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Tracking Antigen‐Specific CD8+ T cells Using MHC Class I Pentamers
Author(s) -
Aleksandrovic Emilija,
Lazarski Christopher,
Bollard Catherine,
Stanojevic Maja,
Hanley Patrick
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.02515
Subject(s) - elispot , cd8 , cytotoxic t cell , antigen , t cell , immunology , peripheral blood mononuclear cell , mhc class i , immunotherapy , antigen presenting cell , biology , medicine , virology , immune system , in vitro , biochemistry
The treatment of choice for many patients with Acute Myeloid Leukemia (AML) is hematopoietic stem cell transplantation, where the patient’s immune system is replaced with that of a matched donor. Due to the conditioning required, cancer relapse and viral infections such as Cytomegalovirus (CMV) are common complications. One potential treatment for relapse and/or viral infection is adoptive immunotherapy with ex vivo expanded T cells targeting either viral or tumor antigens. Prior to infusion into patients, these T cells are typically characterized using ELISpot assays which do not directly assess the presence of distinct T cell clones. Therefore, without a reliable assay to test T cell specificity, optimizing the culture conditions for increased antigen specificity has been challenging. We hypothesized that we could directly assess the specificity of T cell receptors (TCRs) from expanded T cell populations towards their target antigens over time using MHC Class I Pentamers. To test this, we generated CMV‐specific CD8+ T cells from peripheral blood mononuclear cells (PBMCs) from two consenting CMV+ donors and tumor‐specific T cells from one donor on our IRB approved protocol. After 10 days of culture, the average CMV+ T cells expanded 10‐fold and consisted of 22.2% CD4+ (range 15.9–46.6%) and 68.9% CD8+ (range 32.9–77.9%) T cells. Using flow cytometry, we were able to detect pentamer positive cells by day 7 for three distinct epitopes: RPH (58.1%), TPR (8.45%), and NLV (0.36%) in one of the CMV‐specific T cell lines. In addition, ALY pentamer positive T cells were detected as early as day 6 of culture in the tumor T cell lines stimulated with the entire PRAME antigen and single epitope of the same antigen (0.2 and 0.16% respectively), while Survivin (LML) positive T cells were detected on day 13 for cells stimulated with the PRAME/Survivin/WT‐1 pepmix (0.19%). These results indicate that in two CMV‐specific and one tumor‐specific T cell product, we were successful in tracking T cell populations and their specificity towards their target antigens over time. Additional experiments, however, are required to reach statistical significance and demonstrate reproducibility. Since this method can identify virus‐ and tumor‐specific T cells early in the culturing process, it has the potential to be used for culture optimization and monitoring epitope expansion in real time, as well as sorting T cells according to their specific TCRs. Support or Funding Information This project was supported by the Children’s Cancer Foundation grant awarded to Dr. Patrick Hanley.