Spatial proteomics identifies a spectrum of immune dysregulation in acquired bone marrow failure syndromes.

Poor graft function (PGF), manifested by multilineage cytopenias and complete donor chimerism post-allogeneic stem cell transplantation (alloSCT), and acquired aplastic anaemia (AA) are immune-mediated acquired bone marrow (BM) failure syndromes with a similar clinical presentation. In this study, we used spatial proteomics to compare the immunobiology of the BM microenvironment and identify common mechanisms of immune dysregulation under these conditions. Archival BM trephines from patients exhibited downregulation of the immunoregulatory protein VISTA and the M2 macrophage marker and suppressor of T-cell activation ARG1 with increased expression of the immune checkpoint B7-H3 compared to normal controls. Increased CD163 and CD14 expression suggested monocyte/macrophage skewing, which, combined with dysregulation of STING and VISTA, is indicative of an environment of reduced immunoregulation resulting in the profound suppression of hematopoiesis in these two conditions. There were no changes in the immune microenvironment between paired diagnostic AA and secondary MDS/AML samples suggesting that leukaemic clones develop in the impaired immune microenvironment of AA without the need for further alterations. Of the eight proteins with dysregulated expression shared by diagnostic AA and PGF, the diagnostic AA samples had a greater fold change in expression than PGF, suggesting that these diseases represent a spectrum of immune dysregulation. Unexpectedly, analysis of samples from patients with good graft function post-alloSCT demonstrated significant changes in the immune microenvironment compared to normal controls, with downregulation of CD44, STING, VISTA, and ARG1, suggesting that recovery of multilineage haematopoiesis post-alloSCT does not reflect recovery of immune function and may prime patients for the development of PGF upon further inflammatory insult. The demonstrable similarities in the immunopathology of AA and PGF will allow the design of clinical interventions that include both patient cohorts to accelerate therapeutic discovery and translation.

A phase 1 clinical trial of the repurposable acetyllysine mimetic, n-methyl-2-pyrrolidone (NMP), in relapsed or refractory multiple myeloma.

BACKGROUND: N-methyl-2-pyrrolidone (NMP) is an epigenetically active chemical fragment and organic solvent with numerous applications including use as a drug-delivery vehicle. Previously considered biologically inert, NMP demonstrates immunomodulatory and anti-myeloma properties that are partly explained by acetyllysine mimetic properties and non-specific bromodomain inhibition. We therefore evaluated orally administered NMP in a phase 1 dose-escalation trial to establish its maximum tolerated dose (MTD) in patients with relapsed/refractory multiple myeloma (RR-MM). Secondary endpoints were safety, pharmacokinetics (PK), overall response rate and immunological biomarkers of activity. RESULTS: Thirteen patients received NMP at starting doses between 50 and 400 mg daily. Intra-patient dose escalation occurred in five patients, with one attaining the ceiling protocolised dose of 1 g daily. Median number of monthly cycles commenced was three (range 1-20). Grade 3-4 adverse events (AEs) were reported in seven (54%; 95% CI 25-81%) patients. Most common AEs (> 30% of patients) of any grade were nausea and musculoskeletal pain. The only dose limiting toxicity (DLT) was diarrhoea in a patient receiving 200 mg NMP (overall DLT rate 8%; 95% CI 0-36%). Hence, the MTD was not defined. Median progression-free and overall survival were 57 (range 29-539) days and 33 (95% CI 9.7- > 44) months, respectively. The best response of stable disease (SD) was achieved in nine patients (69%; 95% CI 39-91%). PK analysis demonstrated proportional dose-concentrations up to 400 mg daily, with a more linear relationship above 500 mg. Maximum plasma concentrations (Cmax) of 16.7 mg/L at the 800 mg dose were below those predicted to inhibit BET-bromodomains. Peripheral blood immune-profiling demonstrated maintenance of natural killer (NK) cells, and a gene expression signature suggestive of enhanced T, B and NK cell functions; a subject with prolonged exposure manifested sustained recovery of B and NK cells at 12 months. CONCLUSIONS: NMP demonstrated potential disease stabilising and immunomodulatory activity at sub-BET inhibitory plasma concentrations and was well tolerated in RR-MM; an MTD was not determined up to a maximum dose of 1 g daily. Further dose-finding studies are required to optimise NMP dosing strategies for therapeutic intervention.

Dysregulation of immune cell and cytokine signalling correlates with clinical outcomes in myelodysplastic syndrome (MDS).

OBJECTIVES: Myelodysplastic syndromes (MDS) are characterised by ineffective haematopoiesis. Although hypomethylating agents (HMA) have improved survival in higher-risk MDS, most patients eventually succumb to progressive disease. Utilising samples collected prospectively from three MDS clinical trials, we analysed genetic and immunological biomarkers and correlated them with clinical outcomes. METHODS: A hundred and fifty four samples were analysed from 133 de novo MDS patients for T-cell and myeloid cell immunophenotyping and gene expression analysis. Treatments were with HMA or immunomodulatory drug (IMiD) alone or in combination. RESULTS: We observed differences in immune cell subsets between lower- and higher-risk IPSS groups with NKT cells, MDSCs, intermediate-proinflammatory and non-classical monocytes being higher in the latter group, while naïve CD4+ T cells were reduced. Intermediate-proinflammatory monocytes were increased in non-responders and those failing to achieve at least a haematological improvement. Proinflammatory NKT cells were increased at diagnosis for patients failing to derive clinical benefit after 12 months of treatment. Gene expression analysis of paired bone marrow (BM) colony-forming units (CFUs) from diagnosis and 4 cycles post-treatment confirmed that genes involved in cytokine signalling were downregulated in C4 normal colonies. CONCLUSIONS: These findings support the central roles of dysregulation in innate immunity and inflammatory signalling in the pathogenesis of MDS which correlated with clinical outcomes post-treatment.

Venetoclax or Ruxolitinib in Pre-Transplant Conditioning Lowers the Engraftment Barrier by Different Mechanisms in Allogeneic Stem Cell Transplant Recipients.

Allogeneic stem cell transplantation (alloSCT) is utilised to cure haematological malignancies through a combination of conditioning regimen intensity and immunological disease control via the graft versus tumour (GVT) effect. Currently, conventional myeloablative chemotherapeutic or chemoradiation conditioning regimens are associated with significant side effects including graft versus host disease (GVHD), infection, and organ toxicity. Conversely, more tolerable reduced intensity conditioning (RIC) regimens are associated with unacceptably higher rates of disease relapse, partly through an excess incidence of mixed chimerism. Improvement in post-alloSCT outcomes therefore depends on promotion of the GVT effect whilst simultaneously reducing conditioning-related toxicity. We have previously shown that this could be achieved through BCL-2 inhibition, and in this study, we explored the modulation of JAK1/2 as a strategy to lower the barrier to donor engraftment in the setting of RIC. We investigated the impact of short-term treatment of BCL2 (venetoclax) or JAK1/2 (ruxolitinib) inhibition on recipient natural killer and T cell immunity and the subsequent effect on donor engraftment. We identified striking differences in mechanism of action of these two drugs on immune cell subsets in the bone marrow of recipients, and in the regulation of MHC class-II and interferon-inducible gene expression, leading to different rates of GVHD. This study demonstrates that the repurposed use of ruxolitinib or venetoclax can be utilised as pre-transplant immune-modulators to promote the efficacy of alloSCT, whilst reducing its toxicity.

Immune recovery in patients with mantle cell lymphoma receiving long-term ibrutinib and venetoclax combination therapy.

Combination venetoclax plus ibrutinib for the treatment of mantle cell lymphoma (MCL) has demonstrated efficacy in the relapsed or refractory setting; however, the long-term impact on patient immunology is unknown. In this study, changes in immune subsets of MCL patients treated with combination venetoclax and ibrutinib were assessed over a 4-year period. Multiparameter flow cytometry of peripheral blood mononuclear cells showed that ≥12 months of treatment resulted in alterations in the proportions of multiple immune subsets, most notably CD4+ and CD8+ effector and central memory T cells and natural killer cells, and normalization of T-cell cytokine production in response to T-cell receptor stimulation. Gene expression analysis identified upregulation of multiple myeloid genes (including S100 and cathepsin family members) and inflammatory pathways over 12 months. Four patients with deep responses stopped study drugs, resulting in restoration of normal immune subsets for all study parameters except myeloid gene/pathway expression, suggesting long-term combination venetoclax and ibrutinib irreversibly affects this population. Our findings demonstrate that long-term combination therapy is associated with immune recovery in MCL, which may allow responses to subsequent immunotherapies and suggests that this targeted therapy results in beneficial impacts on immunological recovery. This trial was registered at www.clinicaltrials.gov as #NCT02471391.

Parity reduces mammary repopulating activity but does not affect mammary stem cells defined as CD24 + CD29/CD49fhi in mice.

BACKGROUND: Breast cancer (BCa) mortality is decreasing with early detection and improvement in therapies. The incidence of BCa, however, continues to increase, particularly estrogen-receptor-positive (ER +) subtypes. One of the greatest modifiers of ER + BCa risk is childbearing (parity), with BCa risk halved in young multiparous mothers. Despite convincing epidemiological data, the biology that underpins this protection remains unclear. Parity-induced protection has been postulated to be due to a decrease in mammary stem cells (MaSCs); however, reports to date have provided conflicting data. METHODS: We have completed rigorous functional testing of repopulating activity in parous mice using unfractionated and MaSC (CD24midCD49fhi)-enriched populations. We also developed a novel serial transplant method to enable us to assess self-renewal of MaSC following pregnancy. Lastly, as each pregnancy confers additional BCa protection, we subjected mice to multiple rounds of pregnancy to assess whether additional pregnancies impact MaSC activity. RESULTS: Here, we report that while repopulating activity in the mammary gland is reduced by parity in the unfractionated gland, it is not due to a loss in the classically defined MaSC (CD24+CD49fhi) numbers or function. Self-renewal was unaffected by parity and additional rounds of pregnancy also did not lead to a decrease in MaSC activity. CONCLUSIONS: Our data show instead that parity impacts on the stem-like activity of cells outside the MaSC population.

A new role for Notch in the control of polarity and asymmetric cell division of developing T cells.

A fundamental question in biology is how single cells can reliably produce progeny of different cell types. Notch signalling frequently facilitates fate determination. Asymmetric cell division (ACD) often controls segregation of Notch signalling by imposing unequal inheritance of regulators of Notch. Here, we assessed the functional relationship between Notch and ACD in mouse T cell development. To attain immunological specificity, developing T cells must pass through a pivotal stage termed ß-selection, which involves Notch signalling and ACD. We assessed functional interactions between Notch1 and ACD during ß-selection through direct presentation of Notch ligands, DL1 and DL4, and pharmacological inhibition of Notch signalling. Contrary to prevailing models, we demonstrate that Notch signalling controls the distribution of Notch1 itself and cell fate determinants, α-adaptin and Numb. Furthermore, Notch and CXCR4 signalling cooperated to drive polarity during division. Thus, Notch signalling directly orchestrates ACD, and Notch1 is differentially inherited by sibling cells.This article has an associated First Person interview with the first author of the paper.

Recipient BCL2 inhibition and NK cell ablation form part of a reduced intensity conditioning regime that improves allo-bone marrow transplantation outcomes.

Allogeneic hematopoietic stem cell transplantation (alloSCT) is used to treat over 15,000 patients with acute myeloid leukemia (AML) per year. Donor graft-versus-leukemia (GVL) effect can prevent AML relapse; however, alloSCT is limited by significant toxicity related to conditioning intensity, immunosuppression, opportunistic infections, and graft-versus-host disease (GVHD). Reducing the intensity of conditioning regimens prior to alloSCT has improved their tolerability, but does not alter the pattern of GVHD and has been associated with increased rates of graft rejection and relapse. Here, using a murine pre-clinical model, we describe a novel recipient conditioning approach combining reduced intensity conditioning with either genetic or pharmacological inhibition of NK cell numbers that permits efficient donor engraftment and promotes GVL without inducing GVHD. We show that NK cell-specific deletion of Bcl2 or Mcl1 in mice, or pharmacological inhibition of BCL2 impairs radio-resistant NK cell-mediated rejection of allogeneic engraftment and allows reduction of conditioning intensity below that associated with GVHD priming. The combination of reduced intensity conditioning and NK cell targeting in mice allowed successful donor T cell engraftment and protective immunity against AML while avoiding GVHD. These findings suggest that reduced conditioning in combination with targeted therapies against recipient NK cells may allow the delivery of effective alloSCT against AML while reducing the toxicities associated with more intensive conditioning including GVHD.

Transport of stearic acid-based solid lipid nanoparticles (SLNs) into human epithelial cells.

Development of drug delivery systems, as much as the drug molecule itself, is an important consideration for improving drug absorption and bioavailability. The mechanisms by which drug carriers enter target cells can differ depending on their size, surface properties and components. Solid lipid nanoparticles (SLNs) have gained an increased attention in recent years and are the drug carriers of interest in this paper. They are known to breach the cell-membrane barrier and have been actively sought to transport biomolecules. Previous studies by our group, and also other groups, provided an extensive characterization of SLNs. However, few studies have investigated the uptake of SLNs and these have had limited mechanistic focus. The aim of this work was to investigate the pathway of uptake of SLNs by human epithelial cells i.e., lung A549 and cervical HeLa cells. To the best of our knowledge, this is first study that investigates the cellular uptake of SLNs by human epithelial cells. The mechanism of cellular uptake was deciphered using pharmacologic inhibitors (sucrose, potassium-free buffer, filipin and cytochalasin B). Imaging techniques and flow assisted cell sorting (FACS) were used to assess the cellular uptake of SLNs loaded with rhodamine 123 as a fluorescent probe. This study provided evidence that the cellular uptake of SLNs was energy-dependent, and the endocytosis of SLNs was mainly dependent on clathrin-mediated mechanisms. The establishment of entry mechanism of SLNs is of fundamental importance for future facilitation of SLNs as biological or drug carriers.

Divergent lymphocyte signalling revealed by a powerful new tool for analysis of time-lapse microscopy.

We describe a new approach for interactive analysis of time-lapse microscopy, and apply this approach to elucidating whether polarity regulation is conserved between epithelial cells and lymphocytes. A key advantage of our analysis platform, 'TACTICS', is the capacity to visualize individual data points in the context of large data sets, similar to standard approaches in flow cytometry. Scatter plots representing microscopic parameters or their derivations such as polarity ratios are linked to the original data such that clicking on each dot enables a link to images and movies of the corresponding cell. Similar to flow cytometric analysis, subsets of the data can be gated and reanalyzed to explore the relationships between different parameters. TACTICS was used to dissect the regulation of polarization of the cell fate determinant, Numb, in migrating lymphocytes. We show here that residues of Numb that are phosphorylated by atypical protein kinase C (aPKC) to mediate apicobasal polarity in epithelial cells are not required for polarization of Numb in T cells, indicating that the role of aPKC is not conserved between lymphocytes and epithelia.

Ligand binding determines whether CD46 is internalized by clathrin-coated pits or macropinocytosis.

CD46 is a ubiquitous human cell surface receptor for the complement components C3b and C4b and for various pathogens, including the measles virus and human herpes virus 6. Ligand binding to CD46 affects (i) protection of autologous cells from complement attack by breakdown of complement components, (ii) intracellular signals that affect the regulation of immune cell function, (iii) antigen presentation, and (iv) down-regulation of cell surface CD46. Recent evidence indicates that CD46 signaling can link innate and acquired immune function. The molecular mechanisms for these processes and the importance of intracellular trafficking of the receptor have not yet been elucidated. We demonstrate here that, in nonlymphoid cells, CD46 is constitutively internalized via clathrin-coated pits, traffics to multivesicular bodies, and is recycled to the cell surface. However, cross-linking of CD46 at the cell surface, by either multivalent antibody or by measles virus, induces pseudopodia that engulf the ligand in a process similar to macropinocytosis, and leads to the degradation of cell surface CD46. Thus, we have elucidated two pathways for CD46 internalization, which are regulated by the valence of cross-linking of CD46 and which utilize either clathrin-coated pits or pseudopodial extension. This has important implications for CD46 signaling, antigen presentation, CD46 down-regulation, and engulfment of pathogens.

A functional interaction between CD46 and DLG4: a role for DLG4 in epithelial polarization.

Using a yeast two-hybrid screen, we identified a physical interaction between CD46 and DLG4. CD46 is a ubiquitous human cell-surface receptor for the complement components C3b and C4b and for measles virus and human herpesvirus 6. DLG4 is a scaffold protein important for neuronal signaling and is homologous to the Drosophila tumor suppressor DLG. We show that an interaction between CD46 and DLG4 is important for polarization in epithelial cells. Specifically, we show (i) biochemical evidence for an interaction between CD46 and DLG4, (ii) that this interaction is specific for the Cyt1 (but not Cyt2) domain of CD46, (iii) that both CD46 and an alternatively spliced isoform of DLG4 are polarized in normal human epithelial cells, and (iv) that the polarized expression of CD46 in epithelial cells requires the DLG4-binding domain and alters with expression of a truncated form of DLG4. This is the first identification of a direct and cytoplasmic domain-specific interaction between CD46 and an intracellular signaling molecule and provides a molecular mechanism for the polarization of CD46. These data also indicate that, in addition to the known role for DLG4 in neuronal cells, DLG4 may be important for polarization in epithelial cells.