Combined therapy with ibrutinib and bortezomib followed by ibrutinib maintenance in relapsed or refractory mantle cell lymphoma and high-risk features: a phase 1/2 trial of the European MCL network (SAKK 36/13).

BACKGROUND The Bruton's tyrosine kinase inhibitor ibrutinib and the proteasome inhibitor bortezomib have single-agent activity, non-overlapping toxicities, and regulatory approval in mantle cell lymphoma (MCL). In vitro, their combination provides synergistic cytotoxicity. In this investigator-initiated phase 1/2 trial, we established the recommended phase 2 dose of ibrutinib in combination with bortezomib, and assessed its efficacy in patients with relapsed or refractory MCL. METHODS In this phase 1/2 study open in 15 sites in Switzerland, Germany and Italy, patients with relapsed or refractory MCL after ≤2 lines of chemotherapy and both ibrutinib-naïve and bortezomib-naïve received six cycles of ibrutinibb and bortezomib, followed by ibrutinib maintenance. For the phase 1 study, a standard 3 + 3 dose escalation design was used to determine the recommended phase 2 dose of ibrutinib in combination with bortezomib. The primary endpoint in phase 1 was the dose limiting toxicities in cycle 1. The phase 2 study was an open-label, single-arm trial with a Simon's two-stage min-max design, with a primary endpoint of overall response rate (ORR) assessed by CT/MRI. This study was registered with ClinicalTrials.gov, NCT02356458. FINDINGS Between August 2015 and September 2016, nine patients were treated in the phase 1 study, and 49 patients were treated between November 2016 and March 2020 in the phase 2 of the trial. The ORR was 81.8% (90% CI 71.1, 89.8%, CR(u) 21.8%) which increased with continued ibrutinib (median 10.6 months) to 87.3%, (CR(u) 41.8%). 75.6% of patients had at least one high-risk feature (Ki-67 > 30%, blastoid or pleomorphic variant, p53 overexpression, TP53 mutations and/or deletions). In these patients, ibrutinib and bortezomib were also effective with an ORR of 74%, increasing to 82% during maintenance. With a median follow-up of 25.4 months, the median duration of response was 22.7, and the median PFS was 18.6 months. PFS reached 30.8 and 32.9 months for patients with a CR or Cru, respectively. INTERPRETATION The combination of ibrutinib and bortezomib shows durable efficacy in patients with relapsed or refractory MCL, also in the presence of high-risk features. FUNDING SAKK (Hubacher Fund), Swiss State Secretariat for Education, Research and Innovation, Swiss Cancer Research Foundation, and Janssen

An NK Cell Perforin Response Elicited via IL-18 Controls Mucosal Inflammation Kinetics during Salmonella Gut Infection

Salmonella Typhimurium (S. Tm) is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S. Tm infections in knockout mice, cytokine inhibition and -injection experiments revealed that caspase-1 (not -11) dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf(-/-) ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S. Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens

IL-18 modulates the onset of <i>S</i>.Tm-induced intestinal inflammation.

<p>(a) Mature IL-1β and IL-18 protein levels were measured in whole cecum tissue homogenates from C57BL/6 WT mice. Mice were Sm-pretreated and remained uninfected or were infected orally with 5x10⁷ CFU S.Tm for 12h (n = 7 per group); dashed lines indicate the detection limit. (b and c) <i>Il1ab</i>^<i>-/-</i> and <i>Il18</i>^<i>-/-</i> mice and littermate controls were Sm-pretreated and infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 6–9 per group). (b) pathological score; arrows indicate representative mice depicted in panel c, (c) HE-stained cryosections from representative mice of each group; SE = submucosal edema, L = lumen; scale bar = 100μm; left panel: <i>Il1ab</i>^<i>-/-</i>, right panel: <i>Il18</i>^<i>-/-</i>. (d and e) <i>Casp1/11</i>^<i>-/-</i> and <i>Casp11</i>^<i>-/-</i> mice and littermate controls were Sm-pretreated and infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 5–7 per group). (d) Mature IL-18 protein levels in whole cecum tissue lysates from <i>Casp1/11</i>^<i>-/-</i> or <i>Casp11</i>^<i>-/-</i> mice and littermate controls; dashed lines indicate the detection limit. (e) Pathological score. (f) C57BL/6 WT mice were Sm-pretreated, injected intraperitoneally with rIL-18BP or PBS, infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 5 per group) and mucosal inflammation was quantified. (g) C57BL/6 WT mice were Sm-pretreated, injected intraperitoneally with rIL18 or PBS, infected orally with 5x10⁷ CFU <i>S</i>.Tm for 8h (n = 9 per group) and mucosal inflammation was quantified. Data represent the mean ± SD and statistical analysis was performed using the Mann-Whitney-U test (ns = not significant, *: p<0.05; **: p<0.01; ***: p<0.001).</p

IL-18 enhances the migratory potential of NK cells.

<p>(a) Flow cytometric analysis of isolated cecal LP cells from 1:1 <i>Il18r1</i>^-/—CD45.2:WT-CD45.1 mixed bone marrow chimeric mice, Sm-pretreated and infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 9 per group). Single cells were gated on CD45⁺ CD3^- lymphocytes. Quantification of NK1.1⁺ cells from WT or <i>Il18r1</i>^-/- LP cells as distinguished by their congenic markers CD45.1 (WT) and CD45.2 (<i>Il18r1</i>^-/-). (b) Assessment of <i>in vivo</i> cell proliferation via EdU incorporation and flow cytometric analysis of isolated LP cells from C57BL/6 mice, either uninfected or infected orally with 5x10⁷CFU <i>S</i>.Tm for 12h (n = 5–6 per group). Quantification of EdU incorporation in CD3^- NK1.1⁺ cells. (c) 2D Transwell migration assay of splenic NK cells isolated by MACS and stimulated for 3h in presence or absence of 100ng/mL rIL-18. Migration was analyzed towards the indicated concentrations of CXCL9 (n = 4 per group). (d) Flow cytometric analysis of CXCR3 surface expression on splenic NK cells, stimulated for 3h in presence or absence of 100ng/mL rIL-18 (n = 4 per group). Data represent the mean ± SD and statistical analyses were performed using the Mann-Whitney-U test or ordinary one-way ANOVA (ns = not significant, * = p<0.05; ** = p<0.01; *** = p<0.001).</p

Neutrophil recruitment into the infected mucosa is decreased in absence of IL-18 but neutrophil depletion does not seem to be a main driver during the initiation of mucosal inflammation.

<p>(a) <i>Il18</i>^<i>-/-</i> mice and littermate controls were Sm-pretreated, infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 3–4 per group). RNA-Seq was performed on RNA extracted from complete cecum tissue. RNA-Seq analysis: The Volcano plot shows the induction (log₂ fold change) versus the -log₁₀ p-value for all chemokines. Chemokines able to induce neutrophil recruitment are highlighted in green. (b) C57BL/6 WT mice and Il18^-/- mice and littermates were Sm-pretreated and either uninfected (WT) or infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 4–5 per group). <i>Cxcl1 and Cxcl2</i> mRNA levels in whole cecum tissue were measured by RT-qPCR. Results are presented relative to the expression of <i>Actb</i>. (c and d) Flow cytometric analysis of isolated cecal LP cells from Sm-pretreated C57BL/6 WT mice, either uninfected or infected with 5x10⁷ CFU <i>S</i>.Tm (n = 6 per group). Single live cells were gated on CD45⁺CD3^-CD19^-CD11b⁺ cells. (c) Representative dot plots and (d) quantification of Ly-6G⁺ cells. (e and f) Flow cytometric analysis of isolated cecal LP cells from <i>Il18</i>^-/- mice and littermates, Sm-pretreated and orally infected with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 8 per group). Single live cells were gated on CD45⁺CD3^-CD19^-CD11b⁺ cells. (e) Representative dot plots and (f) quantification of Ly-6G⁺ cells. (g and h) C57BL/6 WT mice were injected intraperitoneally with anti-G-CSF (0.4mg/kg; two consecutive days) and anti-Ly-6G (6mg/kg; two days prior to infection) or PBS. Mice were Sm-pretreated and infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 6 per group). (g) Pathological score and (h) representative HE-stained cryosection. Data represent the mean ± SD and statistical analysis was performed using the Mann-Whitney-U test (ns = not significant, *: p<0.05; **: p<0.01).</p

IFNγ expression by NK cells is IL-18-dependent but not required for mounting tissue inflammation during the first 12h of the infection.

<p>(a) Volcano plot of all cytokines differentially expressed in the cecal mucosa of the <i>Il18</i>^<i>-/-</i> mice and littermate shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005723#ppat.1005723.g002" target="_blank">Fig 2A</a>. We plotted log₂ (fold change) against -log₁₀ (p-value). NK cell effector cytokines are highlighted in red. (b-d) <i>Il18</i>^-/- mice and littermate controls were Sm-pretreated and infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h (b) or 18h (c-d). (b) <i>Ifng</i>, <i>Iigp1</i> and <i>Cxcl10</i> transcripts in whole cecum tissue were analyzed by RT-qPCR. Results are presented relative to the expression of <i>Actb</i> (n = 8–9 per group). (c) IFNγ protein concentration in whole cecum tissue lysates (n = 5 per group) as measured by CBA; dashed line = detection limit. (d) Quantification of IFNγ-producing cells by flow cytometric analysis of isolated cecal LP cells (n = 6 per group). (e and f) C57BL/6 mice were infected for 18h with 5x10⁷ CFU <i>S</i>.Tm and cecal LP cells were isolated for staining. Data are shown from one out of three independent experiments. (e) Representative dot plot of IFNγ-expressing cells, pre-gated on single live lymphocytes. (f) FACS-analysis of CD3 and NK1.1 surface marker expression by IFNγ⁺ cell populations. (g) <i>Ifng</i>^-/-, <i>Ifngr1</i>^-/- and littermate controls were Sm-pretreated, infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h and pathological scores were assessed (n = 6–8 per group). (h) Mesenteric lymph node loads as determined by plating of organs from (left) <i>Ifng</i>^-/- and (right) <i>Il18</i>^-/- mice and their littermate controls. Mice were Sm-pretreated and infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h or 72h. Statistical analysis was performed using the Mann-Whitney-U test (ns = not significant, * = p<0.05; ** = p<0.01; *** = p<0.001).</p

IL-18 enhances the recruitment of NK cells into the infected cecum LP thereby stimulating early cecal inflammation.

<p>(a) <i>Il18</i>^<i>-/-</i> mice and littermate controls were Sm-pretreated, infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 3–4 per group). RNA-Seq was performed on RNA extracted from complete cecum tissue. RNA-Seq analysis: The Volcano plot shows the induction (log₂ fold change) versus the -log₁₀ p-value for all chemokines. Chemokines able to induce NK cell recruitment are highlighted in red. (b) C57BL/6 WT mice were Sm-pretreated and either uninfected or infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 4 per group). <i>Cxcl9</i>, <i>Cxcl10</i>, <i>Cxcl11</i>, <i>Ccl3</i> and <i>Ccl4</i> mRNA levels in whole cecum tissue were measured by RT-qPCR. Results are presented relative to the expression of <i>Actb</i>. (c) Same as (b) but comparing cecum tissues from infected <i>Il18</i>^<i>-/-</i> mice vs infected littermate controls. (d and e) Flow cytometric analysis of isolated cecal LP cells from Sm-pretreated C57BL/6 WT mice, either uninfected or infected with 5x10⁷ CFU <i>S</i>.Tm (n = 5 per group). Single live cells were gated on CD45⁺ lymphocytes. (d) Representative dot plots and (e) quantification of NK1.1⁺ CD3^- cells. (f and g) Flow cytometric analysis of isolated cecal LP cells from <i>Il18</i>^-/- mice and littermates, Sm-pretreated and orally infected with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 5–6 per group). Single live cells were gated on CD45⁺ lymphocytes. (f) Representative dot plots and (g) quantification of NK1.1⁺ CD3^- cells. (h) C57BL/6 mice were infected for 12h with 5x10⁷ CFU <i>S</i>.Tm and isolated cecal LP cells of two mice were pooled for staining and isotype control staining; data are shown for one out of three independent experiments. CD45⁺ NK1.1⁺ CD3^- cells were characterized according to their surface expression of KLRG1, NKp46, CD122, TCRγδ and Thy1. (i) C57BL/6 mice were infected for 18h with 5x10⁷ CFU <i>S</i>.Tm. Isolated cecal LP cells of two mice each were pooled for fluorescence activated cell sorting. Live CD45⁺ CD3^- cells were further sorted according to their expression of NK1.1. <i>Tbx21</i>, <i>Eomes</i>, <i>Rorc</i>, <i>Gata3</i>, <i>Prf1 and Sell</i> transcripts were analyzed by RT-qPCR. Results are presented relative to the expression of <i>Actb</i>. (j and k) Flow cytometric analysis of isolated cecal LP cells from Sm-pretreated (j) Casp1/11^-/- or (k) Casp11^-/- mice. Single live cells were gated on CD45⁺ lymphocytes and NK1.1⁺ CD3^- cells were quantified. (l and m) C57BL/6 WT mice were injected intraperitoneally with anti-asialo GM1 antiserum (50μL antiserum/mouse; three consecutive days), anti-NK1.1 (10mg/kg; 2 consecutive days) or PBS and mice were infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 8–10 per group). (l) HE-stained cryosections of anti-asialo GM1 -treated or control mice, (m) pathological score; arrows indicate mice of representative HE-stained cryosections; SE = submucosal edema, L = lumen; scale bar = 100μm. Statistical analysis was performed using the Mann-Whitney-U test (ns = not significant, * = p<0.05; ** = p<0.01).</p

NK cells in the infected cecal mucosa of IL-18-deficient mice are phenotypically immature.

<p>(a and b) Flow cytometric analysis of isolated cecal LP cells from Sm-pretreated C57BL/6 WT mice, either uninfected or infected with 5x10⁷ CFU <i>S</i>.Tm (n = 5 per group). We gated on single live CD45⁺ CD3^- NK1.1⁺ cells. (a) Representative contour plots and (b) quantification of NK cell maturation stages, defined by the surface expression of CD27 and CD11b; arrows indicate the progression of NK-cell maturation. (c and d) Flow cytometric analysis of isolated cecal LP cells from <i>Il18</i>^-/- mice and littermates, Sm-pretreated and orally infected with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 5–6 per group). We gated on single live CD45⁺ CD3^- NK1.1⁺ cells. (c) Representative contour plots and (d) quantification of NK cell maturation stages based on CD27 and CD11b surface expression. Data represent the mean ± SD and statistical analyses were performed using 2way-ANOVA with Sidak’s multiple comparison test (ns = not significant, * = p<0.05; ** = p<0.01; *** = p<0.001).</p

Perforin-deficient mice fail to elicit overt gut inflammation by 12h p.i.

<p>(a and b) <i>Prf1</i>^<i>-/-</i> mice and littermate controls were Sm-pretreated and infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h. (a) Pathological score; arrows indicate representative mice depicted in panel b, (b) HE-stained cryosections from representative mice of each group. (c) <i>Il18</i>^<i>-/-</i> mice and littermates or (d) <i>Prf1</i>^<i>-/-</i> mice and littermates were Sm-pretreated and infected orally with 5x10⁷ CFU <i>S</i>.Tm-p<i>ssaG</i>-GFPmut2 for 12h (n = 4–6 per group). <i>S</i>.Tm cecum tissue counts were determined per 20μm cross-section. Statistical analysis was performed using the Mann-Whitney-U test (** = p<0.01).</p