Antibody-based in vivo leukocyte label for two-photon brain imaging in mice.

Significance: To study leukocyte-endothelial interactions in a living system, robust and specific leukocyte labeling techniques are needed for in vivo two-photon microscopy of the cerebral microvasculature. Aim: We tested fluorophore-conjugated anti-CD45.2 monoclonal antibodies (mAb) to optimize dosing and two-photon imaging parameters for leukocyte labeling in healthy mice and a venous microstroke model. Approach: We retro-orbitally injected anti-CD45.2 mAb at 0.04, 0.4, and 2 mg / kg into BALB/c mice and used flow cytometry to analyze antibody saturation. Leukocyte labeling in the cortical microvasculature was examined by two-photon imaging. We also tested the application of CD45.2 mAb in a pathological leukocyte-endothelial adhesion model by photothrombotically occluding cortical penetrating venules. Results: We found that 0.4 mg / kg of anti-CD45.2 antibody intravenously was sufficient to label 95% of circulating leukocytes. There was no depletion of circulating leukocytes after 24 h at the dosages tested. Labeled leukocytes could be observed as deep as 550 µ m from the cortical surface. The antibody reliably labeled rolling, crawling, and adherent leukocytes in venules around the stroke-affected tissues. Conclusion: We show that the anti-CD45.2 mAb is a robust reagent for acute labeling of leukocytes during in vivo two-photon microscopy of the cortical microvasculature.

Brain capillary obstruction during neurotoxicity in a mouse model of anti-CD19 chimeric antigen receptor T-cell therapy.

Immunotherapy for haematologic malignancies with CD19-directed chimeric antigen receptor T cells has been highly successful at eradicating cancer but is associated with acute neurotoxicity in ∼40% of patients. This neurotoxicity correlates with systemic cytokine release syndrome, endothelial activation and disruption of endothelial integrity, but it remains unclear how these mechanisms interact and how they lead to neurologic dysfunction. We hypothesized that dysfunction of the neurovascular unit is a key step in the development of neurotoxicity. To recapitulate the interaction of the intact immune system with the blood-brain barrier, we first developed an immunocompetent mouse model of chimeric antigen receptor T-cell treatment-associated neurotoxicity. We treated wild-type mice with cyclophosphamide lymphodepletion followed by escalating doses of murine CD19-directed chimeric antigen receptor T cells. Within 3-5 days after chimeric antigen receptor T-cell infusion, these mice developed systemic cytokine release and abnormal behaviour as measured by daily neurologic screening exams and open-field testing. Histologic examination revealed widespread brain haemorrhages, diffuse extravascular immunoglobulin deposition, loss of capillary pericyte coverage and increased prevalence of string capillaries. To measure any associated changes in cerebral microvascular blood flow, we performed in vivo two-photon imaging through thinned-skull cranial windows. Unexpectedly, we found that 11.9% of cortical capillaries were plugged by Day 6 after chimeric antigen receptor T-cell treatment, compared to 1.1% in controls treated with mock transduced T cells. The capillary plugs comprised CD45+ leucocytes, a subset of which were CD3+ T cells. Plugging of this severity is expected to compromise cerebral perfusion. Indeed, we found widely distributed patchy hypoxia by hypoxyprobe immunolabelling. Increased serum levels of soluble ICAM-1 and VCAM-1 support a putative mechanism of increased leucocyte-endothelial adhesion. These data reveal that brain capillary obstruction may cause sufficient microvascular compromise to explain the clinical phenotype of chimeric antigen receptor T-cell neurotoxicity. The translational impact of this finding is strengthened by the fact that our mouse model closely approximates the kinetics and histologic findings of the chimeric antigen receptor T-cell neurotoxicity syndrome seen in human patients. This new link between systemic immune activation and neurovascular unit injury may be amenable to therapeutic intervention.

Activation of Kappa Opioid Receptor Regulates the Hypothermic Response to Calorie Restriction and Limits Body Weight Loss.

Mammals maintain a nearly constant core body temperature (Tb) by balancing heat production and heat dissipation. This comes at a high metabolic cost that is sustainable if adequate calorie intake is maintained. When nutrients are scarce or experimentally reduced such as during calorie restriction (CR), endotherms can reduce energy expenditure by lowering Tb [1-6]. This adaptive response conserves energy, limiting the loss of body weight due to low calorie intake [7-10]. Here we show that this response is regulated by the kappa opioid receptor (KOR). CR is associated with increased hypothalamic levels of the endogenous opioid Leu-enkephalin, which is derived from the KOR agonist precursor dynorphin [11]. Pharmacological inhibition of KOR, but not of the delta or the mu opioid receptor subtypes, fully blocked CR-induced hypothermia and increased weight loss during CR independent of calorie intake. Similar results were seen with DIO mice subjected to CR. In contrast, inhibiting KOR did not change Tb in animals fed ad libitum (AL). Chemogenetic inhibition of KOR neurons in the hypothalamic preoptic area reduced the CR-induced hypothermia, whereas chemogenetic activation of prodynorphin-expressing neurons in the arcuate or the parabrachial nucleus lowered Tb. These data indicate that KOR signaling is a pivotal regulator of energy homeostasis and can affect body weight during dieting by modulating Tb and energy expenditure.