HLA Ligandome Analysis of Primary Chronic Lymphocytic Leukemia (CLL) Cells Under In Vitro Lenalidomide Treatment Confirms Lenalidomide as a Suitable Combination Partner for T-Cell Based Immunotherapy
Authors: Nelde Annika , Kowalewski Daniel J. , Backert Linus , Schuster Heiko , Kanz Lothar , Salih Helmut R. , Rammensee Hans-Georg , Stevanovic Stefan , Stickel Juliane S. 
Published: October 2019

Abstract
Several studies have proven the positive immunomodulatory effect of lenalidomide, a second-generation derivative of thalidomide, on T-cell responses in CLL, in particular regarding the repair of the immune synapse between CLL and T cells (e.g., Ramsay et al. J Clin Invest 2008), as well as the upregulation of costimulatory molecules on CLL cells (e.g., Aue et al. Haematologica 2009). Therefore, lenalidomide seems to be a promising combination partner for T-cell based immunotherapy approaches in CLL patients. We recently conducted a study which directly characterized the antigenic landscape of CLL by mass spectrometric analysis of naturally presented HLA ligands and identified a panel of CLL-specific CD4+ as well as CD8+ T-cell epitopes as suitable targets for T-cell based immunotherapy (Kowalewski et al. PNAS2015). As anti-cancer drugs can have marked effects on the HLA ligandome of tumor cells, it is of great importance to thoroughly characterize and take into account the effects of the immune modulator lenalidomide not only on the effector cells, but also on the antigenic landscape of the target cells.
Here we present a mass spectrometry-based study which longitudinally maps the HLA-presented immunopeptidome and in particular the CLL-associated antigens of primary CLL cells under in vitro lenalidomide treatment. We quantified HLA surface expression on primary CLL cells and autologous B cells (n=4) at t0, t24h and t48h after incubation with 0.5 µM lenalidomide. With regard to HLA class I expression on CD19+CD5+ CLL cells, no significant impact of lenalidomide was observed (fold-change 0.92-1.02, t48h), whereas a slight but also not significant increase of HLA class II molecules after treatment was detectable (fold-change 1.25-1.43, t48h) with absolute molecule counts ranging from 40,000-125,000 class I and 30,000-200,000 class II molecules/cell. Furthermore, we could show that CD19+CD5+ CLL cells (n=4) showed a significantly increased expression of HLA class I molecules compared to normal CD19+CD5-B cells.
Implementing label-free quantification, we then assessed HLA class I ligand presentation during in vitro lenalidomide incubation. We observed a higher plasticity of the HLA ligandome over time compared to treatment with lenalidomide. 2.5±3.0% of HLA class I ligands showed significant modulation (fold change ≥4, p≤0.01) after 24h of mock treatment, whereas only 0.9±1.2% of the ligands were modulated upon lenalidomide treatment. At t48hsimilar proportions of modulation were observed with 4.0±1.7% of HLA ligands significantly altered in their abundance over time (mock control), while lenalidomide treatment only resulted in 0.9±1.2% modulated ligands.
Out of the 6,991 different HLA class I ligands representing 3,983 source proteins identified on primary CLL cells (n=3) by mass spectrometry, we were able to detect 36 (30.5%) of the HLA-matched CLL-associated epitopes described in previous studies on these three samples. Importantly, these CLL-associated antigens showed robust presentation on CLL cells under in vitro lenalidomide treatment. In order to investigate the impact of lenalidomide-treatment on HLA class I allotype distribution within the ligandome, we analyzed the overall frequencies of HLA allotype restrictions of ligands on treated versus untreated primary CLL cells. Thereby, no changes in the HLA allotype distribution were observed.
Analysis of the HLA class II ligandome of primary CLL cells (n=3) revealed a total of 12,026 unique peptides representing 2,333 source proteins. We were able to detect 133 (28.5%) of CLL-associated class II epitopes described in previous studies. Notably, most of the CLL-associated antigens showed robust presentation under lenalidomide therapy. Exemplarily for one patient, we identified 7/66 (10.6%) CLL-associated antigens modulated after 24 h of in vitro lenalidomide treatment, but 10/66 (15.2%) antigens altered over time (mock control).
Taken together our study provides direct insights into the impact of lenalidomide on the HLA ligandome of primary CLL cells. We were able to show that in vitro lenalidomide has no relevant influence on, and rather seems to stabilize the HLA-presented immunopeptidome of primary CLL cells. Importantly, CLL-associated epitopes are stably presented under lenalidomide treatment. Therefore, lenalidomide appears to be an ideal combination partner for T-cell based immunotherapy in CLL patients.

Disclosures
Kowalewski: Immatics Biotechnologies GmbH: Employment. Schuster:Immatics Biotechnologies GmbH: Employment.