B cell profiles, antibody repertoire and reactivity reveal dysregulated responses with autoimmune features in melanoma.

B cells are known to contribute to the anti-tumor immune response, especially in immunogenic tumors such as melanoma, yet humoral immunity has not been characterized in these cancers to detail. Here we show comprehensive phenotyping in samples of circulating and tumor-resident B cells as well as serum antibodies in melanoma patients. Memory B cells are enriched in tumors compared to blood in paired samples and feature distinct antibody repertoires, linked to specific isotypes. Tumor-associated B cells undergo clonal expansion, class switch recombination, somatic hypermutation and receptor revision. Compared with blood, tumor-associated B cells produce antibodies with proportionally higher levels of unproductive sequences and distinct complementarity determining region 3 properties. The observed features are signs of affinity maturation and polyreactivity and suggest an active and aberrant autoimmune-like reaction in the tumor microenvironment. Consistent with this, tumor-derived antibodies are polyreactive and characterized by autoantigen recognition. Serum antibodies show reactivity to antigens attributed to autoimmune diseases and cancer, and their levels are higher in patients with active disease compared to post-resection state. Our findings thus reveal B cell lineage dysregulation with distinct antibody repertoire and specificity, alongside clonally-expanded tumor-infiltrating B cells with autoimmune-like features, shaping the humoral immune response in melanoma.

Quantitation of cardiac troponin I in cancer patients treated with immune checkpoint inhibitors: a case-control study.

OBJECTIVES: Immune checkpoint inhibitors (ICIs) cause a variety of toxicities, including immune-related adverse events (irAEs), but there are no biomarkers to predict their development. Guidelines recommend measuring circulating cardiac troponin I (cTnI) during ICI therapy to detect related cardiotoxicities. Moreover, elevated cTnI has also been associated with worse outcomes in non-cardiac patients, including cancer. Thus here, we investigated whether cTnI levels were higher in patients with irAEs. METHODS: The study consisted of three groups; 21 cancer patients undergoing ICI immunotherapies who presented with irAEs, four patients without irAEs, and 20 healthy controls. Patient samples were assessed at baseline (n=25), during ICI treatment (n=25, median=6 weeks of treatment) and at toxicity (n=6, median=13 weeks of treatment). In addition to blood high sensitivity cardiac troponin I (hs-cTnI), anti-thyroglobulin (TG) and anti-thyroid peroxidase (TPO) antibodies were also quantitated to detect thyroid dysfunction, constituting the second leading toxicity (23.8%) after pneumonitis (28.6%). RESULTS: Four patients with irAEs (n=4/21; 19%) and one without irAEs (n=1/4; 25%) showed higher hs-cTnI levels at any time-point; the remaining had physiological levels. None of these patients developed cardiotoxicity. Concurrent elevated levels of anti-thyroid antibodies and hs-cTnI were detected in one patient with thyroid dysfunction (n=1/5, 20%). However, these antibodies were also elevated in three patients (n=3/16, 19%) with non-thyroid irAEs and in up to 40% of healthy controls. CONCLUSIONS: hs-cTnI was not elevated in patients with irAEs, but larger studies are needed to confirm these observations.

Colony stimulating factor-1 producing endothelial cells and mesenchymal stromal cells maintain monocytes within a perivascular bone marrow niche.

Macrophage colony stimulating factor-1 (CSF-1) plays a critical role in maintaining myeloid lineage cells. However, congenital global deficiency of CSF-1 (Csf1op/op) causes severe musculoskeletal defects that may indirectly affect hematopoiesis. Indeed, we show here that osteolineage-derived Csf1 prevented developmental abnormalities but had no effect on monopoiesis in adulthood. However, ubiquitous deletion of Csf1 conditionally in adulthood decreased monocyte survival, differentiation, and migration, independent of its effects on bone development. Bone histology revealed that monocytes reside near sinusoidal endothelial cells (ECs) and leptin receptor (Lepr)-expressing perivascular mesenchymal stromal cells (MSCs). Targeted deletion of Csf1 from sinusoidal ECs selectively reduced Ly6C- monocytes, whereas combined depletion of Csf1 from ECs and MSCs further decreased Ly6Chi cells. Moreover, EC-derived CSF-1 facilitated recovery of Ly6C- monocytes and protected mice from weight loss following induction of polymicrobial sepsis. Thus, monocytes are supported by distinct cellular sources of CSF-1 within a perivascular BM niche.

BCG vaccination provides protection against IAV but not SARS-CoV-2.

Since the vast majority of species solely rely on innate immunity for host defense, it stands to reason that a critical evolutionary trait like immunological memory evolved in this primitive branch of our immune system. There is ample evidence that vaccines such as bacillus Calmette-Guérin (BCG) induce protective innate immune memory responses (trained immunity) against heterologous pathogens. Here we show that while BCG vaccination significantly reduces morbidity and mortality against influenza A virus (IAV), it fails to provide protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In contrast to IAV, SARS-CoV-2 infection leads to unique pulmonary vasculature damage facilitating viral dissemination to other organs, including the bone marrow (BM), a central site for BCG-mediated trained immunity. Finally, monocytes from BCG-vaccinated individuals mount an efficient cytokine response to IAV infection, while this response is minimal following SARS-CoV-2. Collectively, our data suggest that the protective capacity of BCG vaccination is contingent on viral pathogenesis and tissue tropism.

Adventitial recruitment of Lyve-1- macrophages drives aortic aneurysm in an angiotensin-2-based murine model.

OBJECTIVE: Aortic macrophage accumulation is characteristic of the pathogenesis of abdominal aortic aneurysm (AAA) but the mechanisms of macrophage accumulation and their phenotype are poorly understood. Lymphatic vessel endothelial receptor-1 (Lyve-1+) resident aortic macrophages independently self-renew and are functionally distinct from monocyte-derived macrophages recruited during inflammation. We hypothesized that Lyve-1+ and Lyve-1- macrophages differentially contribute to aortic aneurysm. Approach and results: Angiotensin-2 and ß-aminopropionitrile (AT2/BAPN) were administered to induce AAA in C57BL/6J mice. Using immunohistochemistry (IHC), we demonstrated primarily adventitial accumulation of aortic macrophages, and in association with areas of elastin fragmentation and aortic dissection. Compared with controls, AAA was associated with a relative percent depletion of Lyve-1+ resident aortic macrophages and accumulation of Lyve-1- macrophages. Using CD45.1/CD45.2 parabiosis, we demonstrated aortic macrophage recruitment in AAA. Depletion of aortic macrophages in CCR2-/- mice was associated with reduced aortic dilatation indicating the functional role of recruitment from the bone marrow. Depletion of aortic macrophages using anti-macrophage colony-stimulating factor 1 receptor (MCSF1R)-neutralizing antibody (Ab) reduced the incidence of AAA. Conditional depletion of Lyve-1+ aortic macrophages was achieved by generating Lyve-1wt/cre Csf1rfl/fl mice. Selective depletion of Lyve-1+ aortic macrophages had no protective effects following AT2/BAPN administration and resulted in increased aortic dilatation in the suprarenal aorta. CONCLUSIONS: Aortic macrophage accumulation in AAA derives from adventitial recruitment of Lyve-1- macrophages, with relative percent depletion of Lyve-1+ macrophages. Selective targeting of macrophage subtypes represents a potential novel therapeutic avenue for the medical treatment of AAA.

The changes in systemic monocytes in humans undergoing surgical decompression for degenerative cervical myelopathy may influence clinical neurological recovery.

BACKGROUND: Degenerative cervical myelopathy (DCM) is the most common cause of non-traumatic spinal cord injury worldwide. Surgical decompression is recommended as the preferred treatment strategy for DCM as it halts disease progression and improves neurologic symptoms. We previously demonstrated that neuroinflammation, including monocytes, plays a critical role in the pathobiology of DCM and in ischemic-reperfusion injury (IRI) following surgical decompression. Monocytes are able to enter the spinal cord and brain tissues due to damage to the blood spinal cord and blood brain barrier following injury. Studies have demonstrated that stroke patients and individuals undergoing hip replacement surgery have increased systemic levels of monocytes. Additionally, changes in the signalling responses of monocytes are associated with post-surgical recovery or with ischemic neural tissue damage. Herein, we investigated the role of systemic monocytes as a predictive biomarker for clinical recovery following decompressive surgery for DCM. FINDINGS: There was a 2-fold increase in the number of monocytes in DCM patients at 24 h following decompression as compared to baseline levels, which was associated with a significant improvement in the modified Japanese Orthopedic Association scale (mJOA) at 6-months after surgery (p < .0001). In a mouse model of DCM, depleting acute monocytes reduced the non-classical (Ly6Clow) subset from circulation (p < .05) and resulted in a 1.8-fold increase in CD11b expression in the spinal cord at 5 weeks following decompression. Acute monocyte depletion was accompanied by a modest decline in long-term overground locomotion, as evidenced by significantly reduced hindlimb swing speed. CONCLUSIONS: This work demonstrated that decompressive surgery leads to an acute increase in peripheral monocytes in human DCM patients, which is modestly associated with clinical recovery. We anticipate that this work could contribute to the implementation of routine measurements of blood monocyte subsets, their activation state, and production of cytokines following decompressive surgery. This information could help to select perioperative anti-inflammatory treatments that can enhance the beneficial effects of decompressive surgery and reduce the incidence of post-operative complications, while avoiding a reduction in systemic monocytes.

Methylprednisolone treatment enhances early recovery following surgical decompression for degenerative cervical myelopathy without compromise to the systemic immune system.

BACKGROUND: Degenerative cervical myelopathy (DCM) is caused by degenerative or congenital changes to the discs and soft tissues of the cervical spine, which leads to chronic compression of the spinal cord. The current treatment for moderate to severe DCM consists of surgical decompression, which, while effective in most cases, can result in neuroinflammation and spinal cord reperfusion injury, leading to perioperative neurological complications and suboptimal neurological recovery. The primary objective of this study was to assess, in a translationally relevant animal model of DCM, the efficacy of perioperative methylprednisolone (MP) in enhancing neurological recovery and to evaluate its effect on the inflammatory response following decompression. METHODS: DCM was induced in C57BL/6 mice. Briefly, an aromatic polyether material was implanted underneath the C5-C6 laminae to cause progressive compression of the cervical spinal cord due to focal ossification. Decompressive surgery was undertaken at 12 weeks post initial biomaterial implantation. Animals received one dose of MP (30 mg/kg) or vehicle 30 min before decompression and at 2 weeks after decompression. Acute analysis of secreted cytokines and spinal cord microvasculature was complemented with immunohistochemistry for glial and neuronal cell markers. Locomotor outcomes were measured using the CatWalk system. The composition of circulating white blood cells was analyzed by flow cytometry. RESULTS: A single dose of MP before decompression significantly sped locomotor recovery (*p < 0.05) and reduced the incidence of perioperative motor complications, without affecting the composition of circulating white blood cells. Histological assessment of the spinal cord showed significant neuronal preservation and a modest reduction in parenchymal inflammation. CONCLUSIONS: Our data suggest that MP reduces perioperative neurological complications following decompressive surgery for DCM by protecting neurons from inflammation, without compromising the composition of circulating immune cells. We propose that MP, which is commonly used for neurological disorders including spinal cord injury, be considered as a perioperative adjunct to decompressive surgery to attenuate neurological complications.

Promising neuroprotective strategies for traumatic spinal cord injury with a focus on the differential effects among anatomical levels of injury.

Traumatic spinal cord injury (SCI) is a devastating condition of motor, sensory, and autonomic dysfunction. The significant cost associated with the management and lifetime care of patients with SCI also presents a major economic burden. For these reasons, there is a need to develop and translate strategies that can improve outcomes following SCI. Given the challenges in achieving regeneration of the injured spinal cord, neuroprotection has been at the forefront of clinical translation. Yet, despite many preclinical advances, there has been limited translation into the clinic apart from methylprednisolone (which remains controversial), hypertensive therapy to maintain spinal cord perfusion, and early decompressive surgery. While there are several factors related to the limited translational success, including the clinical and mechanistic heterogeneity of human SCI, the misalignment between animal models of SCI and clinical reality continues to be an important factor. Whereas most clinical cases are at the cervical level, only a small fraction of preclinical research is conducted in cervical models of SCI. Therefore, this review highlights the most promising neuroprotective and neural reparative therapeutic strategies undergoing clinical assessment, including riluzole, hypothermia, granulocyte colony-stimulating factor, glibenclamide, minocycline, Cethrin (VX-210), and anti-Nogo-A antibody, and emphasizes their efficacy in relation to the anatomical level of injury. Our hope is that more basic research will be conducted in clinically relevant cervical SCI models in order to expedite the transition of important laboratory discoveries into meaningful treatment options for patients with SCI.

Delayed decompression exacerbates ischemia-reperfusion injury in cervical compressive myelopathy.

Degenerative cervical myelopathy (DCM) is the most common progressive nontraumatic spinal cord injury. The most common recommended treatment is surgical decompression, although the optimal timing of intervention is an area of ongoing debate. The primary objective of this study was to assess whether a delay in decompression could influence the extent of ischemia-reperfusion injury and alter the trajectory of outcome in DCM. Using a DCM mouse model, we show that decompression acutely led to a 1.5- to 2-fold increase in levels of inflammatory cytokines within the spinal cord. Delayed decompression was associated with exacerbated reperfusion injury, astrogliosis, and poorer neurological recovery. Additionally, delayed decompression was associated with prolonged elevation of inflammatory cytokines and an exacerbated peripheral monocytic inflammatory response (P < 0.01 and 0.001). In contrast, early decompression led to resolution of reperfusion-mediated inflammation, neurological improvement, and reduced hyperalgesia. Similar findings were observed in subjects from the CSM AOSpine North America and International studies, where delayed decompressive surgery resulted in poorer neurological improvement compared with patients with an earlier intervention. Our data demonstrate that delayed surgical decompression for DCM exacerbates reperfusion injury and is associated with ongoing enhanced levels of cytokine expression, microglia activation, and astrogliosis, and paralleled with poorer neurological recovery.