Assessing the diversity of the human ig repertoire after B cell cryopreservation and restimulation

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Cryopreservation of human B cells is widely employed in various clinical and research applications. However, it is still commonly believed that only freshly isolated B cells should be utilized for subsequent analyses to accurately evaluate the natural immunoglobulin (Ig) repertoire in both quantitative and qualitative dimensions. In this study, we use next-generation sequencing to investigate how the Ig repertoire reshapes after the cryopreservation of B cells. Our focus centers on examining the proportional representation of the Ig repertoire after freeze-thawing, both with and without subsequent restimulation. Our research findings encourage scientists to conduct experiments on the Ig repertoire using cryopreserved, patient-derived B cells, underscoring the potential clinical and experimental applications of monitoring the Ig repertoire with cryopreserved B cells.

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Sobre autores

A. Smirnova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: yasha.l@bk.ru
Rússia, 117997 Moscow

L. Ovchinnikova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: yasha.l@bk.ru
Rússia, 117997 Moscow

I. Mamedov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: yasha.l@bk.ru
Rússia, 117997 Moscow

T. Grigoreva

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: yasha.l@bk.ru
Rússia, 117997 Moscow

S. Khazeev

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: yasha.l@bk.ru
Rússia, 117997 Moscow

M. Akhmedova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: yasha.l@bk.ru
Rússia, 117997 Moscow

Y. Lomakin

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Autor responsável pela correspondência
Email: yasha.l@bk.ru
Rússia, 117997 Moscow

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2. Fig. 1. Evaluation of immunoglobulin clonotype diversity under different B-cell cryopreservation conditions. a – Average number of observed IGH (immunoglobulin heavy chain) clonotypes. Unique clonotypes are those that were detected in only one B-cell sample cryopreserved under the specified conditions. Common clonotypes are those that contain identical V, D, J, and C gene segments and the same H-CDR3 nucleotide sequence and are found in at least two different samples. Relative frequency of clonotypes (b) and reads (UMI) (c) obtained for clones with different abundance. Low-frequency clones are rare sequences that were read in the studied sample only once. Clones with multiple reads are sequences with two or more reads. Common clones are sequences found in the test sample and at least one of the freshly isolated B cell samples. Data are based on 3.5 × 104 randomly selected NGS reads for each replicate. Statistical significance of differences between samples was assessed using the Mann–Whitney U test. Significance levels: * p < 0.05 and ** p < 0.005. Legend: Original – B cells lysed in TRIzol immediately after isolation; Cryo – frozen-thawed B cells; 24 h – B cells stimulated in vitro for 24 h after thawing; 72 h – B cells stimulated in vitro for 72 h after thawing

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3. Fig. 2. Effect of B-lymphocyte cryopreservation method on the distribution of immunoglobulin isotypes and the frequency of somatic hypermutations. a – UMI frequency; b – clonotype frequency. Stacked bar graphs showing the distribution of immunoglobulin isotypes in the total B-cell population: freshly isolated (Initial), after freezing and thawing (Cryo), and after stimulation for 24 or 72 h after thawing. c – Distribution of the frequency of somatic hypermutations in the variable domain of the immunoglobulin heavy chain (VH) under different experimental conditions, presented as violin plots. Two biological replicates are presented for each cryopreservation condition (biological replicates for each cryopreservation condition are highlighted in the same color). Data are presented as mean ± standard deviation (SD). Statistical significance of differences between groups was assessed using one-way analysis of variance (ANOVA). Significance levels: * p < 0.05; ** p < 0.005; **** p < 0.00005.

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4. Fig. 3. Characterization of high-abundance clones. a – Distribution of read frequency (UMI) per clone. Clones were classified as high-abundance if their proportion exceeded 0.1% of the total number of reads in the replicate. Statistical significance: one-way ANOVA (**** p < 0.00005). Two biological replicates are presented for each cryopreservation condition (the replicate number is given in brackets to the right of the sample name). Relative distribution of immunoglobulin isotypes among high-abundance clones in B-cell samples obtained under different freeze-thaw conditions based on the number of UMI reads (b) and on the number of detected clonotypes (c). Designations: Original – B cells lysed in TRIzol immediately after isolation; Cryo – frozen-thawed B cells; 24 h – B cells stimulated in vitro for 24 h after thawing; 72 h – B cells stimulated in vitro for 72 h after thawing

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5. Fig. 4. Frequencies of IGH germline genes in B cell samples obtained under different cryopreservation conditions. Scatter plot showing the correlation of variable heavy chain (VH) germline gene usage between two biological replicates of freshly isolated B cells, according to UMI count (a) or clonotype count (b). Frequencies of usage of different V (c), D (r), and J segments (d) of immunoglobulin heavy chains. Heat map displays the proportion of each identified gene segment for each B cell sample, with mean abundances indicated. Key: Original – B cells lysed in TRIzol immediately after isolation; Cryo – frozen-thawed B cells; 24 h – B cells stimulated in vitro for 24 h after thawing; 72 h – B cells stimulated in vitro for 72 h after thawing

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6. Appendix 1
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7. Appendix 2
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