T-Cell Dynamics in Chronic Lymphocytic Leukemia Under

Published OnlineFirst July 2, 2020; DOI: 10.1158/1078-0432.CCR-19-3827

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

T-Cell Dynamics in Chronic Lymphocytic Leukemia under Different Treatment Modalities Anna Vardi1,2,3, Elisavet Vlachonikola1,4, Despoina Papazoglou5, Fotis Psomopoulos1,3, Kostantia Kotta1, Nikolaos Ioannou5, Chrysi Galigalidou1,6, Katerina Gemenetzi1,6, Kostantinos Pasentsis1, Maria Kotouza1, Evdoxia Koravou2, Lydia Scarfo7, Michail Iskas2, Niki Stavroyianni2, Paolo Ghia7, Achilles Anagnostopoulos2, Anastasia Kouvatsi4, Alan G. Ramsay5, Kostas Stamatopoulos1,3, and Anastasia Chatzidimitriou1,3

ABSTRACT ◥ Purpose: Using next-generation sequencing (NGS), we recently (BcRi) preserved pretreatment clones. Extensive comparisons documented T-cell oligoclonality in treatment-na€ve chronic lym- both within CLL as well as against T-cell receptor sequence phocytic leukemia (CLL), with evidence indicating T-cell selection databases showed little similarity with other entities, but instead by restricted antigens. revealed major clonotypes shared exclusively by patients with Experimental Design: Here, we sought to comprehensively CLL, alluding to selection by conserved CLL-associated antigens. assess T-cell repertoire changes during treatment in relation to We then evaluated the functional effect of treatments on T cells (i) treatment type [fludarabine-cyclophosphamide-rituximab and found that (i) R-ID upregulated the expression of activation (FCR) versus ibrutinib (IB) versus rituximab-idelalisib (R-ID)], markers in effector memory T cells, and (ii) both BcRi improved and (ii) clinical response, by combining NGS immunoprofiling, antitumor T-cell immune synapse formation, in marked contrast flow cytometry, and functional bioassays. to FCR. Results: T-cell clonality significantly increased at (i) 3 months Conclusions: Taken together, our NGS immunoprofiling data in the FCR and R-ID treatment groups, and (ii) over deepening suggest that BcRi retain T-cell clones that may have developed clinical response in the R-ID group, with a similar trend detected against CLL-associated antigens. Phenotypic and immune synapse intheIBgroup.Notably,inconstrast to FCR that induced T-cell bioassays support a concurrent restoration of functionality, mostly repertoire reconstitution, B-cell receptor signaling inhibitors evident for R-ID, arguably contributing to clinical response.

Introduction pleiotropic effect in the CLL microenvironment, particularly T cells, which may contribute to their clinical efﬁcacy (7–15). The tumor microenvironment (TME) plays a pivotal role in the T cells are intimately implicated in CLL pathophysiology. Xenograft natural course of chronic lymphocytic leukemia (CLL). This is evi- studies have highlighted the importance of trophic signals provided by denced by ample immunogenetic cues suggesting selection of the T cells for the survival of the malignant clone (16). On the other hand, malignant B-cell clone by a restricted set of antigens, as well as by immunosurveillance by T cells is compromised in CLL, due to, among the remarkable clinical efﬁcacy of drugs interfering with B-cell receptor others, defective cytoskeletal signaling and lytic immune synapse signaling (BcR signaling inhibitors, BcRi; refs. 1–6). Although BcRi formation following tumor immunosuppressive signaling (17–20). were developed to target the clonal B cells, accumulating data suggest a Therefore, delineating the mechanisms driving T-cell tolerance rather than effective antitumor cytotoxic immune responses is highly relevant and could facilitate optimization of immunotherapy for CLL, with the

1 2 aim of eradicating minimal residual disease (21, 22). Institute of Applied Biosciences, CERTH, Thessaloniki, Greece. Hematology Studies employing different methodologies have demonstrated the Department and HCT Unit, G. Papanicolaou Hospital, Thessaloniki, Greece. – 3Department of Molecular Medicine and Surgery, Karolinska Institutet, Stock- existence of T-cell expansions in CLL (23 27). Recently, we investi- holm, Sweden. 4Faculty of Sciences, Department of Genetics, Development and gated the role of antigenic stimulation in shaping the T-cell repertoire Molecular Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece. in treatment-naive CLL using next-generation sequencing (NGS; 5Lymphoma Immunology Group, School of Cancer and Pharmaceutical ref. 28). Our study confirmed T-cell oligoclonality, with major Sciences, Faculty of Life Sciences & Medicine, King's College London, London, (high-frequency) clonotypes persisting and further expanding over- United Kingdom. 6Democritus University of Thrace, Department of Molecular 7 time as well as clonotypes shared among different patients, which Biology and Genetics, Alexandroupolis, Greece. Division of Experimental fi fi Oncology, Universita Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, appeared to be CLL-speci c. Overall, these ndings pointed toward Milan, Italy. selection of T cells by CLL-associated antigenic epitopes. On these grounds, we sought to comprehensively assess the effect of Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). treatment on CLL T-cell repertoire dynamics by combining NGS immunogenetics, flow cytometry and immune synapse bioassays, to Corresponding Author: Anastasia Chatzidimitriou, Institute of Applied Bio- sciences, CERTH, Thessaloniki 57001, Greece. Phone: 3023-1049-8271; investigate associations of quantitative and/or qualitative changes with Fax: 3023-1049-8270; E-mail: [email protected] the type of treatment and clinical response. Considering recent data about collateral effects of BcRi treatment on T cells (7–14, 29), we Clin Cancer Res 2020;XX:XX–XX focused on patients receiving ibrutinib or rituximab-idelalisib which doi: 10.1158/1078-0432.CCR-19-3827 we compared to patients receiving standard chemoimmunotherapy 2020 American Association for Cancer Research. with the fludarabine-cyclophosphamide-rituximab regimen.

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physician, and corresponded to the maximal duration of BcRi Translational Relevance treatment at the time the study was conducted (median time on Chronic lymphocytic leukemia (CLL) T cells display largely BcRi treatment: 20 months), with the single exception of a patient defective antitumor responses. From a clinical perspective, there is who progressed on ibrutinib and proceeded to rituximab-idelalisib a clear need to optimize CLL immunotherapy, especially regarding as second-line treatment. This patient was assessed only at the minimal residual disease eradication. Boosting antitumor T-cell 3-month timepoint while on ibrutinib, at which he had achieved responses could ideally fit this gap, and identifying the best PR. Also, we included 6 bone marrow (BM) samples, collected combination treatment partners is of translational relevance. We along with the respective PB pretreatment sample. A schematic investigated the effect of treatment on CLL T cells in relation to representation of study cohort is provided in Fig. 1A. (i) treatment type (FCR vs. ibrutinib vs. rituximab-idelalisib), and No patient had evidence of infection at sampling. All patients were (ii) clinical response. In contrast to chemoimmunotherapy, B-cell negative for HBV DNA, anti-HCV, and anti-HIV I/II. We carefully receptor signaling inhibitors (BcRi) preserved pretreatment T-cell excluded patients who developed rituximab-related late-onset neu- clones, which expanded further as treatment continued and the tropenia (R-LON), as it is known to be mediated by cytotoxic T-cell clinical response deepened. We found major clonotypes shared clones (30). The local Ethics Review Committee approved the study among different patients, raising the possibility of selection by and written informed consent form was obtained from all individuals conserved, CLL-associated epitopes. A concurrent restoration of in accordance with the Declaration of Helsinki. T-cell functionality with BcRi therapy arguably contributed to clinical response. Overall, these data provide a rationale for design- Next-generation sequencing, definitions, and interpretation ing combination strategies aiming to boost cytotoxic antitumor TRBV–TRBD–TRBJ gene rearrangements were RT-PCR amplified responses. and subjected to paired-end NGS (MiSeq, Illumina), as described previously (28). For all samples, the starting absolute T-cell count exceeded 0.5 106 cells to ensure adequate repertoire profiling depth. Paired-end protocol allowed sequencing of the TRB complemen- In sharp contrast to chemoimmunotherapy, BcRi were found to tarity-determining region 3 (CDR3) twice/read, thus increasing the preserve pretreatment T-cell clones. These clones expanded further as accuracy of results. To further increase the accuracy of results, raw treatment continued and clinical response deepened, particularly in reads were processed through a purpose-built bioinformatics pipeline the case of rituximab–idelalisib. Repertoire comparisons revealed performing: (i) length/quality filtering of raw reads; (ii) merging of major clonotypes shared by different patients with CLL but very few filtered-in paired reads via local alignment; (iii) length/quality filtering “public” clonotypes (i.e., also present in other conditions), suggesting of stitched sequences (28). Detailed length, quality, and overlap rules selection by conserved CLL-associated epitopes. Moreover, phenotyp- are provided in Supplementary Table S2. Importantly, no base calls of ic and immune synapse bioassays supported a concurrent restoration Q-score <30 were allowed in the 75 nucleotide stretch preceding the of T-cell functionality with BcRi therapy, mostly evident for ritux- FGXG motif at the start of TRB FR4, thus further increasing the imab–idelalisib, arguably contributing to clinical response. Taken CDR3 sequencing reliability. Filtered-in sequences were submitted to together, these data provide a rationale for designing combination IMGT/HighV-QUEST (http://www.imgt.org), and metadata was pro- strategies aiming to boost cytotoxic antitumor responses. cessed by a validated bioinformatics algorithm designed for clonotype computation and repertoire analysis (Fig. 1B; refs. 28, 31). Only productive, in-frame TRBV–TRBD–TRBJ gene rearrangements with Materials and Methods functional TRBV genes were included in the analysis. TRBV–TRBD– Patient group TRBJ gene rearrangements carrying TRBV genes with <95% germline We analyzed samples from 28 patients with CLL treated in identity were also discarded as sequences with unacceptable error rate, two centres (Thessaloniki, Greece; Milan, Italy) with FCR (n ¼ 9), given the lack of somatic hypermutation in T cells. IB (n ¼ 15), and/or rituximab–idelalisib (n ¼ 10). Patients were Clonotypes were computed as unique pairs of TRBV genes and selected on the basis of sample availability. Demographic and CDR3 amino acid sequences within a sample. Clonotypes were clinicobiological characteristics are provided in Supplementary considered expanded when they contained ≥2 sequences, otherwise Table S1. For 22 patients, samples were collected over the first line they were considered as “singletons.” The 10 most expanded of treatment, either fludarabine–cyclophosphamide–rituximab or clonotypes within a sample are referred to as “major.” The relative BcRi. For the remaining 6 of 28 patients, we analyzed samples frequency of each clonotype/sample was calculated as the number of collected over consecutive lines of treatment (first-line fludarabine– rearrangements corresponding to the clonotype divided by the cyclophosphamide–rituximab/second-line ibrutinib, n ¼ 3; first-line total number of productive, filtered-in rearrangements for that FCR/second-line rituximab–idelalisib, n ¼ 2; first-line IB/second- particular sample. line rituximab–idelalisib, n ¼ 1). In all cases, samples posttreat- For TRB gene repertoire analysis, clonotypes rather than single ment initiation were analyzed in relation to a respective pretreat- rearrangements were considered to avoid potential biases due to ment initiation sample. In total, we analyzed 98 peripheral expansion following antigenic stimulation, that is, individual TRBV blood (PB) samples: (i) for patients who received fludarabine– gene frequencies within a sample were calculated as the number of cyclophosphamide–rituximab, we analyzed samples collected pre- clonotypes using particular TRBV genes over the total number of treatment (n ¼ 9) and at 3 months after the completion of the 6 clonotypes. Each category of samples (PBMCs, BM) was analyzed cycles (n ¼ 9, all in complete remission); (ii) for patients who separately. received BcRi, we analyzed samples pretreatment (n ¼ 25), at 3months(n ¼ 25), 9 months (n ¼ 17), best clinical response Interpatient and across entities clonotype comparison (median 20 months, n ¼ 13)] while still on continuous BcRi We determined the major clonotypes of all CLL samples included treatment. Best response timepoint was defined by the attending in the study (n ¼ 563 unique clonotypes, i.e., 10 major clonotypes

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Figure 1. A, Schematic representation of the study cohort. The fludarabine–cyclophosphamide–rituximab (FCR) treatment group included 9 patients sampled prior to treatment and 3 months after completion of 6 fludarabine–cyclophosphamide–rituximab cycles. The ibrutinib (IB) group included 15 patients [12 receiving ibrutinib as first-line treatment and 3, coming from the fludarabine–cyclophosphamide–rituximab treatment group, who relapsed after fludarabine–cyclophosphamide– rituximab and received ibrutinib as second-line treatment (the latter in dark gray)]. The rituximab–idelalisib (R-ID) group included 10 patients [7 receiving rituximab– idelalisib as first-line treatment, 2 coming from the fludarabine–cyclophosphamide–rituximab treatment group who relapsed after fludarabine–cyclophosphamide– rituximab and received rituximab–idelalisib as second-line treatment (in light gray without outline), and 1 coming from the ibrutinib treatment group who relapsed on ibrutinib and received rituximab–idelalisib as second-line treatment (in light gray with outline)]. Sampling timepoints for the ibrutinib and rituximab–idelalisib treatment groups were 3 months, 9 months, and at best clinical response while on continuous BcRi treatment. For 6 patients (underlined by a dashed line, ibrutinib, n ¼ 3; rituximab–idelalisib, n ¼ 3), BM samples were also obtained in addition to PB prior to BcRi treatment. All samples were analyzed by NGS. Immune synapse bioassays were performed for 14 patients (marked with a dot; fludarabine-cyclophosphamide-rituximab, n ¼ 3; ibrutinib, n ¼ 6; rituximab–idelalisib, n ¼ 5). Flow cytometry for activation/exhaustion markers was performed for 8 patients (marked with a light gray dot; ibrutinib, n ¼ 4; rituximab–idelalisib, n ¼ 4). B, Schematic representation of the NGS pipeline. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end NGS (MiSeq, Illumina), as described previously (29). Instead of arbitrarily excluding low-frequency clonotypes, which may lead to underestimation of repertoire diversity, both the read-stitching algorithm as well as IRProfiler apply strict quality rules to maximize the accuracy of results.

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of each sample removing duplicate values such as clonotypes 4C in 5% goat serum (Sigma-Aldrich) solution. After washing, which persisted in overtime analysis of the same patient or clono- cell specimens were sealed with 22 32 mm coverslips using types shared among different patients) and compared them across fluorescent mounting (Dako). Images were captured with an CLLpatientsofthiscohort,andagainst:(i)allT-cellclonotypes A1R confocal microscope (Nikon) using a 63 oil objective from our previous NGS study involving treatment-naive CLL with NIS- Elements software Version 5.01 imaging software patients (n ¼ 1,105,728; ref. 28), (ii) T-cell clonotypes from 15 (Nikon) and fluorescence was acquired sequentially. The specificity healthy donors (NGS study by our group, n ¼ 573,651; ref. 32), (iii) of staining was optimized using isotype-control antibodies. Blinded HHV-4 (Epstein–Barr virus, EBV), HHV-5 (cytomegalovirus, confocal images (n ¼ 3 per treatment group) were analyzed using CMV), and BK virus–specific T-cell clonotypes (antiviral T-cell NIS-Elements software Version 5.01 to measure the F-actin (red NGS study by our group, n ¼ 169,502; ref. 33), (iv) nonredundant, fluorescent channel) and perforin (green fluorescent channel) well-annotated, unique T-cell clonotypes retrieved from public polarization at contact sites and immune synapses using relative databases (n ¼ 17,024; refs. 34, 35), and (v) T-cell clonotypes from recruitment index (RRI) analysis.RRIiscalculatedastheratio previous low-throughput immunoprofiling studies (classic subclon- of F-actin/perforin mean fluorescent intensity at the immune ing followed by Sanger sequencing) in clinical monoclonal B-cell synapse area to the respective value at a cellular membrane area lymphocytosis (high-count MBL, n ¼ 545; ref. 36), as well as clinical not involved in an immune synapse (RRI<1indicatesanonpolar- entities associated with clonal T-cell expansions [R-LON (n ¼ 283; ized, dysfunctional immune synapse). RRI values were expressed as ref. 30), chronic idiopathic neutropenia (CIN, n ¼ 576; ref. 37), mean value SEM. large granular T-cell leukemia (T-LGL, n ¼ 932); refs. 38–40]. Statistical analysis and visualization tools Flow cytometry Descriptive statistics for discrete parameters included counts and Multicolor flow cytometry (FACSCanto II cell analyzer, BD Bios- frequency distributions. The significance of bivariate/multivariate ciences) was performed to investigate the expression of activation and relationships between variables was assessed using the Student t exhaustion markers (CD25, CD38, CD69, HLA-DR, and PD1, respec- test, as well as the nonparametric Wilcoxon (for paired values) þ tively) in functionally distinct T-cell subpopulations, namely: (i) CD4 and Mann–Whitney tests. For all comparisons a significance level of þ þ þ þ þ and CD8 naive T cells (CD45RA /CCR7 ), (ii) CD4 and CD8 P ¼ 0.05 was set. þ þ þ central memory T cells (CM, CD45RO /CCR7 ), (iii) CD4 and By applying the Wilcoxon-signed rank test for matched pairs, the þ þ CD8 effector memory T cells (EM, CD45RO /CCR7 ), and power of the NGS analysis ranges from 92% (for the most populated þ þ (iv) CD4 and CD8 terminal effector memory RA cells (TEMRA, comparison, i.e., pre-IB versus IB at 3 months, n ¼ 15 samples) to 53% þ CD45RA /CCR7 ). Eight patients (rituximab–idelalisib, n ¼ 4; for the least populated comparisons, that is, pre- rituximab–idelalisib, ibrutinib, n ¼ 4) pretreatment and at the 3-month timepoint were rituximab–idelalisib at 3 months, rituximab–idelalisib at 9 months analyzed, as shown in Fig. 1A. versus rituximab–idelalisib best response, n ¼ 5 samples). A table with Viably frozen peripheral blood mononuclear cells were thawed and power values for each paired comparison is provided in Supplemen- resuspended in PBS (15 106 cells in 1 mL) and incubated with tary Material (Supplementary Table S3). pretitrated antibodies to cell-surface markers at 4C for 20 minutes. All statistical analyses were performed using the statistical Package CD45 V500, CD3 V450, CD4 PerCP, CD4 PE-Cy7, CD8 PE-Cy7, GraphPad Prism version 5.03 (GraphPad Software, Inc.). For data CCR7 APC-Cy7, CD45RO FITC, CD45RA FITC, CD69 PE, CD25 visualization, either publicly available software (http://circos.ca, APC, CD38 PerCP, HLA-DR PE, and PD1 APC were used (BD https://CRAN.R-project.org) or purpose-built R-tools were utilized. Biosciences). Live lymphocytes were gated by forward and side scatter, CD45-positive plus 7AAD–negative staining. Data were analyzed Data sharing statement using the Kaluza flow cytometry analysis software (Beckman Coulter; Raw sequence data have been submitted to the Sequence Read Supplementary Fig. S1). Archive (SRA); BioProject ID: PRJNA552792 þ An acquisition cut-off of 20,000 CD3 live cells/sample was applied to ensure reliable representation of all studied T-cell subpopulations, as well as uniformity across samples. For T-cell activation/exhaustion Results analysis, results were expressed as the proportion of cells expressing The T-cell repertoire in CLL is skewed both pre- and antigens of interest (percent positive staining). posttreatment initiation in all treatment subgroups, with clonal expansions Immune synapse bioassays, confocal microscopy, and First, we profiled by NGS the TR gene repertoire in terms of quantitative image analysis clonality and TRB gene usage. Overall, 23,262,732 TRBV-TRBD- Tumor cell–T-cell conjugate assays were designed to test the TRBJ sequences were obtained, of which 20,347,768 (87.5%, median ability of CLL T cells to form immune synapses with autologous 155,479/sample) passed filters and were further analyzed. The þ tumor cells. Negatively selected viable CD3 T cells (1 106)from median number of distinct clonotypes/sample was 11,420 (range: untreated, FCR-, or BCRi-treated patients were conjugated with 2,672–54,045); the median number of expanded clonotypes and equal numbers of autologous CLL tumor cells acting as antigen- singletons/sample was 5,287 (range: 772–26,501) and 6,417 (range: presenting cells pulsed with super-antigen cocktail and stained with 1500–32,428), respectively. CMAC blue dye (ThermoFisher Scientific) according to the man- All CLL patient blood samples showed a T-cell oligoclonal profile. ufacturer's instructions. Cell conjugates were incubated for 15 The median T-cell clonality of all analyzed samples, calculated as the fl minutes at 37 C, 5% CO2. Immuno uorescent labeling was done cumulative frequency of the 10 major clonotypes/sample, was 37.5% using Cytofuge2 cell concentrator, as described previously (19). (range 8.1%–70.5%). Antibodies for rhodamine phalloidin (F-actin staining) and per- Nine TRBV genes accounted for almost half of the total reper- forin (Alexafluor 488 conjugated) were applied for 45 minutes at toire both pretreatment and at the 3-month timepoint (46.8% and

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47.6% of the total repertoire, respectively). The relative frequency of (P: 0.99), 39.1% at 9 months (P: 0.44) and 42.1% at best clinical each gene did not particularly differentiate over treatment, response (P: 0.17); Fig. 2]. nor among treatment groups (fludarabine–cyclophosphamide– We also performed analyses where clonality definition was modified rituximab, ibrutinib, rituximab–idelalisib; Supplementary Fig. S2A). in order to include a larger proportion of the repertoire (calculated as The single exception concerned the TRBV28 gene, which was under- the cumulative frequency of the 20 or 50 most expanded clonotypes/ represented in the rituximab-idelalisib group both pretreatment and at sample, instead of 10). These analyses, as well as plots of clonotype the 3-month timepoint [3.0%/3.4% vs. 5.1%/5.1% in the FCR group frequency in relation to its rank in the repertoire, showed that the (P < 0.01) vs. 4.5%/4.6% in the ibrutinib group (P < 0.01), respectively]. gradual increase of clonality resulted from the expansion of the For both the ibrutinib and the rituximab–idelalisib groups, where the major clonotypes, further alluding to their clinicobiological relevance repertoire was studied beyond the 3-month timepoint, the TRBV gene (Supplementary Fig. S3). frequency remained relatively stable overtime and with deepening clinical response (Supplementary Fig. S2B). Differential impact of chemoimmunotherapy versus BcRi on the In conclusion, the T-cell repertoire was skewed in all examined T-cell repertoire subgroups, with restricted TRB gene usage and significant clonal To further study clonal dynamics over treatment, we evaluated expansions. clonal persistence overtime for each treatment group. We found that chemoimmunotherapy (FCR) resulted in T-cell repertoire renewal T-cell clonality increases over treatment and may be associated through ablation and immune reconstitution, whereas BcRi retained to clinical response most major pretreatment T-cell clones (median number of major We then tested clonality dynamics over treatment and explored pretreatment clonotypes that persisted at the 3-month timepoint: 2/10 associations with clinical response. At cohort level (n ¼ 28 patients), for FCR versus 8/10 for ibrutinib vs. 7/10 for rituximab– circulating T-cell clonality increased after treatment [median values idelalisib; Fig. 3A). Overlap analysis of the total repertoire pre- and from 30.3% pretreatment to 38.8% (P < 0.05), 41.9% (P < 0.05) and posttreatment per group showed similar results (Fig. 3B). Further- 46.2% (P < 0.01) at the 3-month, 9-month, and deepest clinical more, in the case of BcRi where the repertoire was studied longitu- response timepoint, respectively]. dinally over treatment, repertoire preservation persisted over time Within different treatment groups, T-cell clonality increased at (Fig. 3C). the 3-month timepoint in the case of fludarabine–cyclophosphamide–rituximab (from 28.8% to 46.9%, P < 0.05) and rituximab– Clonotype comparisons document the existence of idelalisib (from 33.0% to 39.1%, P < 0.01), but not ibrutinib (from “disease-biased” T-cell clonotypes 33.3% to 31.2%, P: 0.99). For the ibrutinib and rituximab–idelalisib To obtain insight into the antigenic specificities of the expanded groups, longitudinal analysis over treatment showed a gradual T-cell clones, we next listed all unique major clonotypes from our increase of T-cell clonality, related to the depth of clinical response, cohort (n ¼ 563 clonotypes) and compared them (i) across patients, reaching statistical significance in the case of rituximab–idelalisib and (ii) against T-cell clonotypes from various entities, as detailed in [from 33.0% pretreatment to 39.1% at 3 months (P < 0.01), 46.0% at the Materials and Methods section. 9months(P < 0.01), and 46.1% at best clinical response (P:0.13)], Thirty-two of the 563 clonotypes were shared among different but not ibrutinib [from 33.3% pretreatment to 31.2% at 3 months patients with CLL (Supplementary Table S4), while 18 of 563 were found in another entity and/or healthy donors (“public” clonotypes”; Fig. 4). In particular, “public” clonotypes corresponded to 9 matches with T-cell clonotypes of herpes virus specificity (CMV, EBV), 2 matches with clonotypes of herpes virus specificity (EBV) that were also found in patients with CIN, and 5 matches with T-cell clonotypes from healthy donors. Importantly, of the 32 major clonotypes that were shared among patients with CLL, only 9 were “public,” whereas the remaining 23 (23/563, 4.1% of all major clonotypes of our cohort) were shared exclusively by patients with CLL, alluding to selection by CLL-associated antigens (“CLL-specific”). Of these 23 “CLL-specific” clonotypes, 3 were shared among 4 different patients, 5 among three different patients, and the remaining 15 among pairs of patients.

Major CLL-specific T-cell clonotypes expand further over treatment Considering the above, we repeated our clonality analysis excluding all “public” clonotypes, with similar results. More specifically, clonality significantly increased at the 3-month timepoint in the case of FCR (from 20.8% to 46.9%, P < 0.01) and rituximab–idelalisib (from 32.9% to 38.8%, P < 0.01), but not ibrutinib (from 33.3% to 31.2%, P ¼ 0.50). Overtime, clonality increased in both the rituximab–idelalisib and ibrutinib group, however, reaching statistical significance only in Figure 2. – fi fl – the former [rituximab idelalisib: from 32.9% to 38.8% at 3 months T-cell clonality signi cantly increases after treatment with udarabine cyclo- P < P < phosphamide–rituximab and over treatment with rituximab–idelalisib. The ( 0.01), 44.4% at 9 months ( 0.01) and 39.5% at best clinical percent cumulative frequency of the major clonotypes is depicted overtime response (P: ns); IB: from 33.3% to 31.2% at 3 months (P:ns), 39.1% at per treatment group in a box–whisker fashion. , P < 0.01. 9 months (P: ns), and 42.1% at best clinical response (P: ns); Fig. 5].

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Figure 3. Fludarabine-cyclophosphamide-rituximab (FCR) results in T-cell repertoire renewal, whereas BcRi treatment retains pretreatment T-cell clones. Venn diagrams are used to depict the overlap between the T-cell repertoire pretreatment and at the 3-month timepoint per treatment group, concerning only the major T-cell clonotypes (A), and the total T-cell repertoire (B). Overlap was calculated as the percentage of clonotypes of the pretreatment repertoire which persisted at 3 months. C, The T-cell repertoire conservation remains over time for the ibrutinib (IB) and rituximab–idelalisib (R-ID) group. The Jaccard index, defined as the size of the intersection divided by the size of the union of the two sample sets [Jaccard similarity coefficient, J(A,B) ¼ |A \ B|/(|A| þ |B|-|A \ B|)], is plotted on the y-axis. Comparisons concern the repertoire of each patient (dot) pre/3 months, pre/9 months, and pre/deepest response, per treatment group. The median overlap (vertical line) remains practically the same over time for the ibrutinib and rituximab–idelalisib group, and significantly higher than for FCR.

Pretreatment major T-cell clonotypes from the peripheral blood Idelalisib-based therapy increases the expression of activation are also found in the bone marrow markers on effector memory T cells For 6 patients (rituximab–idelalisib, n ¼ 3 and ibrutinib, n ¼ 3), NGS immunoprofiling showed that T-cell clonality increased with synchronous pretreatment PB and infiltrated BM samples were avail- BcRis, with major clones persisting and further expanding over time. In able. We performed pairwise comparison of the T-cell repertoires, to some cases, these clones were shared among different patients with CLL investigate for the presence of PB major clonotypes within the but not found in other entities, suggesting that they may have developed respective BM repertoire. Excluding “public” clonotypes, all PB major in response to CLL-specific antigens. Therefore, we next wanted to clonotypes of these patients (n ¼ 56) were identified within the investigate the impact of treatment on T-cell functionality, so as to respective BM repertoires, and 37/56 (66.0%) were listed among the uncover a possible link between these T-cell expansions and antitumor major clonotypes/BM sample. responses that could contribute to the deepening of clinical response.

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Figure 4. “Public” and CLL-speciﬁc clonotypes. Of the 563 major clonotypes of the CLL cohort, 41 (7%) were found in other entities (“public”) or shared among different patients with CLL (“CLL-speciﬁc”).

To this end, we examined distinct T-cell subsets of 8 patients polarization of the cytolytic molecule perforin toward tumor cells in (rituximab–idelalisib, n ¼ 4; ibrutinib, n ¼ 4) pretreatment and at ibrutinib-associated T-cell synapses (P < 0.05; Fig. 6B). For 2 3 months for the expression of activation and exhaustion markers patients (rituximab–idelalisib, n ¼ 1; ibrutinib, n ¼ 1), RRI immune by flow cytometry. While the relative frequencies of these T-cell synapse analysis was also performed at the deepest clinical response þ þ subsets, namely CD4 and CD8 na€ve, central memory (CM), timepoint. These assays revealed no change in T-cell synapse forma- effector memory (EM), and terminal effector memory (TEMRA) T tion at the deepest response timepoint reached with ibrutinib therapy cells, did not significantly fluctuate over treatment (data not (25 months) compared with the 3-month timepoint, that may suggest a shown), the expression of activation markers significantly increased normalization of T-cell function. In contrast, for the patient who posttreatment in the rituximab–idelalisib group compared with the received rituximab–idelalisib, deeper clinical response (12 months) ibrutinib group. More specifically, we noted increased expression of: correlated with a further increase in polarized immune synapse þ (i) CD69 in CD4 EM (median fold change 2.0 vs. 0.5, P < 0.01); (ii) function (Fig. 6D). þ CD25 in CD8 TEMRA (median fold change 1.3 vs. 0.5, P < 0.01), þ and (iii) CD38 in CD8 EM (median fold change 1.6 vs. 0.3, P < 0.05), and TEMRA T cells (median fold change 1.1 vs. 0.3, P < 0.05; Discussion Supplementary Fig. S4). In this work, we sought to investigate the effect of different types of treatment on T cells from patients with CLL. Given the pleiotropic BcRi treatment improves T-cell immune synapse formation with impact of BcRi on the CLL microenvironment, we focused on BcRi as tumor cells currently used for CLL treatment (ibrutinib and rituximab–idelalisib) Given that T-cell dysfunction in CLL has been linked to impaired and compared with standard chemoimmunotherapy (fludarabine– immune synapse formation, we finally investigated the impact of cyclophosphamide–rituximab; ref. 13). Our study cohort was signif- therapy on immune function by characterizing the ability of patient icantly enriched for CLL cases with unmutated IGHV genes, the main T cells to form synapse interactions with autologous baseline CLL cells reason being that these cases are most frequently in need of treatment, in 13 patients pretreatment and at 3 months (fludarabine–cyclophos- especially BcRis. While it cannot directly account for IGHV-mutated phamide–rituximab, n ¼ 3; rituximab–idelalisib, n ¼ 5; IB, n ¼ 5). CLL, all reported findings concern pair-matched samples (samples of Confocal microscopy with quantitative image analysis showed that the same patient over time), therefore we argue that IGHV mutational T cells from FCR-treated patients did not improve their ability to form status is not an influent. synapse interactions (Fig. 6A), whereas both BcRi increased the We report that T-cell clonality significantly increased after treat- formation of polarized F-actin immune synapses with tumor cells ment with FCR and over treatment with rituximab–idelalisib, and, at (rituximab–idelalisib, ibrutinib, P < 0.05; fludarabine-cyclophospha- least in the case of rituximab–idelalisib, this correlated with the depth mide-rituximab, P ¼ 0.99; Fig. 6B and C). We also detected enhanced of clinical response, peaking at 9 months. A similar trend was detected

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Figure 5. Clonality analysis over time, excluding “public” clonotypes. A, Clonality signiﬁcantly increases after treatment with FCR and over treatment with rituximab–idelalisib (R-ID). The percent cumulative frequency of the major clonotypes is depicted over time per treatment group in a box–whisker fashion, after having excluded clonotypes that were also found in other entities/healthy individuals (“public” clonoypes). , P < 0.01. B, Persisting clonotypes expand over time, especially in the case of rituximab–idelalisib. Fishplots depicting how the frequency of each persisting major clonotype changes in relation to the pretreatment timepoint, again having excluded “public” clonotypes. Three characteristic cases of patients receiving rituximab–idelalisib and IB are depicted in the left and right column, respectively. Each ribbon represents a CLL-speciﬁc major clonotype that persists in all three timepoints over treatment (sequentially: pretreatment, at 3 months, 9 months, and deepest response), and the thickness of the ribbon at each timepoint corresponds to the clonotype's relative frequency. The remaining repertoire is shown in white for all cases.

with ibrutinib therapy, however, an increase in clonality occurred later At first glance, the ibrutinib results may seem contradictory to a in the course of treatment (≥9 mo) and did not reach statistical recent study by Yin and colleagues, reporting that ibrutinib therapy significance, possibly due to the smaller number of samples at more resulted in TR repertoire diversification; however, certain experimen- advanced timepoints. tal and analytic differences may explain this (7). From the start, the use

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Figure 6. BcRi treatment restores F-actin immunologic synapses between CLL tumor cells and autologous T cells. Untreated CLL tumor cells (CMAC dyed, blue) were conjugated with autologous negatively selected CD3þ T cells from CLL patient samples obtained before initiation of treatment (pre) and 3 months posttreatment. All conjugates per treatment timepoint were acquired using a confocal microscope. Quantitative image analysis [relative recruitment index (RRI)] was used for the calculation of F-actin (red) and perforin (green) polarization toward the tumor cell in T-cell/B-cell conjugates of fludarabine–cyclophosphamide–rituximab (FCR; n ¼ 3; A), ibrutinib (IB; n ¼ 6; B) and rituximab–idelalisib (R-ID; n ¼ 5; C) treated samples. Bar charts show the mean RRI values of all T-cell/B-cell CLL conjugates per treatment SEM. The confocal images show representative T-cell/B-cell conjugates for all treatments. Original magnification of immunofluorescence images 63. D, F-actin RRI analysis over deepening response for two patients on BcRi. Each dot corresponds to the mean RRI values SEM for treatment timepoint. Differences between pre- and posttreatment samples were assessed by Wilcoxon signed-rank test (, P < 0.05).

of different clonotype definitions renders the results incomparable: Yin the basis of unique amino acid CDR3 sequence and the same IGHV and colleagues reported outcomes on the 1000 most frequent clono- gene. Moreover, Yin and colleagues (7) utilized the ImmunoSEQ types (defined as productive unique sequences per sample), while in platform by Adaptive Biotechnologies, whereas we applied custom- this study we focused on the 10 most frequent clonotypes, defined on ized, purpose-built bioinformatics algorithms with intentionally strict

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length and quality filtering. Of note, the increasing T-cell repertoire mide–rituximab, BcRi improved immune synapse function and the diversity reported by Yin mostly results from an increasing number of recognition of tumor B cells. Interestingly, for the rituximab–idelalisib singletons. When we performed the same type of analysis in our case which was studied beyond 3 months, this synapse restoration cohort, following clonotype definition as per the Yin and colleagues correlated with the depth of clinical response, however definite con- study, we also found an increase of the singleton fraction at the clusions cannot be drawn as this was a single case. Improvement in 9-month timepoint, although not reaching statistical significance. immune synapse formation could be attributed to treatment-induced Besides, Yin and colleagues (7) mention that 6 of 16 dominant reduction in tumor load (as the T-cell defect has been shown to be TRBV-TRBJ usages persist and further expand over 12 months of tumor-induced; refs. 18, 19); however, the fact that this did not occur ibrutinib therapy, and therefore speculate antigenic stimulation, in an following fludarabine–cyclophosphamide–rituximab, the only treat- analogy to our results. Finally, Yin and colleagues (7) comment on ment that led to complete remissions, argues in favor of a BcRi-specific repertoire diversity by focusing on the emergence of new low- effect on T-cell function. Admittedly, the number of cases analyzed for frequency clones over treatment, while, we, on the other hand, focus immune synapse formation was limited, due to the substantial number on the dynamics of the most frequent clones on the consideration of T cells required for all assays performed in this study. Notably, our that these are the most biologically relevant, and dwell into the results are in keeping with a previous report studying the immuno- hypothesis that may have expanded in response to tumor antigens. modulatory impact of fludarabine–cyclophosphamide–rituximab and We do not comment on repertoire diversity; this may be associated rituximab–ibrutinib (43). with infectious risks and complications, as hypothesized by Yin Overall, NGS immunogenetics showed that BcRi retain T-cell and colleagues, but is most probably irrelevant to the dynamics of clones which can expand in parallel to deepening clinical response antitumor clones. and possibly contribute to it. This is further corroborated by pheno- Somewhat expectedly, fludarabine–cyclophosphamide–rituximab typic and functional bioassays that demonstrate concurrent T-cell increased T-cell clonality through repertoire ablation and reconstitu- activation and improvement in synapse recognition of tumor cells. tion, probably reflecting a lymphotoxic effect that is restored overtime. Taken together, this study provides a rationale for designing future Indeed, in a patient who received FCR and was studied prospectively, combinatorial therapeutic strategies aiming to boost antitumor the increase of clonality at 3 months posttreatment was restored to immune responses. In support of this, recent studies have described pretreatment levels by 9 months. In sharp contrast, BcRi did not the ability of both ibrutinib and idelalisib to enhance chimeric antigen reconstitute the T-cell repertoire, retaining pretreatment major T-cell receptor T-cell efficacy in CLL (44, 45). Future identification of the clones during therapy, and this was shown consistently for the longest relevant CLL-associated antigens may eventually pave the way for follow-up period reported so far (median 20 months). stratified treatments by means of engineered T cells or peptide To obtain evidence as to whether the herin identified T-cell vaccines, especially if these epitopes are conserved among patients clones might have expanded in response to CLL-associated antigens with CLL. or not, we performed extensive comparisons against not only CLL/MBL sequence datasets (36, 41), but also datasets from com- Disclosure of Potential Conflicts of Interest mon herpes virus infections (CMV/EBV), healthy individuals, and A. Vardi reports grants from European Hematology Association (European entities mediated by T-cell clones (30, 32–35, 37–40) and found a Hematology Association Physician Scientist Research Grant) during the conduct of significant number of major T-cell clonotypes shared exclusively the study. P. Ghia reports grants and personal fees from AbbVie (advisory boards; among patients with CLL. This is remarkable given: (i) the random research support), personal fees from Adaptive (advisory boards), personal fees from HLA background of our cohort, and (ii) the size of the comparison ArQule (advisory boards), personal fees from BeiGene (advisory boards), grants and personal fees from Gilead (advisory boards; research support), grants and personal dataset (761,968 distinct TRBV-TRBD-TRBJ sequences). Therefore, fi fees from Janssen (advisory boards; research support), personal fees from Juno/ this nding supports the existence of (neo)epitopes that are con- Celgene (advisory boards), personal fees from Loxo (advisory boards), personal fees served across CLL. Along the same line of reasoning, it is worth from MEI (advisory boards), grants and personal fees from Sunesis (advisory boards; noting that, for 3 patients studied over sequential lines of treatment, research support), and personal fees from Acerta/AstraZeneca (advisory boards) we found major clonotypes prior to first-line FCR which disap- outside the submitted work. K. Stamatopoulos reports grants from Janssen during the peared at CR following FCR treatment, but reemerged as major conduct of the study; grants and personal fees from AbbVie, personal fees from Janssen, and personal fees from Gilead outside the submitted work. A. Chatzidimi- clonotypes at first relapse and persisted through second-line treat- triou reports other from RANET Transcan-2, Project Acronym: Novel (supported in ment with BcRi (data not shown). part) and other from Hellenic Precision Medicine Network in Oncology (supported in To investigate the functional impact of treatment on CLL T cells, part) during the conduct of the study. we studied the expression of activation and exhaustion markers on T cells. We found that rituximab–idelalisib upregulates certain Authors’ Contributions activation markers including IL2 receptor (CD25) in effector mem- A. Vardi: Conceptualization, data curation, formal analysis, supervision, funding ory T cells compared with ibrutinib. Although this increase was acquisition, investigation, visualization, methodology, writing-original draft, arithmetically small, one needs to consider the fact that the fre- writing-review and editing. E. Vlachonikola: Data curation, formal analysis, quency of each major clone is relatively low; therefore, even though investigation, methodology. D. Papazoglou: Investigation, methodology, fi writing-original draft. F. Psomopoulos: Software, visualization. K. Kotta: we purposely focused our analysis on well-de ned functional T-cell Investigation, methodology. N. Ioannou: Investigation, methodology, writing- subpopulations, the immunophenotypic activation of antitumor original draft. C. Galigalidou: Investigation. K. Gemenetzi: Investigation. T-cell clones may be “diluted” within the respective subpopulation. K. Pasentsis: Methodology. M. Kotouza: Software, visualization. E. Koravou: It is also possible that CLL T cells undergo phenotypic changes Investigation. L. Scarfo: Resources. M. Iskas: Resources. N. Stavroyianni: within tissue TMEs as well as the PB (42). Resources. P. Ghia: Resources. A. Anagnostopoulos: Resources. A. Kouvatsi: Considering that T-cell dysfunction in CLL manifests in a defective Project administration. A.G. Ramsay: Resources, supervision, writing-original draft, writing-review and editing. K. Stamatopoulos: Conceptualization, ability to form immune synapses with tumor B cells, we investigated resources, supervision, funding acquisition, writing-original draft, writing- the effect of treatment on immune synapse formation capabili- review and editing. A. Chatzidimitriou: Supervision, funding acquisition, ty (18, 19). We found that, in contrast to fludarabine–cyclophospha- project administration.

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Acknowledgments The costs of publication of this article were defrayed in part by the payment of advertisement This work was supported in part by ERANET Transcan-2, Project Acronym: page charges. This article must therefore be hereby marked in Novel, and the Hellenic Precision Medicine Network in Oncology. A. Vardi is accordance with 18 U.S.C. Section 1734 solely to indicate this fact.> recipient of a research fellowship from the European Hematology Association (EHA Physician Scientist Research Grant). The authors thank Theodoros Moysiadis for providing statistical assistance and Tsagiopoulou Maria for providing assistance with Received November 25, 2019; revised April 1, 2020; accepted June 25, 2020; ﬁ ﬁshplot visualizations. published rst July 2, 2020.

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T-Cell Dynamics in Chronic Lymphocytic Leukemia under Different Treatment Modalities

Anna Vardi, Elisavet Vlachonikola, Despoina Papazoglou, et al.

Clin Cancer Res Published OnlineFirst July 2, 2020.

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