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The CD8+ T-cell response is prognostic for survival outcomes in several tumor types. However, whether this extends to tumors in the brain, an organ with barriers to T cell entry, remains unclear. Here, we analyzed immune infiltration in 67 brain metastasis (BrM) and found high frequencies of PD1+ TCF1+ stem-like CD8+ T-cells and TCF1- effector-like cells. Importantly, the stem-like cells aggregate with antigen presenting cells in immune niches, and niches were prognostic for local disease control. Standard of care for BrM is resection followed by stereotactic radiosurgery (SRS), so to determine SRS’s impact on the BrM immune response, we examined 76 BrM treated with pre-operative SRS (pSRS). pSRS acutely reduced CD8+ T cells at 3 days. However, CD8+ T cells rebounded by day 6, driven by increased frequency of effector-like cells. This suggests that the immune response in BrM can be regenerated rapidly, likely by the local TCF1+ stem-like population.

Background: Non-small cell lung cancer (NSCLC) patients with brain metastases have a very poor prognosis. The first line treatments for NSCLC brain metastases are typically surgery and pre- or post-operative stereotactic radiosurgery (SRS) or SRS alone. However, subsequent intra-cranial failure and death is common. Importantly, we have previously shown that a unique immune niche in genitourinary malignancies correlates with the risk for disease recurrence. Accordingly, here we investigated whether this immune niche was present in brain metastases, whether it correlated with patient outcomes, and whether it was modulated by SRS. Methods: Formalin fixed paraffin embedded brain metastases were stained by immunofluorescence for CD8, PD-1, TCF-1, MHC-II and DAPI. Following successful image acquisition using fluorescent whole slide scanning, we used a custom quantitative analysis pipeline to measure the cellularity, relative cellular locations, local cellular density, and marker staining intensity. Results: We identified immune niches consisting of both stem-like TCF-1+ CD8+ T-cells and antigen presenting cells (MHC-II+) in brain metastases. TCF-1+ CD8+ T-cells were found at in areas of higher MHC-II+ cell density than the TCF-1 CD8+negative T-cells. The amount of tumor issue occupied by immune niches correlated with TCF1+ CD8+ T-cell infiltration, as well as with total CD8+ T-cell and MHC-II+ cell infiltration. Importantly, we found that higher MHC-II+ cell density correlated with longer overall survival in this cohort of patients. Additionally, we found this immune niche is preserved following pre-operative SRS. Conclusions: Our study is the first to demonstrate stem-like CD8+ T-cells in brain metastases and that these cells form an immune niche similar to those described extra-cranial tumor sites. Additionally, we report that the immune niche is preserved following SRS, which is highly clinically relevant to the use of combinatorial strategies between SRS and checkpoint blockade. Citation Format: Caroline S. Jansen, Luke del Balzo, Roshan Prabhu, Suzanna Logan, Prasanthi Chappa, Kirtesh Patel, Scott Wilkinson, Ross Lake, Hui-Kuo G. Shu, Jim Zhong, Vishal Dhere, Jeffrey Olson, Adam G. Sowalsky, Mohammad K. Khan, Haydn T. Kissick, Zachary S. Buchwald. An immunologic niche of antigen presenting cells and stem-like CD8+ T-cells is present in non-small cell lung cancer brain metastases [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-041.

The prevention and treatment of acute chest syndrome (ACS) is a major clinical concern in sickle cell disease (SCD). However, the mechanism underlying the pathogenesis of ACS remains elusive. We tested the hypothesis that the hemolysis byproduct hemin elicits events that induce ACS. Infusion of a low dose of hemin caused acute intravascular hemolysis and autoamplification of extracellular hemin in transgenic sickle mice, but not in sickle-trait littermates. The sickle mice developed multiple symptoms typical of ACS and succumbed rapidly. Pharmacologic inhibition of TLR4 and hemopexin replacement therapy prior to hemin infusion protected sickle mice from developing ACS. Replication of the ACS-like phenotype in nonsickle mice revealed that the mechanism of lung injury due to extracellular hemin is independent of SCD. Using genetic and bone marrow chimeric tools, we confirmed that TLR4 expressed in nonhematopoietic vascular tissues mediated this lethal type of acute lung injury. Respiratory failure was averted after the onset of ACS-like symptoms in sickle mice by treating them with recombinant hemopexin. Our results reveal a mechanism that helps to explain the pathogenesis of ACS, and we provide proof of principle for therapeutic strategies to prevent and treat this condition in mice.

BACKGROUND: Increased rates of red blood cell (RBC) alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease. STUDY DESIGN AND METHODS: Transgenic sickle mice, which express human α and βS globin, were transfused with fresh or 14-day-stored RBCs containing the HOD (hen egg lysozyme, ovalbumin, and human Duffyb) antigen; some recipients were inflamed with poly(I : C) before transfusion. Anti-HOD alloantibody responses were subsequently measured by enzyme-linked immunosorbent assay and flow crossmatch; a cohort of recipients had posttransfusion serum cytokines measured by bead array. RESULTS: Both Berkeley and Townes homozygous (SS) and heterozygous (AS) mice had similar rates and magnitude of anti-HOD RBC alloimmunization after fresh HOD RBC transfusion compared with control animals; under no tested condition did homozygous SS recipients make higher levels of alloantibodies than control animals. Unexpectedly, homozygous SS recipients had blunted cytokine responses and lower levels of anti-HOD alloantibodies after transfusion of 14-day stored RBCs, compared with control animals. CONCLUSIONS: In sum, homozygous βS expression and the ensuing disease state are not alone sufficient to enhance RBC alloimmunization to transfused HOD RBCs in two distinct humanized murine models of sickle cell disease under the conditions examined. These data suggest that other factors may contribute to the high rates of RBC alloimmunization observed in humans with sickle cell disease.

Key pathophysiology of sickle cell anaemia includes compensatory erythropoiesis, vascular injury and chronic inflammation, which divert amino acids from tissue deposition for growth/weight gain and muscle formation. We hypothesised that sickle mice maintained on an isoenergetic diet with a high percentage of energy derived from protein (35 %), as opposed to a standard diet with 20 % of energy derived from protein, would improve body composition, bone mass and grip strength. Male Berkeley transgenic sickle mice (S;n8–12) were fed either 20 % (S20) or 35 % (S35) diets for 3 months. Grip strength (BIOSEB meter) and body composition (dual-energy X-ray absorptiometry scan) were measured. After 3 months, control mice had the highest bone mineral density (BMD) and bone mineral content (BMC) (P P = 0·043) attributed this difference to genotype (P = 0·025) and a trend in type of diet (P = 0·067).l-Arginine (l-Arg) supplementation of the 20 % diet was explored, as a possible mechanism for improvement obtained with the 35 % diet. Townes transgenic sickle mice (TS;n6–9) received 0·8, 1·6, 3·2 or 6·4 %l-Arg based on the same protocol and outcome measures used for the S mice. TS mice fed 1·6 %l-Arg for 3 months (TS1.6) had the highest weight gain, BMD, BMC and lean body mass compared with other groups. TS3.2 mice showed significantly more improvement in grip strength than TS0·8 and TS1.6 mice (P l-Arg as part of the mechanism.

Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. Despite aggressive therapy with surgery, radiotherapy, and chemotherapy, nearly all patients succumb to disease within 2 years. Several studies have supported the presence of stem-like cells in brain tumor cultures that are CD133-positive, are capable of self-renewal, and give rise to all cell types found within the tumor, potentially perpetuating growth. CD133 is a widely accepted marker for glioma-derived cancer stem cells; however, its reliability has been questioned, creating a need for other identifiers of this biologically important subpopulation. We used a panel of 20 lectins to identify differences in glycan expression found in the glycocalyx of undifferentiated glioma-derived stem cells and differentiated cells that arise from them. Fluorescently labeled lectins that specifically recognize α-N-acetylgalactosamine (GalNAc) and α-N-acetylglucosamine (GlcNAc) differentially bound to the cell surface based on the state of cellular differentiation. GalNAc and GlcNAc were highly expressed on the surface of undifferentiated cells and showed markedly reduced expression over a 12-day duration of differentiation. Additionally, the GalNAc-recognizing lectin Dolichos biflorus agglutinin was capable of specifically selecting and sorting glioma-derived stem cell populations from an unsorted tumor stock and this subpopulation had proliferative properties similar to CD133(+) cells in vitro and also had tumor-forming capability in vivo. Our preliminary results on a single cerebellar GBM suggest that GalNAc and GlcNAc are novel biomarkers for identifying glioma-derived stem cells and can be used to isolate cancer stem cells from unsorted cell populations, thereby creating new cell lines for research or clinical testing.

Increased rates of red blood cell (RBC) alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease.Transgenic sickle mice, which express human α and β(S) globin, were transfused with fresh or 14-day-stored RBCs containing the HOD (hen egg lysozyme, ovalbumin, and human Duffy(b) ) antigen; some recipients were inflamed with poly(I : C) before transfusion. Anti-HOD alloantibody responses were subsequently measured by enzyme-linked immunosorbent assay and flow crossmatch; a cohort of recipients had posttransfusion serum cytokines measured by bead array.Both Berkeley and Townes homozygous (SS) and heterozygous (AS) mice had similar rates and magnitude of anti-HOD RBC alloimmunization after fresh HOD RBC transfusion compared with control animals; under no tested condition did homozygous SS recipients make higher levels of alloantibodies than control animals. Unexpectedly, homozygous SS recipients had blunted cytokine responses and lower levels of anti-HOD alloantibodies after transfusion of 14-day stored RBCs, compared with control animals.In sum, homozygous β(S) expression and the ensuing disease state are not alone sufficient to enhance RBC alloimmunization to transfused HOD RBCs in two distinct humanized murine models of sickle cell disease under the conditions examined. These data suggest that other factors may contribute to the high rates of RBC alloimmunization observed in humans with sickle cell disease.

Pan-histone deacetylase inhibitors, for example, vorinostat and panobinostat (LBH589; Novartis Pharmaceuticals, East Hanover, NJ), have shown clinical efficacy against advanced cutaneous T-cell lymphoma (CTCL). However, the molecular basis of this activity remains unclear. HDAC7, a class IIA histone deacetylase (HDAC), is overexpressed in thymocytes, where it represses expression of the proapoptotic nuclear orphan receptor Nur77. Here, we demonstrate that treatment with panobinostat rapidly inhibits the in vitro and intracellular activity, as well as the mRNA and protein levels of HDAC7, and induces expression and translocation of Nur77 to the mitochondria. There, Nur77 converts death resistance protein Bcl-2 into a killer protein, promoting cell death of cultured and patient-derived human CTCL cells. Treatment with panobinostat improved survival of athymic nude mice implanted with human CTCL cells. Ectopic expression of Nur77 induced apoptosis and sensitized HH cells to panobinostat, whereas combined knockdown of Nur77 and its family member Nor1 was necessary to inhibit panobinostat-induced apoptosis of CTCL cells. Cotreatment with the Bcl-2/Bcl-xL antagonist ABT-737 decreased resistance and synergistically induced apoptosis of human CTCL cells. These findings mechanistically implicate HDAC7 and Nur77 in sensitizing human CTCL cells to panobinostat as well as suggest that cotreatment with an anti–Bcl-2 agent would augment the anti-CTCL activity of panobinostat.

Acute chest syndrome (ACS) is a major cause of morbidity and mortality in sickle cell disease (SCD). The diagnosis, prevention and treatment of ACS pose major clinical concerns in SCD partly because the mechanism underlying the pathogenesis of this syndrome remains elusive. Our group first reported that excess intravascular hemin causes a lethal acute lung injury (ALI) in transgenic SCD mice reminiscent of ACS (Ghosh and Ofori-Acquah, Blood 116 Suppl 1:944, 2010). Subsequently, large-scale genomics studies by Bean et al., (Blood 120:3822-8, 2012) and Galarneau et al., (Blood, 122:434-42, 2013) have implicated hemin catabolism and inflammation in the pathogenesis of ACS. In addition, we have reported recently that raised plasma free hemin increases the odds of ACS in children with SCD (Adisa et al., Br J Haematol. 2013). Collectively, these studies support a new theme of ACS pathogenesis involving extracellular hemin. In the current study, we validated the respiratory dysfunction of this ACS model, tested the hypothesis that toll-like receptor 4 (TLR4) mediates the associated lung injury, and examined the efficacy of two strategies to treat the condition in mice. Arterial blood gas analysis of SS mice with the ACS-like disease confirmed severe hypoxemia (PaO2; 40.23 ±3.85 mmHg, SO2; 58.72±6.6%, p Disclosures: No relevant conflicts of interest to declare.

Abstract 897 Sickle cell disease is a complex process involving biophysical and biological phenomenon such as microvascular occlusion due to rigid sickle erythrocytes, hemolysis, and aberrant cellular interactions involving endothelial cells and sickle erythrocytes and leukocytes. Indeed, a key aspect of sickle cell pathophysiology is endothelial cell dysfunction. Cardiovascular research in recent years has shown that endothelial cells biologically respond to the local mechanical environment, particularly to the changes in the applied shear stresses (Chiu and Chien, Physiological Reviews, 2011). Interestingly, no studies investigating how the biophysical alterations in sickle cell disease may directly affect endothelial function have been published. The classic view has been that vaso-occlusion is simply due to sickled erythrocytes becoming stuck in microvasculature at low oxygen tensions leading to decreased blood flow and tissue ischemia. However, the mechanical aspects of sickle cell vaso-occlusion themselves, that is, the physical phenomenon of sickling erythrocytes tightly packed in an occluded blood vessel, may directly affect endothelial biology and lead to dysfunction. We hypothesize that these pathologic forces induced by sickling erythrocytes directly lead to dysfunction of endothelial cells, which are mechanosensitive, and contribute to sickle cell pathophysiology. However, these sickling-induced forces and their effects on endothelial cells have been difficult to measure, in part due to a lack of available tools. To that end, we have developed two microfluidic tools to assess the role of sickle-cell vaso-occlusion on endothelial cells. The first device is an in vitro microfluidic platform featuring microchannels the size of post-capillary venules (30 μm) with human endothelial cells cultured within and completely lining the entire inner surface of those microchannels (Figure 1A). This “microvasculature-on-a-chip” enables the visualization of blood cell-endothelial cell interactions during vaso-occlusion under a controlled hemodynamic environment and provides a platform to study the effect of vaso-occlusion on endothelial cells. To date we have characterized this “endothelialized” microfluidic device, showing that endothelial cells are confluent using anti-VE-cadherin immunostaining and adequately generate nitric oxide. Furthermore, we have flowed blood samples from patients with sickle cell disease and found that hydroxyurea treatment both reduces the number of occlusions and increases the mean velocity of the blood traveling through the device, as expected (Figure 1B–E). To decouple whether it is a biochemical or biophysical phenomenon that causes endothelial cell dysfunction during vaso-occlusion, a second micromechanical device was created to quantitatively measure the forces generated by sickling events. The device captures whole blood and will deform outward when forces are applied by the sickle erythrocytes as shown in Figure 2. The membrane above the sickle cells has been coated with 2 μm fluorescent beads which will change focus during deflection. Deflections of one or two beads indicates that a single sickle cell is locally applying force, whereas deflections of large numbers of beads indicates that the cells are collectively applying a pressure to the membrane. The device has been fully fabricated and loaded with blood cells. An accompanying experimental setup enabling the deoxygenation of the device coupled with microscopy has also been created and preliminary tests show successful deoxygenation of sickle erythrocytes from patients with hemoglobin SS disease and the Berkeley sickle cell mouse model. By combining insights gained from each device, future work will determine how the mechanical process of sickling and vaso-occlusion directly affect endothelial function and will lead to a new understanding of sickle cell pathophysiology. Sickle cell vaso-occlusion will be induced in the “endothelialized” microfluidic device while monitoring nitric oxide production and the upregulation of inflammatory markers, such as adhesion molecules and free radicals. The second device will provide quantitative numbers of forces produced by sickling erythrocytes, leading to experiments in which these forces are applied to endothelial cells while monitoring the same metrics. Disclosures: No relevant conflicts of interest to declare.

Abstract 4836 Sickle cell disease (SCD) is a chronic hemolytic and inflammatory disorder characterized by repeated episodes of vaso-occlusion and hemolysis, resulting in oxidative stress and endothelial dysfunction. We have recently demonstrated that the heme scavenging capacity in SCD is severely impaired, highlighting the danger posed by excess heme in this disorder. Paradoxically, heme induces expression of several cyto-protective enzymes including the modifier subunit of glutamate cysteine ligase (GCLM), the rate-limiting enzyme in glutathione (GSH) synthesis, which is a crucial antioxidant in the lung. While the induction of cytoprotective enzymes is thought to attenuate the deleterious effects of heme in SCD the somatic origin of this protection has not previously been defined. Using transgenic mouse models we show for the first time that the level of GCLM in the sickle lung is markedly up-regulated due primarily to enhanced expression of the enzyme in the epithelium and blood mononuclear cells, but not in the endothelium. Based on these findings, we tested the hypothesis that leukocyte-derived GCLM was sufficient to protect the sickle lung from oxidative stress. Thus, bone marrow chimeric SCD mice with GCLM deficiency were generated by transplanting bone marrow from Berkeley SCD transgenic mice into GCLM null mice recipients. We confirmed that the chimeric GCLM-null-SCD mice had a SCD phenotype as determined by >95% engraftment of donor white blood cells, reticulocyte counts, urine osmolality and hemoglobin gel electrophoresis. Whole lung GCLM and total GSH levels in the chimeric mice were identical to the levels in the wild-type SCD mice. Moreover, lung function, as determined by oxygen saturation and breath rate, were identical in the two mouse strains. These results show that loss of GCLM expression in resident lung cells does not compromise production of GSH or the function of the lung in SCD. Disclosures: Ofori-Acquah: Emory University: Patents & Royalties.