Screening TCR-transduced PBMCs for Antigen Reactivity

To measure antigen-specific reactivity of recovered TCR sequences, TCRs were expressed and screened in Jurkat-NFAT-GFP cells as described previously (34 (link)). Paired TCR alpha and beta chains of interest were cloned into a retroviral pMSGV construct as previously described (17 (link)). PBMCs for retroviral transduction were processed and cultured according to our rececent publication(19 (link), 34 (link)). To assess function of the transduced TCRs in human PBMCs, TCR expressing cells were mixed with K562-A2 cells at a ratio of 1:2 (Effector:Target) in the RPMI media and supplemented with 1 μg/ml of anti-CD28/CD49d antibodies (BD Biosciences, 347690) and 1 μg/ml of cognate peptides. For PBMCs, supernatants were collected after 48 hours and analyzed by ELISA (BD Biosciences) to estimate IFN-γ concentration. PBMCs transduced by the vector without a TCR was used as a negative control.

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Koo D., Mao Z., Dimatteo R., Tsubamoto N., Noguchi M., McLaughlin J., Tran W., Lee S., Cheng D., de Rutte J., Sojo G.B., Witte O.N, & Di Carlo D. (2023). Defining T cell receptor repertoires using nanovial-based affinity and functional screening. bioRxiv.

Publication Preprint 2023

anti-CD28/CD49d antibodies

Anti antibodies Antigen Cells Elisa Human Ifn γ Jurkat cells K562 cells Peptides Retroviral Vector

Corresponding Organization : California NanoSystems Institute

Other organizations : Parker Institute for Cancer Immunotherapy

Top 5 similar protocols

Variable analysis

independent variables

Paired TCR alpha and beta chains of interest

dependent variables

Antigen-specific reactivity of recovered TCR sequences
IFN-γ concentration in supernatants

control variables

Retroviral pMSGV construct
RPMI media
1 μg/ml of anti-CD28/CD49d antibodies
1 μg/ml of cognate peptides
PBMCs transduced by the vector without a TCR

positive controls

K562-A2 cells

negative controls

PBMCs transduced by the vector without a TCR

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