Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib Show Expansion of T-Cell Clonotypes Composed of Antitumor Cytotoxic CD8+ T-Cells
Authors: Baptista Maria Joao , Baskar Sivasubramanian , Keyvanfar Keyvan , Gaglione Erika M , Ahn Inhye E , Wiestner Adrian , Sun Clare 
Published: November 2019

We recently showed that ibrutinib induced a more oligoclonal T-cell receptor (TCR) repertoire in patients with chronic lymphocytic leukemia (CLL). Using next generation sequencing (NGS), we found that overrepresented TCRβ clonotypes were mostly patient-specific and that the degree of oligoclonal expansion correlated with CD8+ T-cell counts. Here, we tested the hypothesis that overrepresented clonotypes are CD8+ T-cells that respond to tumor antigens.
Flow cytometric profiling of TCR Vβ (TRBV) chain usage, T-cell subset distribution, and granzyme B (GrB) expression was performed on cryopreserved peripheral blood mononuclear cells obtained at baseline and during response to single-agent ibrutinib (n = 5). Antibodies against TRBV were selected to investigate the ten most abundant TCRβ clonotypes identified by NGS. The cumulative frequency of these clonotypes evaluable by flow cytometry increased at the time of response (mean 31.2% ± SEM 3.9% of all T-cells) compared to baseline (mean 24.1% ± SEM 2.3% of all T-cells). Within each TRBV subpopulation, the proportion of CD4+ T-cells decreased (mean fold-change 0.78 ± SEM 0.04), while the proportion of CD8+ T-cells increased (mean fold-change 1.79 ± SEM 0.36). We further observed that the TRBV clonotypes expanding the most during treatment were comprised exclusively of CD8+ T-cells in every patient. These highly expanded CD8+ clonotypes expressed GrB, consistent with a cytotoxic phenotype.
To evaluate whether expanding CD8+ clonotypes are tumor-specific, T-cells were expanded ex vivo in two conditions: (1) co-culture with autologous CLL cells, CD40L, IL-2, and IL-7 or (2) with anti-CD3/CD28/CD137 beads, IL-2, and IL-7. Overall, T-cells expanded with autologous CLL cells had a higher proportion of CD8+ GrB+ cells than T-cells expanded with anti-CD3/CD28/CD137 beads. The dominant CD8+ clonotypes detected in each patient at the time of response were preferentially expanded by co-culture with autologous CLL cells compared to expansion with beads. The biased expansion of cytotoxic T-cell clonotypes in the CLL-primed T-cell product supports a TCR mediated response to tumor antigens.
Next, cytotoxicity assays were performed by mixing the expanded T-cell products described above with autologous CLL cells at an effector to target ratio of 3:1, 1:1 and 1:3. CLL-primed T-cells killed CLL cells in a dose-dependent fashion. In contrast, bead-expanded T-cells were ineffective at killing CLL cells or even protected tumor cells from spontaneous apoptosis. Thus, tumor-specific cytotoxic T-cells expand during treatment with ibrutinib and are further enriched ex vivo by co-culture with autologous CLL cells.
In summary, increased TCR oligoclonality in patients responding to ibrutinib is driven by the expansion of specific CD8+ clonotypes. These clonotypes expand in co-culture with, and are cytotoxic against, autologous CLL cells. Tumor-specific CD8+ T-cell clonotypes may contribute to the overall treatment response with ibrutinib and supports the investigation of therapeutic strategies combining ibrutinib with T-cell directed immunotherapy.

Disclosures
Baskar: NIH: Patents & Royalties: ROR1 mAb 2A2. Wiestner:Nurix: Research Funding; Acerta: Research Funding; Merck: Research Funding; Pharmayclics: Research Funding.