Activation of Th1 Immunity Within the Tumor Microenvironment is Associated with Clinical Response to Lenalidomide in Chronic Lymphocytic Leukemia^$

Abstract

Immune stimulation contributes to lenalidomide’s anti-tumor activity. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature, autoreactive B cells in secondary lymphoid tissues, blood and bone marrow and progressive immune dysfunction. Previous studies in CLL indicated that lenalidomide can repair defective T-cell function in vitro. Whether T-cell activation is required for clinical response to lenalidomide remains unclear. Here we report changes in the immune microenvironment in patients with CLL treated with single-agent lenalidomide and associate the immunologic effects of lenalidomide with anti-tumor response. Within days of starting lenalidomide, T cells increased in the tumor microenvironment and showed Th1-type polarization. Gene expression profiling of pre-treatment and on-treatment lymph node biopsies revealed upregulation of IFNγ and many of its target genes in response to lenalidomide. The IFNγ-mediated Th1 response was limited to patients achieving a clinical response defined by a reduction in lymphadenopathy. Deep sequencing of T-cell receptor genes revealed decreasing diversity of the T-cell repertoire and an expansion of select clonotypes in responders. To validate our observations, we stimulated T cells and CLL cells with lenalidomide in culture and detected lenalidomide-dependent increases in T-cell proliferation. Taken together, our data demonstrate that lenalidomide induced Th1 immunity in the lymph node that is associated with clinical response.

Introduction

Evading immune destruction is a hallmark of tumor progression (1). Immune cells not only fail to control tumor growth but may in fact sustain proliferation and survival of tumor cells (2). In patients with chronic lymphocytic leukemia (CLL), global gene expression profiling of CD4⁺ and CD8⁺ cells revealed defects involving cell differentiation, cytotoxicity and cytoskeletal pathways (3). Thus, restoration of T-cell anti-tumor immunity represents an attractive treatment strategy to restore immune surveillance (2, 4).

The immunomodulatory drug lenalidomide upregulates co-stimulatory molecules on tumor cells (5, 6) and repairs impaired immunologic synapse formation between T cells and CLL cells (7). Lenalidomide promotes NK-cell mediated killing of tumor cells in vitro (8) and stimulates the production of immunoglobulins by normal B cells (6). The proliferation of CLL cells is also directly inhibited by lenalidomide in culture via a cereblon-dependent induction of the cell-cycle inhibitor p21 (9). Two recent clinical trials showed that maintenance therapy with lenalidomide delayed disease progression without deepening responses (10, 11). In the absence of tumor eradication, the in vivo mechanisms by which lenalidomide exerts activity against CLL are poorly understood.

Here we comprehensively evaluated changes in the T-cell compartment in patients with relapsed or refractory CLL treated with lenalidomide. Our data link interferon-γ (IFNγ) production, T-cell proliferation, and Th1 polarization in the lymph node (LN) microenvironment to clinical response.

Materials and Methods

Patient selection and clinical characteristics

Samples were collected from patients with relapsed CLL or small lymphocytic lymphoma (SLL) treated with lenalidomide under a phase 2 investigator-initiated study (NCT00465127). Between May 2007 and February 2010, 33 patients received lenalidomide at 10 or 20 mg daily cycled 3 weeks on, 3 weeks off for up to 8 cycles (5, 6). The study was approved by the institutional review board at the National Heart, Lung, and Blood Institute, and conducted in accordance with the Declaration of Helsinki. All patients provided written informed consent. The primary endpoint was overall response after 4 cycles as assessed by modified International Workshop on Chronic Lymphocytic Leukemia criteria (12). Lymphadenopathy was assessed by the sum of the product of the greatest diameters of representative lymph nodes with computed tomography (CT). Samples for in vitro studies were collected from patients with treatment naïve CLL after obtaining written informed consent (NCT00923507). Peripheral blood mononuclear cells (PBMCs) and LN core biopsies were collected prior to and on day 8 of therapy and stored as previously described (5).

Gene expression analysis

Total RNA was isolated from CD19⁺ selected PBMCs and LN core biopsies. Microarray analysis was performed on Affymetrix U133 plus 2.0 chips (Santa Clara, CA) as described (13). Biotin-labeled RNA (20 μg) was fragmented to ~200 bp and hybridized to U133 Plus 2.0 chips for 16 hours, washed, and stained on a fluidics station. Affymetrix Expression Console software was used to calculated signal intensities and present calls on the hybridized chips. The signal intensity values of the probe sets were normalized by Robust Multi-Array Average (RMA) across the chips (14). Only probe sets with a present signal on > 5 arrays were selected for analysis. The expression of multiple probe sets corresponding to a gene was averaged. Two-way analysis of variance (ANOVA) was applied to evaluate patient and lenalidomide treatment effects on day 8 relative to day 0. The Benjamini and Hochberg method was used to correct for multiple testing (15). Cluster and Tree View (Eisen Laboratory, Stanford University, Palo Alto, CA) and Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Redwood, CA accessed June 8, 2018) were used for gene expression analysis. The microarray dataset is available on the NCBI GEO website under accession GSE112953. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE112953

Previously described CD4⁺ and CD8⁺ T-cell gene signatures were used for T cell subsets analysis (16–18).

Flow cytometry and immunohistochemistry

Enumeration of CD3⁺ cells and intracellular staining for IFN was performed as previously described (19, 20). IFNγ in the serum was measured using Mesoscale (Gaithersburg, MD).

LN core biopsies were stained with CD3, CD4 and CD8 (Dako, Carpinteria, CA). The number of CD3⁺ cells was scored in five representative high-power fields by a trained pathologist blinded to the samples. Images were captured at 400x fold magnification on an Olympus Bx41 microscope (Center Valley, PA).

T-cell receptor deep sequencing

TCR α- and β-chain deep sequencing was performed to assess lenalidomide-induced clonal expansion of T cells in LN as previously described (21). In brief, 1 μg of total RNA (only 0.75 μg of total RNA available at pre-treatment from subject L2) was used for PCR-based amplification of TRA or TRB gene products with adapter-conjugated primer sets. The template library was amplified by Nextera XT DNA sample prep kit (Illumina, San Diego, CA). Subsequently, the prepared library was analyzed using MiSeq Reagent 600-cycle kit v3 and MiSeq system (Illumina). After deep sequencing, each V, (D), J and C segment of TCR α- and β-chains were mapped to reference sequences in IMGT/GENE-DB (22) and assigned for determination of the complement determining region 3 (CDR3) amino acid sequence as previously described (21). The diversity index (inverse Simpson’s index) of CDR3 sequences was calculated to assess overall diversity and clonality in the TCR α and β clonotypes.

T-cell proliferation assay

PBMC (5×10⁵ cells/mL) were cultured for 3 weeks in RPMI, supplemented with penicillin, streptomycin and glutamine (all Gibco, Grand Island, NY), fetal calf serum (10%, Sigma, St Louis, MO), IL2 (100 u/ml), IL7 (50 IU/ ml) and IL15 (5 IU/ ml, all Peprotech, Rocky Hill, NJ) and in presence (2 μM, dissolved in DMSO (0.1%) or absence (DMSO 0.1%) of lenalidomide (Sequoia Research Products, Berkshire, UK). Cells were once stimulated at a 1:1 ratio with irradiated PBMCs (25 Gy). Then CD3 negative selection (Robosep, Stemcell Technologies, Vancouver, BC, Canada) was performed. CD19⁺ cells were obtained from autologous PBMCs, cultured with lenalidomide (2 μM) or vehicle (DMSO) for 48 hours (CD19 positive selection, Miltenyi, Auburn CA). CD3⁺ cells were stained with 0.5 μM CFSE (carboxyfluorescein succinimidylester Gibco) as described (23). CD3⁺ cells and CD19⁺ cells were co-cultured at a 1:1 ratio at 1×10⁶ cells in RPMI alone (no cytokines, no lenalidomide, no fetal calf serum, 96 well plate). Flow cytometric analysis was performed at 72 hours post-stimulation (Fortessa, BD BioSciences, San Jose, CA) following manufacturer’s instructions. The following experiments were set up: CD3, CD3 (lenalidomide treated), CD3/ CD19, CD3 (lenalidomide treated)/ CD19, CD3 / CD19 (lenalidomide treated), CD3 (lenalidomide treated) / CD19 (lenalidomide treated). Intracellular staining was described before (BD Cytofix/ Cytoperm plus, Fixation/ Permeabilization solution kit with BD GolgiPlug, BD BioSciences, San Jose, CA). 5×10⁵ cells were stained with the following mouse anti-human antibodies: Vivid and CD14 Pacific Blue, CD19 APC, CD4 V500, CD8 H7APC, IFNg PE-Cy7 (all BD BioScience PharMingen, San Jose, CA), and CD3 Efluor605 (Thermofisher eBioscience, San Diego, CA).

Statistical Analysis

Paired t-test was used to compare pre- and on-treatment samples and unpaired t-test was used to compare responders and non-responders. A p-value of < 0.05 was considered statistically significant. Statistical analyses were performed using JMP® 13 (SAS Institute Inc., Cary, NC).

Results

Clinical experience with lenalidomide

Thirty-three patients with relapsed CLL were enrolled on a phase 2 study of lenalidomide at 10 mg or 20 mg daily for 3 weeks followed by 3 weeks off for up to 8 cycles. Patients received a median of 2 prior lines of therapy (range 1–5), including purine analogue in 81% and anti-CD20 monoclonal antibody in 100% of patients. All patients had progressive disease requiring treatment at the time of enrollment. On an intention-to-treat basis, 5 (15%) patients achieved partial remission, 20 patients (60%) had stable disease, and 8 patients (25%) had progressive disease. Twenty-three patients completed 4 cycles of therapy and were evaluated by absolute lymphocyte count (ALC) and CT. Thirteen patients showed a ≥10% reduction in ALC (Supplemental Fig. 1A). Nine patients showed a ≥10% reduction in lymphadenopathy (Supplemental Fig. 1B) and were considered responders for the correlative analyses presented herein.

Lenalidomide activates CLL cells via T-cell derived interferon-γ

To understand the anti-tumor effects of lenalidomide, we performed gene expression profiling on circulating CD19⁺-selected cells from 11 patients treated with lenalidomide. We identified 79 lenalidomide-responsive genes (fold-change ≥ 2, FDR < 0.2 between pre- and Cycle 1 Day 8 samples, Supplemental Table I), of which 67 were upregulated and 12 downregulated (Fig. 1A). Upregulated genes encoded chemokines (CCL3, CCL4), cytokines or cytokine receptors (IL13RA1, TNF, TNFSF13B), signal transduction molecules (STK3, RCAN1, KSR2) and molecules involved in the regulation of apoptosis (DDIT4, PAPRP9, CFLAR). Ingenuity Pathway Analysis identified the IFNγ signaling pathway as the most significantly overrepresented pathway (p = 6.5E-10). Specifically, among 36 known target genes of IFNγ, 6 were upregulated and none were downregulated in response to lenalidomide, suggesting that tumor cells respond to IFNγ. Indeed, serial measurements of serum IFNγ in these patients showed a significant increase as early as day 4, which more than doubled by day 8, and remained elevated during the first 3 weeks on lenalidomide (Fig. 1B). After 3 weeks off lenalidomide, serum IFNγ returned to baseline levels before increasing again with the start of cycle 2.

Figure 1. Induction of a T-cell mediated IFNγ response by lenalidomide.

(A) Heat map of DE genes (fold change ≥ 2, FDR-adjusted p < 0.2) in purified PB CLL cells between paired pre- (Day 0) and on-treatment (Day 8) samples (n = 11). Select genes referred to in the text are indicated. (B) Fold-change in serum IFNγ relative to baseline. Lenalidomide was administered during weeks 1–3 and 6–9. On-treatment serum IFNγ was compared to baseline by paired t-test (* p < 0.05, ** p < 0.01). (C) Detection of intracellular IFNγ in CD4⁺ and CD8⁺ T cells on Day 0 and Day 8 PB samples. Comparisons by paired t-test.

IFNγ is the canonical cytokine of Th1-type tumor immune surveillance and eradication (24, 25). Using flow cytometry, we found an increased proportion of circulating IFNγ⁺ CD4⁺ and CD8⁺ T cells in patients treated with lenalidomide (p = 0.003 and 0.04, respectively, Fig. 1C). While the relative increase was more pronounced in CD4⁺ compared to CD8⁺ T cells, the frequency of IFNγ⁺ cells on day 8 was comparable between the two T-cell subsets, suggesting that both contributed to IFNγ secretion.

Microenvironmental gene expression signature is associated with treatment response

A classic observation in CLL patients starting lenalidomide is the tumor flare reaction (TFR), an often rapid and painful swelling of lymph nodes. TFR is thought to be due to increased T-cell infiltration of the tumor (5, 6). To further dissect the T-cell response induced by lenalidomide, we performed gene expression profiling of pre-treatment and Cycle 1 Day 8 LN samples from the 11 patients described above. Overall, 56 genes were differentially expressed (fold-change ≥ 2, FDR < 0.2, Supplemental Table I) between pre- and on-treatment lymph node samples and suggested a T-cell response induced by lenalidomide. Next, we asked if and how gene expression could differ based on clinical response. We identified 119 differentially expressed genes among 7 responders (Fig. 2A, Supplemental Table II) and none among 4 non-responders. This discrepancy between responders and non-responders suggested that changes in gene expression within the LN could help predict clinical response to lenalidomide.

Figure 2. Lenalidomide modulation of a tissue-specific gene expression profile.

(A) 119 genes identified as lenalidomide-responsive in the 7 patients with decreasing lymphadenopathy are shown for 7 responding (green bar) and 4 non-responding patients (yellow bar). Select genes referred to in the text are indicated. (B) The expression of IFNG and (C) 42 IFNG target genes in response to lenalidomide was significantly different between responders (R) and non-responders (NR) (unpaired t-test). (D) Present absent analysis for all interferons. Yellow = gene expressed, black = gene is not expressed, and red = gene is partially expressed (PA 0.5).

To investigate the relationship between changes in the tumor microenvironment and clinical response, we were particularly interested in the group of 98 genes that were upregulated by lenalidomide in LN samples (Fig. 2A). These genes comprised important immune regulatory molecules including IFNG (Fig. 2B), cytotoxic effector molecules (GZMA, GZMB), lymphocyte activation markers (CD38), and multiple chemokines (e.g. CXCL11). We note that IFNG was the only member of the interferon family consistently expressed across samples (Fig. 2D). Notably, 42 of 98 genes (43%) that were significantly upregulated by lenalidomide in responders compared to non-responders were IFNγ-regulated genes (p = 0.02, Fig. 2C).

Lenalidomide induces a T-cell response in the lymph node

Tumor-infiltrating lymphocytes have been associated with improved survival and response to treatment across multiple cancers (26). We previously reported that the number of T cells in lymph node (LN) biopsies increased in some but not all patients treated with lenalidomide (5). In patients with available pre- and on-treatment LN biopsies, we compared the degree of T-cell infiltration between responders and non-responders. Lenalidomide appeared to induce a more prominent T-cell infiltrate in the LN biopsies of responders than non-responders, but not quite meeting statistical significance likely owing to a small sample size (p = 0.056, Fig. 3A). Additional immunohistochemistry suggested that this T-cell infiltrate was comprised of more CD4⁺ than CD8⁺ T cells (Fig. 3A). Therefore, we compared the expression of CD4⁺ and CD8⁺ T-cell specific gene signatures (16–18, 27–29) between pre- and on-treatment biopsies. The CD4⁺ T-cell specific gene signature was significantly upregulated by lenalidomide while expression of the CD8⁺ T-cell gene signature remained unchanged (Fig. 3B).

Figure 3. Th1 differentiation and oligoclonal expansion of T cells on lenalidomide therapy.

(A) IHC of CD3, CD4 and CD8 in Day 0 and Day 8 LN biopsies of a representative patient. Comparison of Day 8/Day 0 CD3 per high power field (HPF) between responders (R) and non-responders (NR) by unpaired t-test. (B) Fold change in average expression of CD4⁺ and CD8⁺ T-cell subset specific gene signatures on Day 8 and Day 0. Comparisons by paired t-test. (C) Induction of T-bet (TBX21), the transcription factor regulating the Th1 type differentiation program, is significantly higher in R (n = 7) than NR (n = 4). Th2 transcription factor GATA3 was not induced by treatment with lenalidomide irrespective of clinical response. Comparisons by unpaired t-test. (D) Diversity index of the TCRβ repertoire decreased on lenalidomide therapy. The top 10 TCRβ clonotypes on Day 0 and Day 8 are shown in a representative patient.

T cells mediate anti-tumor immunity

The differentiation of CD4⁺ T cells into T helper (Th) 1 or Th2 cells is determined by the opposing transcription factors, T-bet and GATA-3 (30–32). Th1 cells mediate anti-tumor immunity by producing IFNγ, recruiting CD8⁺ T cells, NK cells, and macrophages, and inhibiting angiogenesis (33). To investigate the effect of lenalidomide on Th1/Th2 balance, we analyzed the expression of TBX21 encoding T-bet and GATA3 (31, 34). Lenalidomide induced TBX21 expression in the LN biopsies of responders compared to non-responders (p = 0.008, Fig. 3C), supporting a shift towards a Th1-type immune response. In contrast, GATA3 expression was not different between pre- and on-treatment biopsies or between responders and non-responders (Fig. 3C).

Skewing of the T-cell receptor (TCR) repertoire and the presence of shared clonotypes between patients suggest common antigen selection in CLL (35). In solid tumors, response to immunotherapy with checkpoint inhibitors has been associated with the oligoclonal expansion, and resultant decreased diversity, of tumor-infiltrating T cells (21). To explore the shifts in T-cell diversity on lenalidomide, we performed TCRα and TCRβ deep sequencing on pre- and on-treatment LN biopsies of responders. In the three patients analyzed, the diversity of both TCRα and TCRβ repertoire decreased following treatment with lenalidomide, suggesting expansion of select clonotypes (Fig. 3D, Supplemental Fig. 2).

Lenalidomide increases T-cell proliferation in response to CLL cells in vitro

To dissect the lenalidomide-induced immune response, we performed a set of in vitro assays (Fig. 4). CD3⁺ T cells from CLL patients were stimulated with autologous irradiated CD19⁺ CLL cells and exposed to lenalidomide in vitro or left untreated. Exposure to lenalidomide increased proliferation of both CD4⁺ and CD8⁺ T cells compared to controls (Fig. 4 A, 4B, 4D). The strongest responses were seen when lenalidomide pre-stimulated CD4⁺ T cells were co-cultured with CLL cells (Fig. 4B). Whether the CLL cells were or were not also treated with lenalidomide did not significantly change the rate of CD4⁺ T-cell proliferation. In contrast, exposing CD8⁺ T cells or CLL cells to lenalidomide increased the rate of proliferation of CD8⁺ T cells.

Figure 4. T-cell proliferation and activation in vitro in response to lenalidomide.

(A) Representative flow cytometry dot-plots of T-cell proliferation assays: CD3⁺ cells cocultured with CD19⁺ cells, CD3⁺ cells stimulated with lenalidomide then cocultured with CD19⁺ cells, CD3⁺ cells cocultured with CD19⁺ cells stimulated with lenalidomide, and CD3⁺ and CD19⁺ cells both stimulated with lenalidomide then cocultured. (B and D) Proportion of proliferating CD4⁺ and CD8⁺ cells under the different culture conditions as indicated in (A) across 8 different patient samples. (C and E) Flow cytometric analysis of IFNγ⁺ CD4⁺ and CD8⁺ cells after overnight stimulation (16 hours) of CLL PBMCs with 2 μM of lenalidomide or vehicle (DMSO). Comparisons by paired t-test.

We measured the frequency of IFNγ-producing cells in the CD4⁺ and CD8⁺ T-cell subsets by flow cytometry and found that the addition of lenalidomide to CLL PBMCs induced production of IFNγ from both CD4⁺ and CD8⁺ cells compared to untreated controls (Fig. 4C, 4E), consistent with our observations in lenalidomide-treated patients (Fig. 1C).

Discussion

Tumor-microenvironment interactions support the development and progression of CLL (36). Prior studies have examined the effect of lenalidomide on circulating T-cell subsets in CLL patients (37, 38). However, in vivo analysis of the tumor microenvironment, where the effects of lenalidomide arguably matter most, are needed. Here, our data link immunomodulation of the TME and clinical response to lenalidomide. By gene expression profiling of paired PB and LN samples, we show that transcriptional changes induced by lenalidomide are tissue-specific. Specifically, lenalidomide activated a Th1-type immune response within the TME that was associated with lymph node regression.

Lenalidomide has been shown to reverse several aspects of immune evasion. First, lenalidomide upregulates costimulatory molecules on tumor cells and enhances their immunogenicity (39–41). Second, lenalidomide repairs defective interactions between tumor and T cells (42). Third, lenalidomide induces T-cell secretion of IFNγ and IL-2, which promotes Th1 differentiation (43–45). Last, lenalidomide improves cytotoxic effector function against tumor cells (43, 46). Here we provide a valuable extension of these prior in vitro observations by characterizing the in vivo immune responses induced by lenalidomide within the tumor microenvironment.

Better T-cell function and more CD4⁺ T-cells before treatment initiation have been associated with improved clinical response to lenalidomide (47). Consistent with these findings, we identified an association between the rapid onset of Th1-type immune activation within the tumor microenvironment and treatment response. In responders, we also observed an expansion of certain T-cell clonotypes by TCR repertoire analysis. Because costimulatory molecules on CLL cells are upregulated by lenalidomide (5), we propose that antigenic stimulation may contribute to the clonal expansion of anti-tumor T cells.

How and if lenalidomide fits into the current treatment paradigm for CLL remains unclear. Lenalidomide has single agent activity in treatment naïve (48, 49) and relapsed or refractory (50, 51) CLL. Overall response rates (ORR) ranged between 12% and 72% and complete responses (CR) were seen in less than 20% of patients (48, 51–53). Side effects, including neutropenia and tumor flare reaction, which have been associated with T- and NK-cell activation against CLL cells, were often dose limiting (5, 39, 40). The early termination of a randomized phase 3 trial comparing lenalidomide to chlorambucil highlighted the significant morbidity and mortality associated with lenalidomide use (54). However, there are positive aspects: lenalidomide improves immunoglobulin levels (48), induces long term responses in a subset of patients (53), and prolongs response when given as maintenance therapy (10, 11). In addition, lenalidomide enhances the activity of anti-CD20 antibodies and the combination has become an important treatment regimen for patients with certain B-cell lymphomas (55–57). Thus, judicious incorporation of lenalidomide into treatment regimens may be beneficial and should be weighed against the safety and efficacy of novel immunotherapies.

Activation of anti-tumor immunity has emerged as one of the most promising therapeutic strategies against cancer. In addition to conventional immunotherapies, small molecules, particularly inhibitors of B-cell receptor signaling, also appear to modulate the immune system (58–60). As combinations of immunotherapy and small molecules are being investigated for CLL, it is important to understand their impact on the immune system. We have shown that immunological changes are tissue-specific and that clinically relevant effects may occur primarily within the tumor microenvironment. Characterization of tissue biopsies may therefore be required to identify meaningful biomarkers in ongoing immunotherapy clinical trials.