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Microenvironmental regulation of the IL-23R/IL-23 axis overrides chronic lymphocytic leukemia indolence

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Science Translational Medicine  14 Feb 2018:
Vol. 10, Issue 428, eaal1571
DOI: 10.1126/scitranslmed.aal1571
  • Fig. 1 Evaluation of IL-23R and IL-12Rβ1 chain expression in CLL.

    (A) Two representative chronic lymphocytic leukemia (CLL) cases, with low and high interleukin-23 receptor (IL-23R) expression, respectively, analyzed by flow cytometry. CLL cells were gated as CD19+CD5+ cells. IgG1, immunoglobulin G1. (B) IL-23R and IL-12Rβ1 chain expression evaluated by immunocytochemistry on cytospin smears of CLL peripheral blood samples. DAPI, 4′,6-diamidino-2-phenylindole. (C) IL-12Rβ1 chain expression in 57 consecutive cases featuring different IL-23R chain expression. (D) Expression of IL-23R and IL-12Rβ1 by CD5+CD19 [T cells + natural killer cells (T + NK cells)] from the peripheral blood of the same CLL cases shown in (C) (n = 57). Statistical comparisons were carried out by Wilcoxon tests. Asterisks indicate statistically significant P values (P < 0.05). (E) Kaplan-Meier curves comparing time to first treatment of IL-23Rhigh (n = 97) or IL-23Rlow (n = 122) CLL cases. Statistical significance of associations between individual variables and survival was calculated using the log-rank test. (F) Cox multivariate analysis showing that IL-23R chain expression maintains an independent prognostic impact in the presence of other prognostic indicators (P = 0.014). HR, hazard ratio; CI, confidence interval; cMBL, clinical monoclonal B lymphocytosis; IGHV, immunoglobulin heavy chain variable region; ZAP-70, zeta chain–associated protein kinase 70.

  • Fig. 2 Analysis of CLL lymph nodes.

    (A) Two representative cases (of the 16 examined) expressing low (n = 10) or high (n = 6) IL-23R chain (magnification, ×200). (B) Double-marker immunofluorescence (IF) analysis of IL-23R chain (left) or IL-12Rβ1 chain (right) and CD20 in a representative lymph node displaying high expression of the IL-23R chain (n = 6 of 16) (magnification, ×200). (C) Double-marker IF analysis of IL-23R and IL-12Rβ1 showing foci of higher and lower IL-12Rβ1 expression (dashed circles; magnification, ×100). (D) Double-marker IF analysis of IL-12Rβ1 and CD68 showing higher expression of IL-12Rβ1 in foci with higher CD68-expressing cell density. (E) Double-marker IF analysis showing different representative areas of the same lymph node with lower (top) and higher (bottom) IL-12Rβ1 expression and corresponding CD40L expression (magnification, ×200). (B to E) Microphotographs are relative to one representative lymph node (of six evaluated) displaying IL-23Rhigh chain. IF microphotographs are representative of analyses on at least 5 for low-power magnification (×100) or 10 for high-power magnification (×200 and ×400) microscopic fields performed on each lymph node sample.

  • Fig. 3 In vitro induction of IL-12Rβ1 chain expression.

    (A) Peripheral mononuclear CLL cells from two representative CLL cases (IL-23Rlow and IL-23Rhigh, respectively) were cocultured with CD40L-expressing NIH-3T3 (CD40L-TC) or control NIH-3T3 cells expressing an empty vector (Mock) for 48 hours and then analyzed for IL-23R complex expression by flow cytometry. Only viable cells were gated, as indicated (left), and of these, CD5+CD19+ cells (middle) were analyzed for IL-23R chain expression (right). For further details, see fig. S1. SSC-H, side scatter height; FSC-H, forward scatter height. (B) Time course analysis of IL-23R complex (top), IL-23R (middle), and IL-12Rβ1 (bottom) chains evaluated by flow cytometry before (T0) and after CD40L engagement in 13 CLL cases. See table S3 for a summary of patient features. The P values shown are relative to 48-hour cell cultures (Wilcoxon test). (C) Time course experiments showing a representative test on a CLL case (PD601). Peripheral blood mononuclear cells (PBMCs) were cultured in medium alone or in the presence of CD3/CD28 beads and IL-2, and cells were harvested at the indicated times. Analysis of the IL-23R chain expression (bottom) was carried out on CLL cells gated for CD5 and CD19 (top). (D) IL-23R-complex expression by the CLL cells of 14 cases analyzed after a 120-hour coculture, as described in (C). P value shown (***P = 0.0001) refers to the three pairs analyzed (Wilcoxon test). (E) CD38 expression on CLL cells after a 120-hour culture in presence of autologous activated T cells and (F) evaluation of IL-23R complex expression on CD38-positive or CD38-negative cells (n = 11 CLL cases) (Wilcoxon test). (G) Ki67 expression by IL-23R complex–positive and IL-23R complex–negative CLL cells, respectively. CLL cells, cultured in the presence of activated autologous T cells as in (D), were stained for the indicated receptor chain. CD5+CD19+ CLL cells were gated and evaluated for both Ki67 and IL-23R chain expression. (H) Mean and SEM of Ki67 expression by the cells from six CLL cases treated and analyzed as described in (G) (Wilcoxon test). (I) Double-marker IF analysis showing different representative areas of the same lymph node with lower (left) and higher (right) IL-12Rβ1 expression and corresponding Ki67 expression (magnification, ×200). IF microphotographs are relative to a representative lymph node (of six evaluated) displaying IL-23Rhigh chain and of at least 10 microscopic fields performed on each lymph node sample.

  • Fig. 4 IL-23 production by CLL clones.

    (A) Immunohistochemistry (IHC) evaluation of two representative cases of CLL lymph node specimens (of the 16 examined) producing higher (n = 6) or lower (n = 10) levels of IL-23 (magnification, ×200). (B) Double-marker IF and confocal microscopy analysis of IL-23R chain and IL-23 or IL-12Rβ1 chain and IL-23 or DAPI of a representative CLL lymph node displaying IL-23Rhigh chain (of six analyzed) showing production of IL-23 cytokine by IL-23R complex–expressing cells (magnification, ×200). Microphotographs in (A) and (B) are representative of analyses of at least 10 microscopic fields performed on each lymph node sample. (C) IL-23 production in supernatants of cells from 18 CLL cases cultured in the presence of CD40L-TC or control (Mock) fibroblasts. (D) IL-23 production by CLL cells induced by contact with activated autologous T cells. PBMCs from seven CLL cases were cultured in the presence of CD3/CD28 beads and IL-2 or in medium alone for 120 hours. At the end of this period, CLL cells were depleted of T cells by negative selection and placed in culture for an additional 24 hours before measuring IL-23 concentrations (in picograms per milliliter) in the supernatant. P values of the difference between stimulated CLL cells and controls are indicated (Wilcoxon test). Asterisks indicate statistically significant P values (P < 0.05).

  • Fig. 5 Functional analysis of IL-23 signaling in CLL cells.

    (A) Flow cytometry determination of viable cells [annexin V/propidium iodide (PI)–negative cells] or of (B) Ki67-positive CLL cells. PBMCs from CLL patients were cultured with CD3/CD28 beads + IL-2 (120 hours). Subsequently, CLL cells were purified by negative selection and recultured for 48 hours in the presence or absence of IL-23 (100 ng/ml) or of IL-23 neutralizing monoclonal antibody (mAb) (αIL-23p19) in the indicated combinations. (C and D) PBMCs from CLL patients were cultured with CD3/CD28 beads + IL-2 for 120 hours as in (A) and (B). CLL cells were subsequently purified by negative selection and recultured in the presence of the indicated kinase inhibitors with or without IL-23. Cell viability and Ki67 expression were determined after a 48-hour culture. Each dot represents a test on a different CLL case. Mean ± SEM are given. Statistical analysis was carried out using Wilcoxon (A to C) or Mann-Whitney U tests (D). Asterisks indicate statistically significant P values (P < 0.05). (E) Immunoblotting analysis of signal transducer and activator of transcription 3 phosphorylation (pSTAT3) and Bruton’s tyrosine kinase phosphorylation (pBTK) in purified CLL cells (from case RD0468; 52% IL-23R–positive cells) activated with T cells as in (A), purified and stimulated at 37°C with IL-23 for the indicated times. (F) Phosphorylation of BTK and STAT3 was assessed in freshly isolated CLL cells after exposure to anti-μ antibody (Gαμ-Ab), anti-δ antibody (Gαδ-Ab), or a combination of both, at 37°C for 5 min. CTR, control. (G) Summary of data from five tests of STAT3 phosphorylation on different CLL cases: Data are presented as pSTAT3/total STAT3 (tSTAT3) ratio after a 10-min and 30-min incubation with IL-23. (H) CLL cells (from case AM609; 53% IL-23R–positive cells) were induced to express the IL-23R complex as in (A), purified and exposed to IL-23 or medium alone for the indicated times, and studied for the presence of pSTAT3 by flow cytometry (right). Left: Results obtained in CLL cells for which the preactivation step was omitted (PBMCs from CLL patients were cultured without CD3/CD28 beads; CLL cells were purified and then exposed to IL-23 or medium alone for 1 hour). The degree of pSTAT3 was evaluated on viable cells gated as in Fig. 3A and as detailed in fig. S1. CD19 expression was also detected to verify the purity of the CLL cell isolation. (I) Summary of data on pSTAT3 induction (determined by flow cytometry) after a 48-hour exposure to IL-23 in activated and purified CLL cells [as in (A)] from five different CLL cases. (J and K) CLL cells from case GE1-CC45 (36% IL-23R–positive cells) were activated and purified as in (A) and cultured in the presence or absence of the indicated inhibitors for 48 hours, and pSTAT3-positive (J) or pBTK-positive (K) cells were measured by flow cytometry.

  • Fig. 6 NSG mice model engrafted with CLL cells.

    A total of 50 × 106 PBMC cells from case PM608 were injected into three NOD/Shi-scid,γcnull (NSG) mice intravenously. Mice were sacrificed and analyzed 4 weeks after the inoculum. (A) Flow cytometry analysis shows that the injected cells were IL-23Rlow (6.3% positive cells). (B) IHC analysis of tissue slices from engrafted mice showing spleen (top) and liver (bottom) infiltration by CLL cells stained by the indicated anti-human mAb (magnification, ×40). (C) IHC (top, magnification, ×400) and IF (middle and bottom, magnification, ×40) staining of paraffin-embedded tissue slice sections showing expression of IL-23R chains in areas characterized by cell proliferation (Ki67+ cells). (D) Flow cytometry analysis of the IL-23R complex in CLL cells in mouse spleen. PE, phycoerythrin. (E) Confocal microscopy image of the mouse spleen. The arrows indicate neoplastic CLL cells (human CD20+) producing human IL-23 (IL-23p19+) [magnifications, ×200 (left) and ×630 (right)]. (F) IL-23 chains in xenografted CLL cells measured by flow cytometry. Human IL-23 chains were detected by intracellular staining with αIL-23p19 and αIL-12/IL-23p40 antibodies on gated human CD45+CD19+ CLL cells. Microphotographs are representative of analyses on at least 10 for high-power magnification (×200 to ×630) microscopic fields performed on each mouse spleen tissue sample.

  • Fig. 7 Treatment with neutralizing IL-23 antibody (αIL-23p19) inhibits CLL growth and proliferation in the NSG mouse model.

    (A) Treatment scheme of αIL-23p19 or isotype CTR mAb in NSG mice. Mice were injected with 50 × 106 CLL cells previously stimulated with autologous activated T cells. After 4 to 6 weeks, blood samples were evaluated for the presence of circulating leukemic cells by flow cytometry before treatment with four doses of αIL-23p19 or isotype CTR mAbs every 2 days. At the end of the experiments, mice were sacrificed, and cell suspensions from the spleen, liver, bone marrow (BM), and peripheral blood were analyzed by flow cytometry for the percentage of neoplastic B cells (human CD5+CD19+ cells / total human CD45+ cells). (B) The mean and SEM of 18 mice injected with CLL cells from three different cases (PA0146, GC0626, and GE1-BA101) are shown (Wilcoxon test). (C) Low-magnification image (top) of longitudinal sections of paraffin-embedded mouse spleen (CLL PA0146) double-stained for CD20 and Ki67. Spleen from mouse treated with isotype CTR (left) or αIL-23p19 (right) mAb. Analysis at higher magnification (×400) is shown (bottom). (D) IHC analysis of IL-23p19 in spleens of mice treated as indicated in (A). (E and F) Demonstration of apoptotic neoplastic cells in both treated and control mice spleen by CD45/CD19/CD5/annexin V staining and flow cytometry. (G) Demonstration of Ki67-positive cycling cells by flow cytometry and (H) by confocal microscopy (magnification, ×200) in spleens of isotype CTR (top) or αIL-23p19 mAb-treated mice (bottom). Microphotographs are representative of analyses on at least 10 for high-power magnification (×200, ×400) microscopic fields performed on each mouse tissue sample. The scatter dot plots in (B), (F), and (I) show data for each mouse, and mean values are calculated for each treatment mice group; statistical comparisons were carried out by Wilcoxon test. SSC-A, side scatter area.

  • Fig. 8 αIL-23p19 induces regression of the leukemic clone for a prolonged period.

    Mice were injected with 100 × 106 CLL cells per mouse (CLL GE2-RL201). After 2 weeks, blood samples were evaluated for the presence of circulating leukemic cells before treatment with four doses of αIL-23p19 or isotype CTR mAb every 2 days. Three weeks after the last mAb dose, mice were sacrificed, and the spleen, liver, BM, and peripheral blood were analyzed by IHC and flow cytometry. (A and B) Low-magnification image of longitudinal sections of paraffin-embedded mouse spleen stained with αCD20-Ab. The insets show infiltrating foci observed at higher magnification (×200). Black peaks indicated by the arrows in the graph insets show the presence of B cell receptor gene rearrangement of the leukemic clone as assessed by fragment length analysis. Microphotographs are representative of analyses on at least 10 microscopic fields performed on each mouse spleen tissue sample. (C) Spleen, liver, BM, and peripheral blood samples analyzed by flow cytometry for percentage of neoplastic B cells (CD5+CD19+ cells). The mean and SEM of 12 mice injected with CLL cells from two different cases (SD36 and GE2-RL201) are shown (Wilcoxon test).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/428/eaal1571/DC1

    Materials and Methods

    Fig. S1. Gating strategy to analyze IL-23R complex induction after activation by CD40L-expressing fibroblasts.

    Fig. S2. IL-12Rβ1 mRNA production after CLL cell activation by CD40L-expressing fibroblasts.

    Fig. S3. Expression of a complete IL-23R by CLL cells after depletion of IL-23R–positive cells and coculture with autologous activated T cells.

    Fig. S4. Demonstration of intracytoplasmic IL-23 chains in CLL cells.

    Fig. S5. HS5 stromal cells do not express CD40L.

    Fig. S6. Enhanced IL-23R complex expression by coculture of PBMC with NLC.

    Fig. S7. IL-23R complex expression by CLL cells cocultured with stromal cells or NLCs.

    Fig. S8. Relationship between the BCR signaling pathway and IL-23R complex expression in CLL cells.

    Fig. S9. Silencing of IL-23A or IL-23R genes by siRNA.

    Fig. S10. STAT3 phosphorylation induced by CLL cell exposure to IL-23.

    Fig. S11. NSG mice engrafted with CLL cells.

    Fig. S12. Minimal residual disease detection in mice tissues after treatment with αIL-23p19.

    Fig. S13. Anti–IL-23p19 mAb does not induce antibody-dependent cytotoxicity.

    Fig. S14. Schematic representation of the IL-23R complex/IL-23 axis in CLL.

    Table S1. Relationship between IL-23R chain expression and prognostic parameters.

    Table S2. Univariate and multivariate bootstrapping validated Cox regression analysis of TTFT.

    Table S3. Features of the CLL cases used in coculture experiments with CD40L-expressing fibroblasts (CD40L-TC).

    Table S4. Summary of the features of all CLL cases whose cells were used for xenograft tests.

    Table S5. Primary data.

    References (5053)

  • Supplementary Material for:

    Microenvironmental regulation of the IL-23R/IL-23 axis overrides chronic lymphocytic leukemia indolence

    Giovanna Cutrona,* Claudio Tripodo, Serena Matis, Anna Grazia Recchia, Carlotta Massucco, Marina Fabbi, Monica Colombo, Laura Emionite, Sabina Sangaletti, Alessandro Gulino, Daniele Reverberi, Rosanna Massara, Simona Boccardo, Daniela de Totero, Sandra Salvi, Michele Cilli, Mariavaleria Pellicanò, Martina Manzoni, Sonia Fabris, Irma Airoldi, Francesca Valdora, Silvano Ferrini, Massimo Gentile, Ernesto Vigna, Sabrina Bossio, Laura De Stefano, Angela Palummo, Giovanni Iaquinta, Martina Cardillo, Simonetta Zupo, Giannamaria Cerruti, Adalberto Ibatici, Antonino Neri, Franco Fais, Manlio Ferrarini, Fortunato Morabito

    *Corresponding author. Email: giovanna.cutrona{at}hsanmartino.it

    Published 14 February 2018, Sci. Transl. Med. 10, eaal1571 (2018)
    DOI: 10.1126/scitranslmed.aal1571

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Gating strategy to analyze IL-23R complex induction after activation by CD40L-expressing fibroblasts.
    • Fig. S2. IL-12Rβ1 mRNA production after CLL cell activation by CD40L-expressing fibroblasts.
    • Fig. S3. Expression of a complete IL-23R by CLL cells after depletion of IL-23R–positive cells and coculture with autologous activated T cells.
    • Fig. S4. Demonstration of intracytoplasmic IL-23 chains in CLL cells.
    • Fig. S5. HS5 stromal cells do not express CD40L.
    • Fig. S6. Enhanced IL-23R complex expression by coculture of PBMC with NLC.
    • Fig. S7. IL-23R complex expression by CLL cells cocultured with stromal cells or NLCs.
    • Fig. S8. Relationship between the BCR signaling pathway and IL-23R complex expression in CLL cells.
    • Fig. S9. Silencing of IL-23A or IL-23R genes by siRNA.
    • Fig. S10. STAT3 phosphorylation induced by CLL cell exposure to IL-23.
    • Fig. S11. NSG mice engrafted with CLL cells.
    • Fig. S12. Minimal residual disease detection in mice tissues after treatment with αIL-23p19.
    • Fig. S13. Anti–IL-23p19 mAb does not induce antibody-dependent cytotoxicity.
    • Fig. S14. Schematic representation of the IL-23R complex/IL-23 axis in CLL.
    • Table S1. Relationship between IL-23R chain expression and prognostic parameters.
    • Table S2. Univariate and multivariate bootstrapping validated Cox regression analysis of TTFT.
    • Table S3. Features of the CLL cases used in coculture experiments with CD40L-expressing fibroblasts (CD40L-TC).
    • Table S4. Summary of the features of all CLL cases whose cells were used for xenograft tests.
    • References (5053)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S5 (Microsoft Excel format). Primary data.

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