Extracellular vesicles and PD-L1 suppress macrophages, inducing therapy resistance in TP53-deficient B-cell malignancies.

Genetic alterations in the DNA damage response (DDR) pathway are a frequent mechanism of resistance to chemoimmunotherapy (CIT) in B-cell malignancies. We have previously shown that the synergy of CIT relies on secretory crosstalk elicited by chemotherapy between the tumor cells and macrophages. Here, we show that loss of multiple different members of the DDR pathway inhibits macrophage phagocytic capacity in vitro and in vivo. Particularly, loss of TP53 led to decreased phagocytic capacity ex vivo across multiple B-cell malignancies. We demonstrate via in vivo cyclophosphamide treatment using the Eµ-TCL1 mouse model that loss of macrophage phagocytic capacity in Tp53-deleted leukemia is driven by a significant downregulation of a phagocytic transcriptomic signature using small conditional RNA sequencing. By analyzing the tumor B-cell proteome, we identified a TP53-specific upregulation of proteins associated with extracellular vesicles (EVs). We abrogated EV biogenesis in tumor B-cells via clustered regularly interspaced short palindromic repeats (CRISPR)-knockout (KO) of RAB27A and confirmed that the EVs from TP53-deleted lymphoma cells were responsible for the reduced phagocytic capacity and the in vivo CIT resistance. Furthermore, we observed that TP53 loss led to an upregulation of both PD-L1 cell surface expression and secretion of EVs by lymphoma cells. Disruption of EV bound PD-L1 by anti-PD-L1 antibodies or PD-L1 CRISPR-KO improved macrophage phagocytic capacity and in vivo therapy response. Thus, we demonstrate enhanced EV release and increased PD-L1 expression in TP53-deficient B-cell lymphomas as novel mechanisms of macrophage function alteration in CIT resistance. This study indicates the use of checkpoint inhibition in the combination treatment of B-cell malignancies with TP53 loss.

Macrophage-derived IL-6 trans-signalling as a novel target in the pathogenesis of bronchopulmonary dysplasia.

RATIONALE: Premature infants exposed to oxygen are at risk for bronchopulmonary dysplasia (BPD), which is characterised by lung growth arrest. Inflammation is important, but the mechanisms remain elusive. Here, we investigated inflammatory pathways and therapeutic targets in severe clinical and experimental BPD. METHODS AND RESULTS: First, transcriptomic analysis with in silico cellular deconvolution identified a lung-intrinsic M1-like-driven cytokine pattern in newborn mice after hyperoxia. These findings were confirmed by gene expression of macrophage-regulating chemokines (Ccl2, Ccl7, Cxcl5) and markers (Il6, Il17A, Mmp12). Secondly, hyperoxia-activated interleukin 6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signalling was measured in vivo and related to loss of alveolar epithelial type II cells (ATII) as well as increased mesenchymal marker. Il6 null mice exhibited preserved ATII survival, reduced myofibroblasts and improved elastic fibre assembly, thus enabling lung growth and protecting lung function. Pharmacological inhibition of global IL-6 signalling and IL-6 trans-signalling promoted alveolarisation and ATII survival after hyperoxia. Third, hyperoxia triggered M1-like polarisation, possibly via Krüppel-like factor 4; hyperoxia-conditioned medium of macrophages and IL-6-impaired ATII proliferation. Finally, clinical data demonstrated elevated macrophage-related plasma cytokines as potential biomarkers that identify infants receiving oxygen at increased risk of developing BPD. Moreover, macrophage-derived IL6 and active STAT3 were related to loss of epithelial cells in BPD lungs. CONCLUSION: We present a novel IL-6-mediated mechanism by which hyperoxia activates macrophages in immature lungs, impairs ATII homeostasis and disrupts elastic fibre formation, thereby inhibiting lung growth. The data provide evidence that IL-6 trans-signalling could offer an innovative pharmacological target to enable lung growth in severe neonatal chronic lung disease.

Kinase activity profiling reveals contribution of G-protein signaling modulator 2 deficiency to impaired regulatory T cell migration in rheumatoid arthritis.

The ability of regulatory T (Treg) cells to migrate into inflammatory sites is reduced in autoimmune diseases, including rheumatoid arthritis (RA). The reasons for impaired Treg cell migration remain largely unknown. We performed multiplex human kinase activity arrays to explore possible differences in the post-translational phosphorylation status of kinase related proteins that could account for altered Treg cell migration in RA. Results were verified by migration assays and Western blot analysis of CD4+ T cells from RA patients and from mice with collagen type II induced arthritis. Kinome profiling of CD4+ T cells from RA patients revealed significantly altered post-translational phosphorylation of kinase related proteins, including G-protein-signaling modulator 2 (GPSM2), protein tyrosine kinase 6 (PTK6) and vitronectin precursor (VTNC). These proteins have not been associated with RA until now. We found that GPSM2 expression is reduced in CD4+ T cells from RA patients and is significantly downregulated in experimental autoimmune arthritis following immunization of mice with collagen type II. Interestingly, GPSM2 acts as a promoter of Treg cell migration in healthy individuals. Treatment of RA patients with interleukin-6 receptor (IL-6R) blocking antibodies restores GPSM2 expression, thereby improving Treg cell migration. Our study highlights the potential of multiplex kinase activity arrays as a tool for the identification of RA-related proteins which could serve as targets for novel treatments.

Macrophage-Mediated Antibody Dependent Effector Function in Aggressive B-Cell Lymphoma Treatment is Enhanced by Ibrutinib via Inhibition of JAK2.

Targeted inhibition of Bruton's Tyrosine Kinase (BTK) with ibrutinib and other agents has become important treatment options in chronic lymphocytic leukemia, Waldenström's Macroglobulinemia, Mantle cell lymphoma, and non-GCB DLBCL. Clinical trials combining small molecule inhibitors with monoclonal antibodies have been initiated at rapid pace, with the biological understanding between their synergistic interactions lagging behind. Here, we have evaluated the synergy between BTK inhibitors and monoclonal antibody therapy via macrophage mediated antibody dependent cellular phagocytosis (ADCP). Initially, we observed increased ADCP with ibrutinib, whilst second generation BTK inhibitors failed to synergistically interact with monoclonal antibody treatment. Kinase activity profiling under BTK inhibition identified significant loss of Janus Kinase 2 (JAK2) only under ibrutinib treatment. We validated this potential off-target effect via JAK inhibition in vitro as well as with CRISPR/Cas9 JAK2-/- experiments in vivo, showing increased ADCP and prolonged survival, respectively. This data supports inhibition of the JAK-STAT (Signal Transducers and Activators of Transcription) signaling pathway in B-cell malignancies in combination with monoclonal antibody therapy to increase macrophage-mediated immune responses.