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

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; CFSE^dim), slow responders (SR; CFSE^brightCD69^pos) and non-responders (NR; CFSE^brightCD69^neg) 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 50^th percentile of total TCRB reads being 11.8 +/- 6.5%, 17.7 +/- 8.5%, and 20.2 +/- 1.8% of unique reads, respectively (p<0.01). Candidate AR/GVL TCRB clonotypes, specified as those enriched in MLR-responder populations compared to pre-stimulation samples, were validated by comparison with pre-stimulation frequency-matched clonotypes. The mean frequency of AR/GVL TCRB clonotypes in blood samples post-HCT were 10- to 100-fold greater than control clonotypes, depending on the patient (Fig 1C). AR/GVL T cells were present within the post-HCT PBMC TCRB repertoires at mean frequencies of 5x10^-4 at day +90 rising to 2x10^-3 by day +360 post-HCT in 3/4 patients experiencing at least 2 year remissions post-HCT. In patients experiencing early relapse (within 1yr post-HCT), no candidate AR/GVL clonotypes were identified in 1/3 patients and 2/3 exhibited AR/GVL clonotypes at roughly one log lower frequencies (mean 8x10^-5 at day +90 and 4x10^-4 at day 360) than those with longer remissions (Fig 1D). One patient who experienced fatal post-DLI steroid-refractory GVHD exhibited striking changes in AR/GVL clonotype prevalence following DLI (Fig 1D).

Figure 1

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MLR T cell populations are sorted by FACS (A). Clonotypic compositions of rapid responder (RR), slow responder (SR), and non-responder (NR) cells are determined (B). Candidate AR/GVL clonotypes are validated bioinformatically (C). Three representative patient courses demonstrate MRD quantification (red) and presence of AR/GVL clonotypes (black) in a patient with late relapse, absence in a patient with early relapse, and clonotypic transition in a patient with post-DLI GVHD (D).

Figure 1

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MLR T cell populations are sorted by FACS (A). Clonotypic compositions of rapid responder (RR), slow responder (SR), and non-responder (NR) cells are determined (B). Candidate AR/GVL clonotypes are validated bioinformatically (C). Three representative patient courses demonstrate MRD quantification (red) and presence of AR/GVL clonotypes (black) in a patient with late relapse, absence in a patient with early relapse, and clonotypic transition in a patient with post-DLI GVHD (D).

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In vitro MLR between donor T cells and CpG-stimulated CLL cells selects clonotypically distinct T cell populations with an oligoclonal RR population. Persistence of adoptively transferred candidate AR/GVL clones identified by MLR appears to correlate with likelihood of maintaining clinical remission beyond 2 years in CLL patients undergoing RIC allo-HCT. Failure to adoptively transfer AR/GVL clonotypes may be associated with early treatment failure.

Klinger:Sequenta, Inc.: Employment, Research Funding. Moorhead:Sequenta, Inc.: Employment, Research Funding. Zheng:Sequenta, Inc.: Employment, Research Funding. Faham:Sequenta Inc.: Employment, Stockholder Other.