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Distinct immune composition in lymph node and peripheral blood of CLL patients is reshaped during venetoclax treatment

Iris de Weerdt, Tom Hofland, Renate de Boer, Johan A. Dobber, Julie Dubois, Denise van Nieuwenhuize, Mehrdad Mobasher, Fransien de Boer, Mels Hoogendoorn, Gerjo A. Velders, Marjolein van der Klift, Ester B. M. Remmerswaal, Frederike J. Bemelman, Carsten U. Niemann, Sabina Kersting, Mark-David Levin, Eric Eldering, Sanne H. Tonino and Arnon P. Kater

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Few cytotoxic lymphocytes in the LN of CLL patients. Lymphocyte analysis in PB and LN samples from untreated CLL patients (PB, n = 42; LN, n = 28) and HCs (PB, n = 10; LN, n = 5) by flow cytometry. Percentage of CLL (CD5+CD19+) cells (A) and T (CD3+) cells (B) within lymphocytes. (C) Ratio of CD4+/CD8+ T cells. Percentage of Vδ1+ cells (D) and Vδ2+ cells (E) within T cells. (F) Percentage of NK (CD56bright and CD16+) cells within lymphocytes (CLL PB, n = 21; CLL LN, n = 5). Data are presented as mean (bar) and individual patients (circles and triangles). *P < .05, **P < .01, ***P < .001, ****P <. 0001, Wilcoxon matched-pairs signed-rank test (A), 1-way ANOVA followed by Sidak’s multiple-comparisons test (B-F).

  • Figure 2.

    CLL cells reside in an immunosuppressive context in the LN. Lymphocyte analysis in PB and LN samples from untreated CLL patients (PB, n = 42; LN, n = 28) and HCs (PB, n = 10; LN, n = 5) by flow cytometry. Percentage of Tfh (CXCR5+PD-1+) cells (A) and Tregs (CD25+FoxP3+) (B) within CD4+ T cells. (C) Correlation between the percentage of Tregs of CD4+ T cells in the CLL LN and the absolute leukocyte count (ALC). (D) Percentage of PD-1+ cells within CD8+ T cells. ***P < .001, ****P < .0001, 1-way ANOVA followed by Sidak’s multiple-comparisons test (A-B,D), Pearson correlation analysis (C).

  • Figure 3.

    Depletion of nonmalignant lymphocytes during venetoclax-obinutuzumab treatment. Lymphocyte analysis in PB of patients at baseline (n = 36), after preinduction with obinutuzumab (n = 5), and after 1 year of treatment with venetoclax-obinutuzumab (n = 11) by flow cytometry. Absolute number of B cells (A) and CD4+ and CD8+ T cells (B) in PB. (C) Absolute number of NK (CD56bright and CD16+) cells in PB (baseline, n = 20). (D) Ratio of CD56bright (CD56brightCD16)/CD56dim (CD56dimCD16+) cells (baseline, n = 6). Horizontal lines indicate reference values. *P < .05, **P < .01, 1-way ANOVA followed by Sidak’s multiple-comparisons test.

  • Figure 4.

    Tumor-submissive T-cell subsets decrease during venetoclax-obinutuzumab treatment. Lymphocyte analysis in PB of patients at baseline (n = 11), after preinduction with obinutuzumab (n = 5), and after 1 year of treatment with venetoclax-obinutuzumab (n = 11) by flow cytometry. Percentage of Tfh (CXCR5+PD-1+) cells (A) and Tregs (CD25+FoxP3+) (B) within CD4+ T cells. (C) Percentage of PD-1+ cells within CD8+ T cells. (D) IgG levels in plasma at baseline and after 1 year of venetoclax-obinutuzumab treatment. (E) Cytokine production by CD4+ T cells after stimulation of PBMCs with PMA/ionomycin for 4 hours. Cytokine production and CD107a expression by CD8+ T cells (F) and NK cells (G) after stimulation of PBMCs with PMA/ionomycin for 4 hours. *P < .05, **P < .01, ***P < .001, 1-way ANOVA followed by Sidak’s multiple-comparisons test.

  • Figure 5.

    Depletion of nonmalignant lymphocytes when venetoclax is combined with ibrutinib. Lymphocyte analysis in PB of patients at baseline, after preinduction with ibrutinib, and after 1 year of treatment with venetoclax-ibrutinib (n = 10) by flow cytometry. Absolute number of B cells (A) and CD4+ and CD8+ T cells (B) in PB. (C) Absolute number of NK (CD56bright and CD16+) cells in PB. (D) Ratio of CD56bright (CD56brightCD16) cells vs CD56dim (CD56dimCD16+) cells. Horizontal lines indicate reference values. *P < .05, **P < .01, 1-way ANOVA followed by Sidak’s multiple-comparisons test.

  • Figure 6.

    Fewer Tregsand PD-1+CD8+T cells after treatment with venetoclax-ibrutinib. Lymphocyte analysis in PB of patients at baseline, after preinduction with ibrutinib, and after 1 year of treatment with venetoclax-ibrutinib (n = 10) by flow cytometry. Percentage of Tfh (CXCR5+PD-1+) cells (A) and Tregs (CD25+FoxP3+) (B) within CD4+ T cells. (C) Percentage of PD-1+ cells within CD8+ T cells. (D) IgG levels in plasma at baseline and after 1 year of venetoclax-ibrutinib. (E) Cytokine production by CD4+ T cells after stimulation of PBMCs with PMA/ionomycin for 4 hours. Cytokine production and CD107a expression by CD8+ T cells (F) and NK cells (G) after stimulation of PBMCs with PMA/ionomycin for 4 hours. *P < .05, **P < .01, 1-way ANOVA followed by Sidak’s multiple-comparisons test.

Tables

  • Table 1.

    Patient characteristics

    HCsHOVON139 baseline PBHOVON139 baseline LNHOVON139 1-y PBHOVON141 baseline PBHOVON141 1-y PB
    N104128111010
    Males, %5075.780.881.830.030.0
    Age, y63 (45-78)71 (57-80)71 (57-79)71.5 (60-78)68 (52-76)68 (52-76)
    CMV positive, %83414864Not knownNot known
    ALC, ×109/LNot known98.6 (3.5-420.7)97.3 (18.0-399.0)4.8 (2.8-9.9)129.8 (20.0-248.9)4.8 (3.9-7.6)
    TTFT, moN/A28.2 (0.7-244.6)28.1 (0.7-244.6)18.9 (1.7-244.6)Not knownNot known
    Prior treatment, nN/AN/AN/AN/A1 (1-3)1 (1-3)
    Prior chemotherapy, %N/AN/AN/AN/A100100
    TTT, moN/AN/AN/AN/A42.8 (13.8-141.8)42.8 (13.8-141.8)
    Rai stage III-IV, %N/A57.144.036.48080
    IgVH status U-CLL, %N/A55.955.627.34040
    • Unless otherwise noted, data are median (range).

    • ALC, absolute leukocyte count; CMV, cytomegalovirus; N/A, not applicable; TTFT, time to first treatment; TTT, time from last treatment to current treatment; U-CLL, CLL with unmutated immunoglobulin heavy chains.