The Bruton’s tyrosine kinase inhibitor ibrutinib is a highly effective, new targeted therapy for chronic lymphocytic leukemia (CLL) that thwarts leukemia cell survival, growth, and tissue homing. The effects of ibrutinib treatment on the T cell compartment, which is clonally expanded and thought to support the growth of malignant B cells in CLL, are not fully characterized. Using next-generation sequencing technology, we characterized the diversity of TCRβ-chains in peripheral blood T cells from 15 CLL patients before and after 1 y of ibrutinib therapy. We noted elevated CD4+ and CD8+ T cell numbers and a restricted TCRβ repertoire in all pretreatment samples. After 1 y of ibrutinib therapy, elevated peripheral blood T cell numbers and T cell–related cytokine levels had normalized, and T cell repertoire diversity increased significantly. Dominant TCRβ clones in pretreatment samples declined or became undetectable, and the number of productive unique clones increased significantly during ibrutinib therapy, with the emergence of large numbers of low-frequency TCRβ clones. Importantly, broader TCR repertoire diversity was associated with clinical efficacy and lower rates of infections during ibrutinib therapy. These data demonstrate that ibrutinib therapy increases diversification of the T cell compartment in CLL patients, which contributes to cellular immune reconstitution.

Chronic lymphocytic leukemia (CLL), the most common leukemia in adults in Western societies, is characterized by the expansion of long-lived CD5+ mature monoclonal B lymphocytes in the blood and lymphatic tissues (1).The expression and function of the BCR are central to disease pathogenesis and prognosis (2). Activation of BCR signaling occurs in the secondary lymphatic tissues, where CLL cells proliferate in areas called pseudofollicles or proliferation centers (3). In these areas, CLL cells are interspersed with T cells, which colocalize with Ki67+ proliferating CLL cells, suggesting that T cells provide help to CLL cells to promote their expansion (46). Peripheral blood (PB) T cell numbers in untreated CLL patients are elevated and oligoclonal (4, 7), and emerging data indicate that these CLL T cells expand in an Ag-dependent fashion in a process that resembles normal adaptive immune responses to Ag (810). Prior studies reported on T cell repertoire skewing and oligoclonality in CLL patients based on flow cytometric analysis, spectratyping (9, 1113), and TCRβ sequencing (10). TCRαβ diversity is generated by random rearrangements of V and J segments in the TCRα gene and V, D, and J segments in the TCRβ gene, concurrent with nontemplated nucleotide insertions and deletions at the junctions (N-region); the resulting NDN region along with short segments of the flanking V and J genes make up CDR3 of the TCR, which is primarily responsible for recognition of antigenic peptides (14). Prior studies in CLL established T cell dysfunction (anergy), which was linked to increased susceptibility to infections (4, 1517), and defective immunologic synapse formation between T cells and CLL cells or other APCs, resulting in impaired cytotoxicity against the malignant B cells (15, 16) and an “exhausted” T cell phenotype from chronic antigenic stimulation (4, 8, 17).

Ibrutinib is an orally bioavailable irreversible inhibitor of Bruton’s tyrosine kinase (BTK), a central BCR signaling molecule (2). In patients with CLL, ibrutinib is given continuously, and it characteristically causes rapid shrinkage of enlarged lymph nodes, along with a transient increase in PB CLL cells due to redistribution of tissue-resident CLL cells into the PB (1820). With longer ibrutinib treatment, the vast majority of patients achieve durable remissions (1921). We recently reported that T cell numbers in CD4 and CD8 subsets normalized during ibrutinib-based therapy (19). It is unknown whether ibrutinib affects the T cell compartment via direct or indirect mechanisms. Ibrutinib’s inhibition of IL-2 inducible kinase (ITK), a member of the TEC kinase family that plays an important role in TCR signaling, T cell polarization, adhesion, and migration, may argue for direct drug effects (22). Because of a conserved Cys in the kinase domain of ITK that is identical to BTK, ITK is also inhibited by ibrutinib and, hence, may affect T cell function, especially in Th2-polarized CD4+ T cells (23).

Given the importance of T cells in CLL pathophysiology and the effects of ibrutinib treatment on PB T cell numbers, we studied ibrutinib treatment–induced changes in the T cell compartment in detail, using high-throughput TCRβ-chain sequencing to analyze the TCRβ repertoire in CLL patients before and after 12 mo of ibrutinib therapy, along with profiling of T cell–related cytokines.

This study was conducted using samples from patients treated by protocols that were reviewed and approved by the Institutional Review Board at MD Anderson Cancer Center in accordance with the Declaration of Helsinki; all patients provided informed consent for this study. Patients met clinical and immunophenotypic criteria for CLL and were treated at the MD Anderson Cancer Center between February 2012 and April 2015 with ibrutinib monotherapy or ibrutinib plus rituximab (ClinicalTrials.gov, NCT01520519 and NCT02007044). PB samples were collected before and after 3, 6 and 12 mo of ibrutinib treatment. Plasma samples were collected after centrifugation and stored at −80°C until further use. PBMCs were isolated via density gradient centrifugation over Ficoll-Paque (GE Healthcare, Pittsburgh, PA) and frozen in FBS (Life Technologies, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% DMSO (Sigma-Aldrich, St. Louis, MO) for storage in liquid nitrogen until further use. Clinical characteristics and laboratory data from the 29 patients analyzed in this study were collected using Clinic Station Version 3.4.4, an institutional electronic medical record system. Of the 29 patients, samples from 15 patients underwent T cell repertoire analysis using purified CD3+ T cells (Table I); serial samples from 14 patients were analyzed for changes in plasma cytokine levels. Additionally, T cell repertoire analyses from five age-matched healthy control samples (median age: 60 y, range 49–71 y; three females and two males) were obtained from the immunoSEQ platform database (Adaptive Biotechnologies, Seattle, WA). Baseline characteristics of the 29 patients are summarized in Supplemental Table I. Age-matched control plasma samples were purchased from ProteoGenex (Culver City, CA) for measurement of each cytokine in a cohort of age-matched healthy controls (median age: 62 y, range: 60–70 y, five females and seven males).

Table I.
Clinical characteristics of CLL patients with TCR repertoire analysis (n = 15)
Characteristicn (%)a
Sex  
 Female 7 (46.7) 
 Male 8 (53.3) 
Age (y; median [range]) 65 (48–75) 
Rai stage  
 0 0 (0.0) 
 I 3 (20.0) 
 II 3 (20.0) 
 III 2 (13.3) 
 IV 7 (46.7) 
Cytogenetic abnormalities (fluorescence in situ hybridization)  
 Del 17p 6 (40.0) 
 Del 11q 4 (26.6) 
 Trisomy 12 1 (6.7) 
 Diploid 1 (6.7) 
 Del 13q 3 (20.0) 
Survival status  
 Alive 15 (100.0) 
 Deceased 0 (0.0) 
Ig V region H chain  
 Mutated 1 (6.7) 
 Unmutated 13 (86.6) 
 Unknown 1 (6.7) 
CD38  
 Positive 5 (33.3) 
 Negative 10 (66.7) 
ZAP-70  
 Positive 9 (60.0) 
 Negative 4 (26.7) 
 Unknown 2 (13.3) 
Disease status  
 CR 3 (20.0) 
 PR 12 (80.0) 
Relapsed/refractory 12 (80.0) 
 No. of previous treatments 2 (1–4) 
 Time from last treatment to ibrutinib therapy (y; median [range]) 3 (1.5–13) 
 Previous purine analog 10 (66.7) 
 Previous alkylating agent 11 (73.3) 
 Previous anti-CD20 mAb 11 (73.3) 
 Previous lenalidomide 0 (0) 
Characteristicn (%)a
Sex  
 Female 7 (46.7) 
 Male 8 (53.3) 
Age (y; median [range]) 65 (48–75) 
Rai stage  
 0 0 (0.0) 
 I 3 (20.0) 
 II 3 (20.0) 
 III 2 (13.3) 
 IV 7 (46.7) 
Cytogenetic abnormalities (fluorescence in situ hybridization)  
 Del 17p 6 (40.0) 
 Del 11q 4 (26.6) 
 Trisomy 12 1 (6.7) 
 Diploid 1 (6.7) 
 Del 13q 3 (20.0) 
Survival status  
 Alive 15 (100.0) 
 Deceased 0 (0.0) 
Ig V region H chain  
 Mutated 1 (6.7) 
 Unmutated 13 (86.6) 
 Unknown 1 (6.7) 
CD38  
 Positive 5 (33.3) 
 Negative 10 (66.7) 
ZAP-70  
 Positive 9 (60.0) 
 Negative 4 (26.7) 
 Unknown 2 (13.3) 
Disease status  
 CR 3 (20.0) 
 PR 12 (80.0) 
Relapsed/refractory 12 (80.0) 
 No. of previous treatments 2 (1–4) 
 Time from last treatment to ibrutinib therapy (y; median [range]) 3 (1.5–13) 
 Previous purine analog 10 (66.7) 
 Previous alkylating agent 11 (73.3) 
 Previous anti-CD20 mAb 11 (73.3) 
 Previous lenalidomide 0 (0) 
a

Unless otherwise noted.

We used the MILLIPLEX map Human High Sensitivity T Cell Panel (EMD Millipore, Billerica, MA) to measure plasma levels of 21 cytokines before and after 3, 6, and 12 mo of ibrutinib therapy. The kit includes specific components for quantification of human IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-21, IL-23, TNF-α, IFN-γ, GM-CSF, CCL3 (MIP-1α), CCL4 (MIP-1β), CXCL11 (ITAC), CCL20 (MIP-3α), and fractalkine. The assay was performed according to the manufacturer’s instructions. The resulting raw data were acquired using a Luminex 200 plate reader and analyzed with the associated Bio-Plex Manager 4.0 software (Bio-Rad, Hercules, CA).

To analyze changes in TCR repertoire during ibrutinib therapy, we applied next-generation sequencing technology to characterize TCRβ CDR3 sequences before and after 12 mo of ibrutinib monotherapy (n = 10) or ibrutinib plus rituximab therapy (n = 5). To obtain purified T cell DNA templates, PBMCs were thawed, and CD3+ T cells were stained with a mouse mAb anti-human CD3 conjugated to allophycocyanin, clone UCHT1 (BD Biosciences, San Jose, CA) and purified by FACS using a FACSAria II (BD Biosciences); the gating strategy consisted of a first gate on live cells, followed by second and third gates on single cells (forward scatter–A versus forward scatter–H and side scatter–A versus side scatter–H, respectively). The purity of the sorted CD3+ population was >98% for all samples. Then, genomic DNA was extracted from purified CD3+ T cells using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions, and quantification and purity assessment were performed using a 1000 NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE). The input template DNA amount for next-generation sequencing was 1.2 μg per sample. Bias-controlled multiplexed PCR amplification, high-throughput sequencing, and TCRβ CDR3 region analysis via a bioinformatics pipeline were performed using the immunoSEQ platform (Adaptive Biotechnologies) (24, 25). The total sequences per sample were composed of productive sequences (“in frame”) and nonproductive sequences (including “out of frame” and “has stop,” which indicates a stop codon within the CDR3 region that was generated during VDJ recombination). Incomplete and out-of-frame rearrangements were filtered out. All productive unique sequences (clonotypes) per sample were ranked in four groups classified according to their frequencies (based on the ratio of read counts per clone/input cell numbers) to analyze the features of every TCRβ clonotype. Group 4 refers to the 1000 most frequent clones (top 1000) within each sample; group 3 refers to clones with frequency ranging from 0.0005% up to the top 1000; group 2 refers to clones with frequency ranging from singleton up to 0.0005%; and group 1 denotes singleton clones that were from nearly single cells (with read counts < 10). Next, to observe how the frequency changed for each patient in terms of distribution of clones among groups 1–4 from pretreatment to 1 y after ibrutinib therapy, all TCRβ-productive unique clones from each patient were considered as a whole, and each clone was assigned to one of three classes: “new,” “undetected,” and “persistent.” “New” indicates that the clones appeared after ibrutinib therapy and were not present before treatment; “undetected” refers to clones that were present before treatment and then disappeared after therapy; and “persistent” indicates that clones existed both before and after treatment.

All statistical analyses were performed using GraphPad Prism version 6.00 for Mac (GraphPad, La Jolla, CA). The results were expressed as mean ± SEM or median and range, as appropriate. Comparisons of proportions and variables between different groups were performed with the Mann–Whitney U test, Wilcoxon matched-paired signed-rank test, and paired or unpaired t test, as appropriate. Pearson correlation was used to analyze the correlation between univariates. Using a two-sided analysis, p ≤ 0.05 was considered statistically significant.

Prior to ibrutinib treatment, we noted elevated counts of CD3+ lymphocytes (mean ± SEM, 5458 ± 971 per microliter, n = 26), which dropped to 4531 ± 1401 per microliter after 3 mo of ibrutinib therapy (n = 11) and to 3486 ± 713 per microliter after 6 mo (n = 25), and they decreased significantly to 1876 ± 209 per microliter after 12 mo (n = 27, p = 0.0009, Fig. 1A). Accordingly, CD4+ and CD8+ T cell counts synchronously decreased with ibrutinib therapy over time (r = +0.981, p = 0.0189, Supplemental Fig. 1A). The mean CD4+ T cell count decreased from 2209 ± 337 per microliter prior to treatment (n = 27) to 1601 ± 346 per microliter after 3 mo (n = 11), to 1378 ± 194 per microliter after 6 mo (n = 25, p = 0.0157), and to 788 ± 59 per microliter after 12 mo of ibrutinib therapy (n = 27, p < 0.0001, Fig. 1A). CD8+ T cell counts decreased from 2711 ± 372 per microliter prior to therapy (n = 27) to 2224 ± 674 per microliter after 3 mo (n = 11), to 1994 ± 421 per microliter after 6 mo (n = 25), and to 1068 ± 169 per microliter after 12 mo (n = 27, p < 0.0001, Fig. 1A). We noted significant negative correlations between CD3+, CD4+, and CD8+ T cell counts and duration of ibrutinib therapy (CD3+ T cells: r = −0.998, p = 0.0016; CD4+ T cells: r = −0.980, p = 0.0199; and CD8+ T cells: r = −0.995, p = 0.0055, Supplemental Fig. 1A). The decreases in T cell counts were accompanied by a reduction in CD19+ CLL cell counts (Supplemental Fig. 1B).

FIGURE 1.

T cell counts and the levels of related plasma cytokines decreased significantly during ibrutinib therapy. (A) T cell subsets decreased over time during ibrutinib therapy. (B) Levels of plasma Th1-, Th2-, and Th17-type cytokines and chemotactic factors declined significantly compared with pretreatment levels. The bars represent mean values, and the dashed lines indicate the normal ranges. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001. Ctrl, age-matched control group; ns, not significant; Pre, pretreatment.

FIGURE 1.

T cell counts and the levels of related plasma cytokines decreased significantly during ibrutinib therapy. (A) T cell subsets decreased over time during ibrutinib therapy. (B) Levels of plasma Th1-, Th2-, and Th17-type cytokines and chemotactic factors declined significantly compared with pretreatment levels. The bars represent mean values, and the dashed lines indicate the normal ranges. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001. Ctrl, age-matched control group; ns, not significant; Pre, pretreatment.

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The majority of the plasma cytokine and chemokine concentrations, which were elevated pretreatment in CLL patients compared with healthy volunteers as reported previously (26), were reduced significantly after 3 mo of ibrutinib therapy. IL-6 and IL-8 were the only exceptions, with a moderate increase at 3 mo and a subsequent decrease (Supplemental Fig. 1C). Th1-, Th2-, and Th17-type cytokines remained low after 3, 6, 9, and 12 mo of continuous treatment (Fig. 1B). Because IFN-γ and IL-4 are the important differentiation factors for Th1 and Th2 T cells, respectively (23), the IFN-γ/IL-4 ratio can be used as an approximation of the Th1/Th2 balance. The mean IFN-γ/IL-4 ratio increased from 2.48 ± 0.83 at baseline to 2.94 ± 1.23 after 12 mo of therapy (n = 14, p = 0.708), suggesting that IFN-γ–producing Th1 cells become more prevalent during ibrutinib treatment (23). Consistent with previous studies (19, 27), plasma CCL3 and CCL4 levels were decreased significantly 3 mo after ibrutinib therapy, and remained low during the follow-up of 12 mo (Fig. 1B).

TCRβ sequences were generated with equal amounts of input template DNA that were extracted from matched numbers of purified CD3+ T cells in pretreatment samples and samples collected after 1 y of ibrutinib therapy (Table I). Consequently, we did not observe any significant difference in total productive sequence counts between pre- and posttreatment samples (p = 0.792, Fig. 2A). In contrast, we noted a significant increase in total unique TCRβ sequences from 58,783 ± 6,505 (n = 15) per sample before treatment to 80,945 ± 9,211 (n = 15) after 1 y of ibrutinib therapy (p = 0.0055, Supplemental Fig. 1C). More importantly, absolute productive unique sequence counts, an indicator of richness of clones, also increased significantly from 47,227 ± 5,258 (n = 15) per sample before treatment to 65,482 ± 7,633 (n = 15) after 1 y of ibrutinib therapy (p = 0.0056, Fig. 2B), with a median increase of 76.1% (range 0.53–115.46%, Supplemental Fig. 1D, 1E). However, these counts were still significantly lower than those in age-matched healthy controls (p < 0.0001), suggesting that it may take >1 y before the process of TCR diversity recovery is complete. Furthermore, to quantify TCR diversity we analyzed the relative productive unique sequences (i.e., ratio of productive unique sequences/productive total sequences) and confirmed that TCRβ repertoire diversity increased significantly 1 y after ibrutinib therapy relative to pretreatment (p = 0.0215, Fig. 2C).

FIGURE 2.

TCRβ repertoire diversity increased after 1 y of ibrutinib therapy. (A) There was no significant difference in the numbers of productive total TCRβ sequences between samples taken before and after 1 y (12 m) of ibrutinib therapy. In contrast, the number of absolute productive unique sequences, representing the richness of the clones (B), and relative productive unique sequences (C) increased significantly after 1 y (12 m) of ibrutinib therapy. *p < 0.05, **p < 0.01, ****p < 0.0001. ns, not significant; Pre, pretreatment.

FIGURE 2.

TCRβ repertoire diversity increased after 1 y of ibrutinib therapy. (A) There was no significant difference in the numbers of productive total TCRβ sequences between samples taken before and after 1 y (12 m) of ibrutinib therapy. In contrast, the number of absolute productive unique sequences, representing the richness of the clones (B), and relative productive unique sequences (C) increased significantly after 1 y (12 m) of ibrutinib therapy. *p < 0.05, **p < 0.01, ****p < 0.0001. ns, not significant; Pre, pretreatment.

Close modal

To further characterize TCRβ repertoire restoration during ibrutinib therapy, clonotypes within the TCR libraries from each sample were assigned to four groups according to frequency (Fig. 3A). We found that there was significantly greater diversity in T cell repertoire after 1 y of ibrutinib therapy, because the fraction of “top 1000” clones declined from 2.49 ± 0.26% (n = 15) in pretreatment samples to 1.94 ± 0.30% (p = 0.0222), and the fraction of “singleton” clones increased significantly from 27.35 ± 1.43% (n = 15) to 33.32 ± 2.10% (p = 0.0069, Fig. 3B, 3C). The diversity in the CDR3 regions of the TCRβ-chains is generated by nontemplated nucleotide insertions at the Vβ–Dβ and Dβ–Jβ junctions. Accordingly, we noted more nucleotide insertions in the CDR3 regions in clones with low-frequency TCRβ sequences than in clones with high-frequency sequences (Supplemental Fig. 2A), together with greater CDR3 length (Supplemental Fig. 2B). Collectively, these results indicate that patients acquire more low-frequency clones with greater diversification of TCR repertoire during ibrutinib therapy.

FIGURE 3.

High-frequency TCRβ clones diminished and low-frequency T cell clones increased during ibrutinib therapy. (A) Graphic representation of the model used for assignment of clones according to their frequency. (B and C) TCRβ repertoire diversity increased significantly after 1 y of ibrutinib therapy compared with pretreatment samples, because the fraction of high-frequency clones declined significantly, and the fractions of “singleton” clones increased significantly. *p < 0.05, **p < 0.01, ****p < 0.0001. ns, not significant.

FIGURE 3.

High-frequency TCRβ clones diminished and low-frequency T cell clones increased during ibrutinib therapy. (A) Graphic representation of the model used for assignment of clones according to their frequency. (B and C) TCRβ repertoire diversity increased significantly after 1 y of ibrutinib therapy compared with pretreatment samples, because the fraction of high-frequency clones declined significantly, and the fractions of “singleton” clones increased significantly. *p < 0.05, **p < 0.01, ****p < 0.0001. ns, not significant.

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For analysis of correlations between TCRβ repertoire diversity and established prognostic markers, individual TCRβ repertoire diversity was stratified based on patients’ ZAP-70 and CD38 expression, prior treatment, cytogenetic abnormalities, disease duration, Rai stage, age, β2-microglobulin (β2M) levels, and absolute lymphocyte counts at pretreatment and at 1 y. Before ibrutinib therapy, there was no significant difference in TCRβ repertoire diversity between patients with low- and high-risk prognostic markers (Supplemental Fig. 3A). We also did not find any significant impact of ZAP-70, CD38, disease duration, disease status, or prior treatment (ibrutinib versus ibrutinib plus rituximab) on TCRβ repertoire diversification (Supplemental Fig. 3). However, we noted that patients who achieved complete remission (CR) and who had no prior treatment had broader TCR repertoire diversification after 1 y of ibrutinib therapy than did patients who achieved partial remission (PR) and had received prior treatment (both p = 0.0482, Fig. 4A, Supplemental Fig. 3A). Significantly higher TCR repertoire diversification during ibrutinib therapy also was noted in younger patients (age < 65 y, p = 0.0298) and in patients with lower β2M levels (<4.5 mg/l, p = 0.0303), lower Rai stages (stage I or II, p = 0.0107), and lower pretreatment CD3+ and CD8+ T cell counts (p = 0.0099 and p = 0.029, respectively, Supplemental Fig. 3A). However, these correlations are based on relatively small numbers of patients and, therefore, need to be interpreted with caution and should be validated in a larger cohort.

FIGURE 4.

Broader TCR repertoire diversification was associated with clinical response and lower infection rates during ibrutinib therapy. (A) Patients who achieved CR had greater TCR repertoire diversification than did patients who achieved PR after 1 y of ibrutinib therapy. (B) Broader TCR repertoire diversity was associated with lower rates of infections during the first 6 mo of ibrutinib therapy, and TCR repertoire diversity increased significantly after 1 y of ibrutinib therapy in patients with lower rates of infection. *p < 0.05. ns, not significant.

FIGURE 4.

Broader TCR repertoire diversification was associated with clinical response and lower infection rates during ibrutinib therapy. (A) Patients who achieved CR had greater TCR repertoire diversification than did patients who achieved PR after 1 y of ibrutinib therapy. (B) Broader TCR repertoire diversity was associated with lower rates of infections during the first 6 mo of ibrutinib therapy, and TCR repertoire diversity increased significantly after 1 y of ibrutinib therapy in patients with lower rates of infection. *p < 0.05. ns, not significant.

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Next, we explored the impact of TCR repertoire diversity on the risk for infection in CLL patients. Twelve of fifteen patients (80%) developed 27 cases of infections during a median follow-up time of 25 mo (range 22–26 mo). Infections were more frequent during the first 6 mo, with an average rate of 16.7 infections per 100 patient-months compared with 4.2 thereafter. Respiratory tract infections were the most common (51.9%), followed by skin (14.8%) and genitourinary (11.1%) infections. We noted a significant correlation between broader TCR repertoire diversity and lower rates of infections during the first 6 mo of ibrutinib therapy (p = 0.0425, Fig. 4B); accordingly, TCR repertoire diversity increased significantly after 1 y in patients with lower rates of infections (p = 0.0308, Fig. 4B).

Sixteen and twenty-two Vβ-Jβ segment usages with >20,000 reads were found before and after 1 y of ibrutinib therapy, respectively. Of the top 16 Vβ-Jβ segment usages before treatment, six usages (Vβ 06-05/Jβ 01-02, Vβ 02-01/Jβ 01-01, Vβ 06-05/Jβ 01-01, Vβ 06-01/Jβ 01-05, Vβ 05-06/Jβ 01-03, and Vβ 07-09/Jβ 02-07) had increased further at 1 y after ibrutinib therapy. These findings suggest that usages of Vβ-Jβ subfamilies, skewed before treatment, were improved and increased after 1 y of ibrutinib therapy, and in the meantime, antigenic stimulation (by unknown Ags) promotes the persistence or overexpression of certain Vβ-Jβ subfamilies during therapy.

To track each productive unique clone in CLL patients from pretreatment to 1 y of ibrutinib therapy, we categorized all productive unique clones from each patient into new, undetected, or persistent T cell clones. We detected a large number of new clones that emerged during 1 y of ibrutinib therapy (median 53.16%, range 34.67–65.91%) (Fig. 5). Interestingly, the majority of these new clones (median 61.95%, range 41.57–81.07%) were small-sized clones, with frequency < 0.0005% (groups 2 and 1, Supplemental Fig. 2C), suggesting that these represent newly generated T cell clones rather than being derived from T cells present before treatment. In contrast, 31.14% (median) of pretreatment clones (range 23.96–53.69%) were undetectable after 1 y of ibrutinib therapy (Fig. 5). These were primarily high-frequency clones (frequency > 0.0005%, groups 4 and 3, Supplemental Fig. 2D). Moreover, a median 75% of the top 1000 pretreatment clones (range 63.9–86.6%) declined in frequency, and 5.60% (range 1.60–15.10%) became undetectable. Increases were seen in only 18% of these clones (range 10.90–29.50%, Fig. 5). A total of 11.43% (range 5.98–21.89%) of T cell clones persisted after 1 y of ibrutinib therapy and were derived primarily from high-frequency clones; of these, a median of 45.48% (range 30.79–50.23%) had increased in frequency, and 54.52% (range 49.77–69.21%) had decreased (Fig. 5). Collectively, these results indicate that the vast majority of dominant TCRβ clones that were present before treatment decreased or disappeared during ibrutinib therapy, whereas, at the same time, a large number of new low-frequency T cell clones, presumably naive T cells, emerged.

FIGURE 5.

Longitudinal evaluation of the shift in T cell clones from pretreatment to 1 y after ibrutinib therapy. The dominant clones present before treatment decreased in frequency or vanished, whereas a large number of new clones emerged 1 y after ibrutinib therapy.

FIGURE 5.

Longitudinal evaluation of the shift in T cell clones from pretreatment to 1 y after ibrutinib therapy. The dominant clones present before treatment decreased in frequency or vanished, whereas a large number of new clones emerged 1 y after ibrutinib therapy.

Close modal

This deep-sequencing analysis of the TCRβ repertoire in CLL patients undergoing ibrutinib therapy revealed a significant diversification of the TCRβ repertoire during ibrutinib therapy. At the same time, the elevated numbers of PB T cells in pretreatment samples, affecting the CD4 and CD8 subsets, normalized. More specifically, we noted a significant increase in the numbers of productive unique TCRβ clones after 1 y of ibrutinib therapy compared with the matched pretreatment samples. Furthermore, this increase in TCRβ productive unique sequences and the emergence of large numbers of new T cell clones with low frequency, together with a major reduction or deletion of the majority of clonally expanded high-frequency T cell clones that were present before therapy, corroborate recovery of T cell repertoire diversification during ibrutinib therapy in CLL patients.

T cell immune dysfunction is a common feature in CLL that was linked to increased susceptibility to infectious and autoimmune complications (15, 16, 28). Studies of T cell repertoire and function in CLL patients established a clonal expansion of T cells with increased numbers of CD4+ and CD8+ T cells, elevated levels of T cell–related cytokines, and restrictive usages of specific Vβ-Jβ subfamilies driven by Ag selection, resulting in a skewed TCR repertoire (4, 912, 17, 29). The synchronous decline in blood CLL and T cell numbers and the associated decrease in plasma chemokine and cytokine levels during ibrutinib therapy (Fig. 1) support the concept of coevolution and interdependence of T cells and CLL cells (4, 23, 27). With regard to the decreases in plasma cytokine levels during ibrutinib therapy, more direct effects of ibrutinib have to be considered. Through inhibition of BTK and off-target enzymes, such as ITK, ibrutinib can directly alter the activity of T cells (23), NK cells (30, 31), and monocytes/macrophages (27), resulting in reduced cytokine secretion by these cells. The functional role of these clonally expanded T cells in CLL patients remains controversial; both protumoral and tumor-suppressive activities were discussed (3235). In addition to CLL-promoting CD4+ cells (32, 33), other studies characterized CTLs that recognize CLL-specific idiotype peptides or other tumor-associated Ags (34, 35) and identified leukemia-associated mono/oligoclonal T cells within the circulating T cell compartment (7). However, these tumor-associated Ag-specific T cell clones apparently cannot effectively eliminate the malignant B cells, which may be related to T cell exhaustion.

Notably, ibrutinib therapy in CLL results in a decline and normalization of elevated CD4+ and CD8+ T cell numbers, and inhibition of T cell–related chemokine and cytokine production (19, 23, 27) also alters the composition of T cell subsets by exerting a Th1-selective pressure and inhibiting differentiation of the Th17 T cell subset that was skewed in CLL, as described previously (23, 27, 36), indicating a normalized T cell state. More importantly, TCRβ repertoire diversification was reconstituted after 1 y of ibrutinib therapy, which is reminiscent of changes in the T cell compartment after allogeneic hematopoietic stem cell transplantation, for which large numbers of new TCRβ sequences and diverse T cell clonotypes were also seen, with a simultaneous decline or deletion of TCRβ clones that were dominant before allogeneic hematopoietic stem cell transplantation (3739). These findings suggest that, over time, effective ibrutinib therapy reduces TCR repertoire skewing and T cell clonal expansion, and patients acquire a more normal TCR repertoire. Moreover, these findings are also interesting in the context of clinical findings showing a major decrease in infectious complications in CLL patients treated with ibrutinib once remissions were achieved (22, 28, 40). Interestingly, we also noted that the rate of infection was relatively low in patients with broader TCR repertoire diversity, especially during the first 6 mo of ibrutinib treatment, when infections were more frequent. Moreover, patients that achieved CR had higher TCR repertoire diversity than did PR patients after 1 y of ibrutinib therapy, which indicates that deeper remission allows for better recovery of TCR repertoire diversity.

Additionally, we found broader TCR repertoire diversification during ibrutinib therapy in patients with low-risk prognostic markers (e.g., in younger patients and those with low β2M levels and lower Rai stages). Therefore, changes in TCR repertoire diversity during ibrutinib therapy reflect, to some extent, treatment response and patient characteristics and may have an impact on longer-term outcome. However, given the relatively small number of cases analyzed in this study, these findings need to be corroborated in a larger cohort of patients.

In summary, our data demonstrate that ibrutinib therapy promotes the recovery of TCR repertoire diversity in CLL patients. The emergence of a large number of new TCRβ clones, together with the increased diversity of the TCR repertoire, indicate that CLL patients acquire a broader TCR Vβ repertoire after 1 y of ibrutinib therapy. These findings strengthen the concept of coevolution of CLL cells with T cells and provide novel insight into ibrutinib’s effects on the T cell compartment in CLL patients.

We thank Sunita Patterson for outstanding manuscript edits and Benjamin Hayes for assistance with sample and data collection.

This work was supported by a Leukemia and Lymphoma Society Scholar Award in Clinical Research (to J.A.B.), a Cancer Center Support Grant (National Cancer Institute Grant P30 CA016672), The University of Texas MD Anderson Cancer Center Moon Shots Program in Chronic Lymphocytic Leukemia, and the National Natural Science Foundation of China (Grant 81000921).

The online version of this article contains supplemental material.

Abbreviations used in this article:

BTK

Bruton’s tyrosine kinase

CLL

chronic lymphocytic leukemia

CR

complete remission

ITK

IL-2 inducible kinase

β2M

β2-microglobulin

PB

peripheral blood

PR

partial remission.

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J.A.B. and S.O. received research funding from Pharmacyclics. The other authors have no financial conflicts of interest.

Supplementary data