Longitudinal High-Throughput T Cell Repertoire Profiling of Chronic Lymphocytic Leukemia Patients Under Different Types of Treatment: Implications for Combination Strategies
Authors: Vardi Anna , Vlachonikola Elisavet , Ioannou Nikolaos , Psomopoulos Fotis , Kotta Konstantia , Papazoglou Despoina , Kotouza Maria , Koravou Evangelia , Galigalidou Chrysi , Gemenetzi Katerina , Pasentsis Kostas , Iskas Michalis , Stavroyianni Niki , Anagnostopoulos Achilles , Ramsay Alan G. , Stamatopoulos Kostas , Hadzidimitriou Anastasia 
Published: October 2019

Abstract
Using next-generation sequencing (NGS), we recently documented the clonal architecture of the T cell repertoire in treatment-naive chronic lymphocytic leukemia (CLL), with ample immunogenetic evidence indicating selection by restricted antigens. Our preliminary NGS study in 16 patients pre- and 3-month post-treatment indicated a differential impact of standard chemoimmunotherapy (FCR) versus B cell receptor signaling inhibitors (BcRi) on CLL T cells. Prompted by these observations, here we sought to comprehensively assess CLL T cell repertoire changes over treatment in relation to both treatment type and clinical response by combining NGS immunoprofiling, flow cytometry and functional assays.
NGS profiling of the T cell receptor (TR) gene repertoire was performed in 28 CLL patients who received FCR (n=9), ibrutinib (IB, n=15) and/or rituximab-idelalisib (R-ID, n=10) at successive timepoints (pre, +3mo, +9mo and at deepest clinical response, total samples: n=113). TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end NGS. Raw reads were processed through a purpose-built, validated bioinformatics pipeline, culminating to 20,347,768 productive, filtered-in TRB sequences (median 155,479/sample). For repertoire analysis, clonotypes (i.e. rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence) were considered (median 11,420 distinct clonotypes/sample).
All cases displayed significant clonal T cell expansions both pre- and post-treatment [median clonality, measured as the cumulative frequency of the 10 most expanded (major) clonotypes/sample: 30.3% and 39.6%, respectively]. Median clonality significantly increased at +3mo in the FCR (29.0% to 46.9%, p<.001) and R-ID group (33.0% to 39.1%, p<.001), but not in the IB group (33.3% to 31.2%, p>.05). Overtime analysis revealed a gradual increase of clonality over deepening clinical response (pre-, +3mo, +9mo, deepest response) in the R-ID group (33.0% to 39.1% to 46.0% to 46.1%, respectively; p<.001), but only a trend in this respect for IB (33.3% to 31.2% to 33.8% to 42.0%; p>.05). Considering that FCR resulted in T cell repertoire reconstitution whereas BcRis retained pre-treatment clones, we then focused on major clones persisting over treatment and found that they significantly expanded in the R-ID group, peaking at +3mo (p<.01). Cross-comparison across all CLL patients and against 767,438 unique TRB sequences retrieved from multiple public databases (HSV infections, T-cell lymphoproliferations, autoimmune disorders, healthy individuals), revealed 23/563 major clonotypes shared exclusively among CLL patients, alluding to selection by conserved CLL-related antigens. We then sought to test the functional effect of treatments on T cells. To this end, we evaluated activation markers on CLL T cell subpopulations for 8 CLL patients (R-ID, n=4; IB, n=4) pre- and +3mo post-treatment by flow cytometry and found statistically significant upregulation of T cell activation markers for R-ID compared to IB, particularly for: (i) CD69 in CD4+ effector memory T cells (p<.01); (ii) CD25 in CD8+ TEMRA T cells (p=0.006); and, (iii) CD38 in CD8+ effector memory T cells (p<.05) and CD8+ TEMRA T cells (p<.05). We also investigated the ability of CD3+ T cells, purified from 13 patients pre- and +3mo post-treatment (FCR, n=3; R-ID, n=5; IB, n=5), to form immune synapses with autologous pre-treatment CD19+ tumor cells. Quantitative relative recruitment index (RRI) analysis for F-actin showed that both R-ID (p<.01) and IB (p<.05) treated T cells form polarized immune synapses in contrast to FCR (p>.05).
Taken together, NGS immunoprofiling suggests that BcRis retain T cell clones that may have developed in response to CLL-related antigens, which in the case of R-ID expand and peak at +3mo. Phenotypic and immune synapse bioassays support a concurrent restoration of functionality, mostly evident for R-ID, arguably contributing to clinical response. Overall, this data provides rationale for designing combination strategies, e.g. of R-ID with immunomodulating drugs, aiming to boost cytotoxic anti-tumor responses. Moreover, identifying the relevant neoepitopes may eventually pave the way for stratified treatments by means of engineered T cells or peptide vaccines, especially if these epitopes are conserved among CLL.

Disclosures
Vardi: Janssen: Honoraria; Gilead: Research Funding. Gemenetzi:Gilead: Research Funding. Ramsay:MedImmune: Research Funding; Roche Glycart AG: Research Funding; Celgene Corporation: Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Hadzidimitriou:Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding.