2 | 1 | 1 | 2 | 1 | 1 | +1 | +5 | |||
312G | 2 | 1 | +4 | +5 |
mab | Reactivity Recombinant Proteins Immunoblotting | Reactivity m3T3 hLa Immunoblotting | Immuno-Precipitation m3T3 hLa | Immuno-Fluorescence PFA | Immuno-Fluorescence MeOH | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
rhLa | rmLa | HeLa | 3T3 | hLa | mLa | hLa | mLa | HeLa | 3T3 | HeLa | 3T3 | |
SW5 | +++ | ++ | +++ | - | +++ | - | ++ | - | +++ | - | ++ | - |
7B6 | +++ | ++ | +++ | - | ++ | - | - | - | (+) | - | (+) | - |
5B9 | +++ | +++ | +++ | +++ | +++ | +++ | - | - | ++ | - | +++ | +++ |
24BG7 | +++ | + | +++ | + | ++ | + | + | - | ++ | ++ | - | - |
22A | +++ | - | +++ | - | + | - | ++ | - | ++ | - | - | - |
27E | +++ | + | +++ | +++ | ++ | ++ | + | + | +++ | +++ | +++ | + |
312B | +++ | +++ | +++ | +++ | + | ++ | +++ | +++ | +++ | +++ | ++ | + |
2F9 | +++ | +++ | ++ | ++ | + | + | - | - | + | + | (+) | - |
32A | +++ | - | ++ | - | (+) | - | - | - | (+) | - | - | - |
16C | + | * | * | * | * | * | * | * | C | C | C | C |
13C5B | + | * | * | * | * | * | * | * | C | C | C | C |
312G | - | * | * | * | * | * | * | * | C | C | C | C |
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Bachmann, M.P.; Bartsch, T.; Bippes, C.C.; Bachmann, D.; Puentes-Cala, E.; Bachmann, J.; Bartsch, H.; Arndt, C.; Koristka, S.; Loureiro, L.R.; et al. T Cell Mediated Conversion of a Non-Anti-La Reactive B Cell to an Autoreactive Anti-La B Cell by Somatic Hypermutation. Int. J. Mol. Sci. 2021, 22, 1198. https://doi.org/10.3390/ijms22031198
Bachmann MP, Bartsch T, Bippes CC, Bachmann D, Puentes-Cala E, Bachmann J, Bartsch H, Arndt C, Koristka S, Loureiro LR, et al. T Cell Mediated Conversion of a Non-Anti-La Reactive B Cell to an Autoreactive Anti-La B Cell by Somatic Hypermutation. International Journal of Molecular Sciences. 2021; 22(3):1198. https://doi.org/10.3390/ijms22031198
Chicago/Turabian StyleBachmann, Michael P., Tabea Bartsch, Claudia C. Bippes, Dominik Bachmann, Edinson Puentes-Cala, Jennifer Bachmann, Holger Bartsch, Claudia Arndt, Stefanie Koristka, Liliana R. Loureiro, and et al. 2021. "T Cell Mediated Conversion of a Non-Anti-La Reactive B Cell to an Autoreactive Anti-La B Cell by Somatic Hypermutation" International Journal of Molecular Sciences 22, no. 3: 1198. https://doi.org/10.3390/ijms22031198