Alloreactive T Cell Clonotypes Identified By In Vitro Mixed Lymphoid Reaction and High-Throughput Sequencing Exhibit Increased Frequency In Peripheral Blood Samples From Patients Following Allogeneic Hematopoietic Cell Transplantation For Chronic Lymphocytic Leukemia

• Published in: Blood 2013-11, Vol.122 (21), p.149-149
• Main Authors: Logan, Aaron C., Krampf, Mark R., Klinger, Mark, Moorhead, Martin, Zheng, Jianbiao, Armstrong, Randall, Faham, Malek, Weinberg, Kenneth I, Miklos, David B.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V122.21.149.149

Allogeneic hematopoietic cell transplantation (allo-HCT) provides long-term immunologic disease control for a substantial portion of patients with hematologic malignancies. Chronic lymphocytic leukemia (CLL) is sensitive to graft-versus-leukemia (GVL) effects as evidenced by responses to reduced-intensity conditioning (RIC) allo-HCT, and to donor lymphocyte infusions (DLI) for post-HCT relapse. To identify potential alloreactive (AR)/GVL T cells, we performed in vitro mixed lymphocyte reactions (MLRs) between recipient CLL cells and donor T cells derived from blood apheresis products acquired for DLI. Responder and non-responder T cell populations from MLRs and recipient post-HCT blood samples underwent T cell receptor beta (TCRB) high-throughput sequencing (HTS). The prevalence of candidate AR/GVL TCRB clonotypes at various times following HCT was quantified and correlated with CLL disease burden and graft-versus-host disease (GVHD). CLL cells isolated from cryopreserved PBMC aliquots of 7 patients who experienced post-HCT relapse were pre-stimulated in vitro for 72 hours with CpG oligodeoxynucleotides in X-VIVO 15 supplemented with IL-4, IL-7, BAFF, and GM-CSF. Upregulation of CD80, CD86, CD40L, MHCI, and/or MHCII was confirmed by flow cytometry. Donor T cells were isolated from cryopreserved DLI using pan-T cell isolation beads (Miltenyi), labeled with CFSE, and incubated with CpG-stimulated CLL cells for 7 days. Upon the conclusion of the MLR incubation, T cell populations were sorted into rapid responders (RR; CFSEdim), slow responders (SR; CFSEbrightCD69pos) and non-responders (NR; CFSEbrightCD69neg) and RNA was isolated from each cell population. RNA was then amplified and TCRB sequenced using the LymphoSIGHT platform (Sequenta). PBMC samples collected and cryopreserved pre-HCT and regularly following HCT and DLI were also subjected to TCRB-HTS. RR cells comprised 11.5 +/- 9.2% of MLR T cells, whereas SR were 4.2 +/- 3.5% and NR were 84.3 +/- 10.1% (Fig 1A). RR, SR, and NR populations demonstrated clonotypic exclusion with a mean 4.4% +/- 5.5% coincidence between populations (Fig 1B). TCRB diversity in the RR population was more restricted compared with diversity in the SR and NR populations, with the mean number of clonotypes comprising the top 50th percentile of total TCRB reads being 11.8 +/- 6.5%, 17.7 +/- 8.5%, and 20.2 +/- 1.8% of unique reads, respectively (p