High-Throughput Single-Cell Sequencing of both <i>TCR-β</i> Alleles

Hosoya, Takamitsu; HongYang, Li; Ku, Chia-Jui; Wu, Qingqing; Engel, James Douglas

doi:10.4049/jimmunol.1800774

Cited by 7 publications

(8 citation statements)

References 20 publications

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“…Pre-existing immunity has also been demonstrated for the bacterial CAS9 protein and could dramatically limit genome editing tools such as CRISPR/Cas9 technology ( 136 ). All these new therapeutic approaches might benefit from already off-the-shelf technologies to investigate the T cell response and from recent advances in single-cell analysis ( 137 ) and next-generation sequencing ( 138 ).…”

Section: Perspectivesmentioning

confidence: 99%

Specificity of the T Cell Response to Protein Biopharmaceuticals

et al. 2020

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The anti-drug antibody (ADA) response is an undesired humoral response raised against protein biopharmaceuticals (BPs) which can dramatically disturb their therapeutic properties. One particularity of the ADA response resides in the nature of the immunogens, which are usually human(ized) proteins and are therefore expected to be tolerated. CD4 T cells initiate, maintain and regulate the ADA response and are therefore key players of this immune response. Over the last decade, advances have been made in characterizing the T cell responses developed by patients treated with BPs. Epitope specificity and phenotypes of BP-specific T cells have been reported and highlight the effector and regulatory roles of T cells in the ADA response. BP-specific T cell responses are assessed in healthy subjects to anticipate the immunogenicity of BP prior to their testing in clinical trials. Immunogenicity prediction, also called preclinical immunogenicity assessment, aims at identifying immunogenic BPs and immunogenic BP sequences before any BP injection in humans. All of the approaches that have been developed to date rely on the detection of BP-specific T cells in donors who have never been exposed to BPs. The number of BP-specific T cells circulating in the blood of these donors is therefore limited. T cell assays using cells collected from healthy donors might reveal the weak tolerance induced by BPs, whose endogenous form is expressed at a low level. These BPs have a complete human sequence, but the level of their endogenous form appears insufficient to promote the negative selection of autoreactive T cell clones. Multiple T cell epitopes have also been identified in therapeutic antibodies and some other BPs. The pattern of identified T cell epitopes differs across the antibodies, notwithstanding their humanized, human or chimeric nature. However, in all antibodies, the non-germline amino acid sequences mainly found in the CDRs appear to be the main driver of immunogenicity, provided they can be presented by HLA class II molecules. Considering the fact that the BP field is expanding to include new formats and gene and cell therapies, we face new challenges in understanding and mastering the immunogenicity of new biological products.

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Section: Perspectivesmentioning

confidence: 99%

Specificity of the T Cell Response to Protein Biopharmaceuticals

et al. 2020

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“…Single cells were sorted directly into 10 mL lysis buffer (1x Herculase II reaction buffer, 20 mg/ml Proteinase K (Thermofisher), 0.1% Triton X-100 (Sigma-Aldrich)) and digested (1 hour at 55 C followed by 15' at 95 C) as described elsewhere (Hosoya et al, 2018). The first PCR was performed in a 20 mL reaction (1x Herculase II buffer, 50 nM each primer, 250 mM dNTPs and 0.4 mL Herculase II fusion DNA Polymerase).…”

Section: Analysis Of Vdjb Rearrangements In Single Cellsmentioning

confidence: 99%

“…The second PCR was performed using 1 mL of the first PCR product in the same conditions. Primers used in both PCR rounds were the same as described (Hosoya et al, 2018) (Table S5). Successful PCRs were confirmed by running 5 mL of the second PCR product in agarose gels.…”

Section: Analysis Of Vdjb Rearrangements In Single Cellsmentioning

confidence: 99%

Self-renewal of double-negative 3 early thymocytes enables thymus autonomy but compromises the β-selection checkpoint

et al. 2021

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Self-renewal of double-negative 3 early thymocytes enables thymus autonomy but compromises the b-selection checkpoint Graphical abstract Highlights d Thymus autonomy depends on DN3e thymocytes that secure T cell differentiation d DN3e in autonomy self-renew, retain DNA label, and have enhanced proliferation d Thymus autonomy preserves the hallmarks of thymopoiesis at the single-cell level d Aberrant cells with active notch that bypassed b-selection emerge in autonomy

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“…Single cells were sorted directly into 10 μl lysis buffer (1x Herculase II reaction buffer, 20 mg/ml Proteinase K (Thermofisher), 0.1 % Triton X100 (Sigma-Aldrich)) and digested (1 hour at 55 ºC followed by 15' at 95ºC) as described elsewhere 26 . The first PCR was performed in a 20 μl reaction (1x Herculase II buffer, 50 nM each primer, 250 μM dNTPs and 0.4 μl Herculase II fusion DNA Polymerase).…”

Section: Analysis Of Vdj Rearrangements Of Tcrβ In Single Cellsmentioning

confidence: 99%

“…The second PCR was performed using 1 μl of the first PCR product in the same conditions. Primers used in both PCR rounds were the same as described 26 (and supplementary table 5). Successful PCRs were confirmed by running 5 μl of the second PCR product in agarose gels.…”

Section: Analysis Of Vdj Rearrangements Of Tcrβ In Single Cellsmentioning

confidence: 99%

Self-renewal of double negative 3 (DN3) early thymocytes allows for thymus autonomy but compromises the β-selection checkpoint

Paiva

Sousa

Ramos

et al. 2020

Preprint

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T lymphocyte differentiation in the thymus relies on high cellular turnover, which is enforced by cell competition. If deprived of competent progenitors, the thymus can maintain thymopoiesis autonomously for several weeks but this bears a high risk of leukemia. Here we show that double negative 3 early (DN3e) thymocytes can acquire stem cell like properties, which enables them to maintain thymopoiesis. Specifically, DN3e proved to be long-lived, they proliferated and differentiated in vivo, they were necessary for autonomous thymopoiesis, and included DNA-labelretaining cells. Single cell RNAseq revealed a transcriptional program of thymopoiesis similar in autonomy and the controls. Nevertheless, a new population was identified in thymus autonomy that was enriched for an aberrant Notch target gene signature and bypassed the b-selection checkpoint.In sum, DN3e have the potential to self-renew and differentiate in vivo if cell competition is compromised but this enables the accumulation of atypical cells, probably leading to leukemia.

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High-Throughput Single-Cell Sequencing of both TCR-β Alleles

Cited by 7 publications

References 20 publications

Specificity of the T Cell Response to Protein Biopharmaceuticals

Specificity of the T Cell Response to Protein Biopharmaceuticals

Self-renewal of double-negative 3 early thymocytes enables thymus autonomy but compromises the β-selection checkpoint

Self-renewal of double negative 3 (DN3) early thymocytes allows for thymus autonomy but compromises the β-selection checkpoint

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