Association of Regular Thrombus Surface Phenotype With Complete Recanalization in First-Line Contact Aspiration Thrombectomy for Basilar Artery Occlusion

Objective: To assess whether angiographic thrombus surface phenotype has an impact on efficacy of contact aspiration (CA) thrombectomy in patients with basilar artery occlusion (BAO).Methods: From January 2016 to December 2019, consecutive stroke patients with a BAO and first-line CA were analyzed in this retrospective study. We assessed baseline and imaging characteristics and treatment and clinical outcomes. We rated thrombus surface phenotype on pre-treatment digital subtraction angiography in a three-reader-consensus setting. Primary outcome was complete recanalization (modified treatment in cerebral ischemia [mTICI] 3 and arterial occlusive lesion [AOL] 3) after first-line CA without additionally stent retriever passes. Data analysis was stratified according to thrombus surface phenotype and complete first-line recanalization.Results: Seventy-eight patients met the inclusion criteria. Median age was 74 years (IQR 64–80), 64% were male, and median baseline NIHSS score was 24 (IQR 7–32). Thirty patients had a regular and 16 patients had an irregular thrombus phenotype. Thrombus surface was not assessable in 32 patients. In patients with a regular phenotype, complete recanalization was more often achieved compared to irregular and non-ratable phenotypes (50 vs. 18.8% and 21.9%; p = 0.027). Patients with a regular phenotype [odds ratio [OR] 8.3; 95% confidence interval [CI]: 1.9–35.8; p = 0.005], cardioembolic stroke (OR 12.1, 95% CI: 2.0–72.8; p = 0.007), and proximal end of the thrombus in the middle basilar artery segment (OR 5.2, 95% CI: 1.0–26.6; p = 0.046) were more likely to achieve complete recanalization after first-line CA without rescue therapy.Conclusion: The efficacy of CA may differ according to the angiographic thrombus surface phenotype in patients with BAO. A regular phenotype is associated with higher rates of complete recanalization in first-line CA. However, assessment of thrombus phenotype is frequently not feasible in BAO.

Towards understanding the cell surface phenotype, metabolic properties and immune functions of resident macrophages of the peritoneal cavity and splenic red pulp using high resolution quantitative proteomics

Background: Resident macrophages (Mϕs) are distributed throughout the body and are important for maintaining tissue homeostasis and for defence against infections. Tissue Mϕs are highly adapted to their microenvironment and thought to mediate tissue-specific functions involving metabolism and immune defence that are not fully elucidated. Methods: We have used high resolution quantitative proteomics to gain insights into the functions of two types of resident tissue Mϕs: peritoneal cavity Mϕs and splenic red pulp Mϕs. The cellular expression levels of many proteins were validated by flow cytometry and were consistently in agreement with the proteomics data. Results: Peritoneal and splenic red pulp macrophages displayed major differences in cell surface phenotype reflecting their adaptation to different tissue microenvironments and tissue-specific functions. Peritoneal Mϕs were shown to be enriched in a number of key enzymes and metabolic pathways normally associated with the liver, such as metabolism of fructose, detoxification, nitrogen homeostasis and the urea cycle. Supporting these observations, we show that peritoneal Mϕs are able to utilise glutamine and glutamate which are rich in peritoneum for urea generation. In comparison, splenic red pulp Mϕs were enriched in proteins important for adaptive immunity such as antigen presenting MHC molecules, in addition to proteins required for erythrocyte homeostasis and iron turnover. We also show that these tissue Mϕs may utilise carbon and nitrogen substrates for different metabolic fates to support distinct tissue-specific roles. Conclusions: This study provides new insights into the functions of tissue Mϕs in immunity and homeostasis. The comprehensive proteomics data sets are a valuable resource for biologists and immunologists.

A first-in-class ex vivo combination between cytokine-induced memory like (CIML) NK cells and a CD38 targeting antibody recruiting molecule (ARM) as a novel approach to target NK cells without cellular engineering for the treatment of multiple myeloma.

8523 Background: Cytokine-induced memory-like (CIML) NK cells differentiate after a brief preactivation with interleukin-12 (IL-12), IL-15, and IL-18 and exhibit enhanced responses to cytokine or activating receptor restimulation for several weeks to months. Antibody Recruiting Molecules (ARM) are bifunctional molecules that simultaneously bind a given tumor marker and engage endogenous IgG antibodies, therefore binding and activating NK cells via their FcR and targeting them to kill tumors cells. A first in class therapy that has the potential to improve the activity of CIML NK cells by combining them with KP1237, a CD38-ARM is presented for the treatment of newly diagnosed multiple myeloma (MM) patients who are minimal residual disease positive (MRD+) and eligible for autologous stem cell transplant (ASCT). Methods: Specific killing of CD38 expressing MOLP-8 cells by CIML NK cells alone or in combination with KP1237 was measured. Additionally, NK and CIML NK cells from healthy volunteers and MM patients at various stages of the disease were tested for activity against autologous and allogeneic tumor cell targets. In these experiments, surface phenotype and activation status of NK cells were determined. Results: A statistically significant increase in specific killing was observed when KP1237 was used in combination with CIML NK cells in an ADCC assay against CD38 expressing MOLP-8 (p = 0.0105, mean 61.8 ± SD 9.1 and mean 83.7 ± SD 13.58 for CIML NK alone and CIML NK+KP1237, respectively). This effect was accompanied by an increase in plasma membrane retention of CD107a. Surface phenotype of NK and CIML NK cells from healthy volunteers and MM patients was compared since the functional activity of NK cells from such patients is debated. We report surface expression of CD56, CD16, CD57, NKG2D, Nkp44, Nkp46, CD107a, KIR2DL2/3/DS2, and CD69 as well as frequencies of CD57hi CD56+ mature versus CD57lo CD56+ immature NK cell subsets. Further, autologous CIML NK cells + KP1237 exhibit cytotoxicity towards patient plasma cells. Conclusions: The activity of CIML NK cells towards CD38 expressing MM target cells is improved by the addition of KP1237 avoiding the need for cellular engineering with chimeric antigen receptors. This led to the development of the combination drug product consisting of patient autologous CIML NK cells, KP1237 and IVIG. Clinical development is under way for (MRD+) MM patients eligible for ASCT.

Could Increased Expression of Hsp27, an “Anti-Inflammatory” Chaperone, Contribute to the Monocyte-Derived Dendritic Cell Bias towards Tolerance Induction in Breast Cancer Patients?

Dendritic cells (DCs) are the most efficient antigen-presenting cells and link the innate immune sensing of the environment to the initiation of adaptive immune responses, which may be directed to either acceptance or elimination of the recognized antigen. In cancer patients, though DCs would be expected to present tumor antigens to T lymphocytes and induce tumor-eliminating responses, this is frequently not the case. The complex tumor microenvironment subverts the immune response, blocks some effector mechanisms, and drives others to support tumor growth. Chronic inflammation in a tumor microenvironment is believed to contribute to the induction of such regulatory/tolerogenic response. Among the various mediators of the modulatory switch in chronic inflammation is the “antidanger signal” chaperone, heat shock protein 27 (Hsp27), that has been described, interestingly, to be associated with cell migration and drug resistance of breast cancer cells. Thus, here, we investigated the expression of Hsp27 during the differentiation of monocyte-derived DCs (Mo-DCs) from healthy donors and breast cancer patients and evaluated their surface phenotype, cytokine secretion pattern, and lymphostimulatory activity. Surface phenotype and lymphocyte proliferation were evaluated by flow cytometry, interferon- (IFN-) γ, and interleukin- (IL-) 10 secretion, by ELISA and Hsp27 expression, by quantitative polymerase chain reaction (qPCR). Mo-DCs from cancer patients presented decreased expression of DC maturation markers, decreased ability to induce allogeneic lymphocyte proliferation, and increased IL-10 secretion. In coculture with breast cancer cell lines, healthy donors’ Mo-DCs showed phenotype changes similar to those found in patients’ cells. Interestingly, patients’ monocytes expressed less GM-CSF and IL-4 receptors than healthy donors’ monocytes and Hsp27 expression was significantly higher in patients’ Mo-DCs (and in tumor samples). Both phenomena could contribute to the phenotypic bias of breast cancer patients’ Mo-DCs and might prove potential targets for the development of new immunotherapeutic approaches for breast cancer.

Cellular investigations

This chapter covers cellular investigations, including identification of cell surface phenotype, intracellular proteins, cellular function, secreted products, and abnormal constituents. Techniques are described, along with principles for testing and interpretation of the results.

Plastic fantastic innate lymphoid cells

In this issue of JEM, Nagasawa et al. (https://doi.org/10.1084/jem.20190490) have undertaken a detailed study of the cell surface phenotype, transcriptional profile, and cytokine secretion of ILC progenitor populations in human peripheral blood and tissues and describe markers that highlight the heterogeneity of these chameleons.