Abstract 5189: High throughput single-cell based cloning reveals functional diversity of T cell receptors targeting minor histocompatibility antigen
Authors: Ito Sawa , Panousis Constantinos G. , Rowe Alexander M. , Ventura Kedwin , Roy Jennifer D. , Stras Stephanie , Brocca-Cofano Egidio , Kim Josh , Shlomchik Mark J. , Shlomchik Warren D. 
Published: June 2022

Abstract
Hematopoietically-restricted minor histocompatibility antigens (miHAs) specific T cells can mediate graft-versus-leukemia and promote engraftment with a low risk of graft-vs-host disease in allogeneic stem cell transplantation (alloSCT). Thus the miHA are ideal targets for adoptive T cell immunotherapy for the recipient of alloSCT. We developed a novel single-cell based high-throughput technology (TCXpress) for cloning T cell receptors (TCRs) and successfully cloned multiple TCRs reactive against the miHA, HA-1 from a parous woman who was naturally immunized to HA-1 through pregnancy. We identified an HLA-A*02:01 woman homozygous for the non-immunogenic HA-1 alleles (R/R) who had pregnancies from an HA-1(H/R) father. TCRs were cloned from single-cell-sorted HA-1 dextramer+ (dexHA-1+) T cells from unstimulated peripheral blood mononuclear cells (PBMCs) and subsequently from CD8 cells cultured with HA-1 peptide-pulsed antigen-presenting cells (APCs). TCRs were re-expressed in reporter cells and analyzed for dextramer binding and CD69 upregulation after culture with peptide-pulsed APCs. TCR sequencing was performed to define CDR3 diversity. 48 dexHA-1+CD8+ T cells were single-cell sorted from 3.9 x10e7 PBMCs of a parous woman. Using TCXpress technology, we cloned 38 TCRs from 48 single-cell dexHA-1+CD8+ T cells. Of these 38, 16 unique TCRs, when expressed in Jurkat cells, were functionally reactive against HA-1(H) peptide by ELISpot. These TCRs had a broad range of EC50s as measured by CD69 upregulation when cultured with HA-1(H) peptide-pulsed T2 cells. CD8+ T cells from the same donor were expanded with autologous HA-1(H)-peptide pulsed APCs for one week. 704 additional TCRs were cloned from dexHA-1+ cells from these cultures. 440 clones were confirmed to bind HA-1 dextramer when expressed in CD8-expressing 293 cells. TCR sequencing of these 440 TCRs identified six additional unique anti-HA-1 TCRs. Several TCRs, when re-expressed in primary CD8+ T cells, killed HA-1+ target cells. TCR sequencing revealed that almost all dexHA-1+ CD8+ T cells used TRBV7-9, consistent with other anti-HA-1 TCR clones in previous reports. In summary, TCXpress technology has yielded 22 unique anti-HA-1 TCRs with a broad functional affinity from a single donor in only two experiments. Our data also highlight the wide range of TCR affinities that can arise from a natural immune response against a single allopeptide/HLA complex. We aim to apply this technology to clone and characterize TCRs against other miHAs, particularly those with expression relatively restricted to hematopoietic cells.
Citation Format: Sawa Ito, Constantinos G. Panousis, Alexander M. Rowe, Kedwin Ventura, Jennifer D. Roy, Stephanie Stras, Egidio Brocca-Cofano, Josh Kim, Mark J. Shlomchik, Warren D. Shlomchik. High throughput single-cell based cloning reveals functional diversity of T cell receptors targeting minor histocompatibility antigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5189.