Supplementary Figure S2. BALB/c mice were inoculated with TUBO 3d before adoptive serum transfer of 25 µg equivalent α-neu Ab from mice previously treated with cryo + CpG or pNeuE2 vaccination, naïve control serum, or 25 µg monoclonal Ab4 (n=3-5) as a single tail vein injection in a total volume of 200 µL with PBS. TUBO tumor volume 30 days after inoculation. **P<0.01

Supplementary Figure Legend from Classification of Chemotherapeutic Agents Based on Their Differential In vitro Effects on Dendritic Cells

Supplementary Figure 1 from Classification of Chemotherapeutic Agents Based on Their Differential <i>In vitro</i> Effects on Dendritic Cells

Supplementary Figure S1. (A) Untreated TUBO - tumor volume (mm3) and α-neu IgG (Ab4 equivalent μg/mL) are plotted on the left and right y-axis respectively. (B) Experimental Scheme - BALB/c females were vaccinated with pNeuE2 on day 0 and 2 (n= 4). Data representative of three independent experiments.

Supplementary Table 1, Figure Legends 1-2 from Dual Therapeutic Efficacy of Vinblastine as a Unique Chemotherapeutic Agent Capable of Inducing Dendritic Cell Maturation

Supplementary Figure 2 from Dual Therapeutic Efficacy of Vinblastine as a Unique Chemotherapeutic Agent Capable of Inducing Dendritic Cell Maturation

Supplementary Table 1 from Classification of Chemotherapeutic Agents Based on Their Differential <i>In vitro</i> Effects on Dendritic Cells

Supplementary Figure S3. (A) Experimental scheme. Sera and tumor measurements were collected from BALB/c mice on days 0, 8, 17, 24, and 30 (n= 9). (B) α-Her2 IgG levels were quantified from sera and correlated with tumor volume. Mean D2F2/E2 volume (mm3) and α-Her2 IgG (Ab5 equivalent μg/mL) are plotted on the left and right y-axis respectively. (C) Three mice were euthanized at day 17 to collect spleens for IFN-γ ELISPOT. Splenocytes were isolated and stimulated with either EKB or KB 3T3 lines for 48hr (n=3/group). **P<0.01 Unpaired t-test.

Supplementary Movie 1 from Dual Therapeutic Efficacy of Vinblastine as a Unique Chemotherapeutic Agent Capable of Inducing Dendritic Cell Maturation

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal — used as a measure of priming 2) in vivo neutrophil labeling — achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Rapid enhancement of phagocyte functionality is a hallmark of neutrophil priming. GeneChip analyses unveiled elevated CD54, dectin-2, and IL-1β mRNA expression by neutrophils isolated from inflammatory sites. In fact, CD54 and dectin-2 protein expression was detected on neutrophils recovered from skin, peritoneal, and lung inflammation lesions but not on those in bone marrow or peripheral blood. Neutrophils increased CD54 and dectin-2 mRNA during migration in Boyden chambers and acquired CD54 and dectin-2 surface expression after subsequent exposure to GM-CSF. Neutrophils purified from IL-1β promoter-driven DsRed-transgenic mice acquired DsRed signals during cell migration or exposure to GM-CSF. CD54 and dectin-2 were expressed by DsRed(+) (but not DsRed(-)) neutrophils in GM-CSF-supplemented cultures, and neutrophils recovered from inflammatory sites exhibited strong DsRed signals. The dynamic process of neutrophil priming was studied in chemically induced inflammatory skin lesions by monitoring DsRed expression using confocal microscopy. A majority (>80%) of Ly6G(+) neutrophils expressed DsRed, and those DsRed(+)/Ly6G(+) cells exhibited crawling motion with a higher velocity compared with their DsRed(-)/Ly6G(+) counterparts. This report unveils motile behaviors of primed neutrophils in living animals. We propose that neutrophil priming occurs in a sequential manner with rapid enhancement of phagocyte functionality, followed by CD54 and dectin-2 mRNA and protein expression, IL-1β promoter activation, and accelerated motility. Not only do these findings provide a new conceptual framework for our understanding of the process of neutrophil priming, they also unveil new insights into the pathophysiology of many inflammatory disorders that are characterized by neutrophil infiltration.

Accumulating lines of evidence now demonstrate that neutrophils can participate in adaptive immune responses directly or indirectly. Even more striking is their potential to acquire phenotypic and functional properties that are typically reserved for professional APCs. These newly emerging concepts of neutrophil heterogeneity and plasticity now challenge the classic view of neutrophils as terminally differentiated leukocytes fully committed to phagocyte functionality. Here, we present a brief overview of our current understanding of neutrophil plasticity by focusing on the acquisition of DC-like properties in culture and at sites of inflammation. Human and murine neutrophils acquire surface expression of MHC II, costimulatory molecules, and other surface markers of DCs when cultured in the presence of selected cytokines. The resulting populations also exhibit potent APC activities to present various antigens to T cells. "Unusual" neutrophils expressing DC markers have been detected in inflammatory lesions in human patients and mouse disease models. These findings imply that acquisition of DC-like properties by neutrophils at the sites of inflammation may represent a key process for linking the innate and adaptive arms of immune responses.

Spinal cord injury (SCI) causes the release of danger signals by stressed and dying cells, a process that leads to neuroinflammation. Evidence suggests that inflammation plays a role in both the damage and repair of injured neural tissue. We show that microglia at sites of SCI rapidly express the alarmin interleukin (IL)-1α, and that infiltrating neutrophils and macrophages subsequently produce IL-1β. Infiltration of these cells is dramatically reduced in both IL-1α−/− and IL-1β−/− mice, but only IL-1α−/− mice showed rapid (at day 1) and persistent improvements in locomotion associated with reduced lesion volume. Similarly, intrathecal administration of the IL-1 receptor antagonist anakinra restored locomotor function post-SCI. Transcriptome analysis of SCI tissue at day 1 identified the survival factor Tox3 as being differentially regulated exclusively in IL-1α−/− mice compared with IL-1β−/− and wild-type mice. Accordingly, IL-1α−/− mice have markedly increased Tox3 levels in their oligodendrocytes, beginning at postnatal day 10 (P10) and persisting through adulthood. At P10, the spinal cord of IL-1α−/− mice showed a transient increase in mature oligodendrocyte numbers, coinciding with increased IL-1α expression in wild-type animals. In adult mice, IL-1α deletion is accompanied by increased oligodendrocyte survival after SCI. TOX3 overexpression in human oligodendrocytes reduced cellular death under conditions mimicking SCI. These results suggest that IL-1α-mediated Tox3 suppression during the early phase of CNS insult plays a crucial role in secondary degeneration. SIGNIFICANCE STATEMENT The mechanisms underlying bystander degeneration of neurons and oligodendrocytes after CNS injury are ill defined. We show that microglia at sites of spinal cord injury (SCI) rapidly produce the danger signal interleukin (IL)-1α, which triggers neuroinflammation and locomotor defects. We uncovered that IL-1α−/− mice have markedly increased levels of the survival factor Tox3 in their oligodendrocytes, which correlates with the protection of this cell population, and reduced lesion volume, resulting in unprecedented speed, level, and persistence of functional recovery after SCI. Our data suggest that central inhibition of IL-1α or Tox3 overexpression during the acute phase of a CNS insult may be an effective means for preventing the loss of neurological function in SCI, or other acute injuries such as ischemia and traumatic brain injuries.

AbstractPercutaneous cryoablation is a minimally invasive procedure for tumor destruction, which can potentially initiate or amplify antitumor immunity through the release of tumor-associated antigens. However, clinically efficacious immunity is lacking and regional recurrences are a limiting factor relative to surgical excision. To understand the mechanism of immune activation by cryoablation, comprehensive analyses of innate immunity and HER2/neu humoral and cellular immunity following cryoablation with or without peritumoral CpG injection were conducted using two HER2/neu+ tumor systems in wild-type (WT), neu-tolerant, and SCID mice. Cryoablation of neu+ TUBO tumor in BALB/c mice resulted in systemic immune priming, but not in neu-tolerant BALB NeuT mice. Cryoablation of human HER2+ D2F2/E2 tumor enabled the functionality of tumor-induced immunity, but secondary tumors were refractory to antitumor immunity if rechallenge occurred during the resolution phase of the cryoablated tumor. A step-wise increase in local recurrence was observed in WT, neu-tolerant, and SCID mice, indicating a role of adaptive immunity in controlling residual tumor foci. Importantly, local recurrences were eliminated or greatly reduced in WT, neu tolerant, and SCID mice when CpG was incorporated in the cryoablation regimen, showing significant local control by innate immunity. For long-term protection, however, adaptive immunity was required because most SCID mice eventually succumbed to local tumor recurrence even with combined cryoablation and CpG treatment. This improved understanding of the mechanisms by which cryoablation affects innate and adaptive immunity will help guide appropriate combination of therapeutic interventions to improve treatment outcomes. Cancer Res; 74(19); 5409–20. ©2014 AACR.

Neutrophils contribute to innate host immunity by functioning as professional phagocytes, whereas dendritic cells (DCs) are prototypic antigen presenting cells (APCs) responsible for the induction of adaptive immune responses. We have demonstrated recently that neutrophils trans-differentiate into a unique population, termed “neutrophil-DC hybrids,” expressing surface markers of both neutrophils and DCs and exhibiting dual functionality of both phagocytes and APCs. Although the hybrid cells emerged in significant numbers in murine bone marrow (BM) culture in the presence of GM-CSF, mechanisms regulating their development remained mostly unknown. In this study, we tested a total of 61 cytokines for their potentials to regulate neutrophil-DC hybrid formation using a newly developed BM micro-culture system combined with semi-automated FACS analysis. Several cytokines including GM-CSF were found to promote the generation of neutrophil-DC hybrids defined by the phenotype of CD11c+/MHC II+/Ly6G+. When tested in the presence of GM-CSF, hybrid cell development was enhanced by IL-4 and suppressed by interferon-γ (IFNγ) in dose-dependent fashions. We next determined in vivo impacts of IL-4 and IFNγ on the development of neutrophil-DC hybrids in thioglycollate-induced peritonitis lesions. Intraperitoneal administrations of IL-4/anti-IL-4 antibody complex (IL-4C) significantly increased the number of hybrids recovered from the lesions. By contract, recovery of hybrids was reduced by recombinant IFNγ. With regard to function, those hybrid cells recovered from IL-4C-treated mice and IFNγ-treated mice showed potent abilities to capture E.coli. These observations imply that emergence of neutrophil-DC hybrids in inflammatory sites is tightly regulated by local cytokine milieus.

In this issue of Blood, Janelsins et al report that substance P (SP)–treated dendritic cells (DCs) efficiently home to lymph nodes, where they induce inflammatory DCs to produce interleukin-12 (IL-12), thereby promoting type 1 polarized immunity.