Sustained Club Cell Injury in Mice Induces Histopathologic Features of Deployment-Related Constrictive Bronchiolitis.

Histopathologic evidence of deployment-related constrictive bronchiolitis (DRCB) has been identified in soldiers deployed to Southwest Asia. While inhalational injury to the airway epithelium is suspected, relatively little is known about the pathogenesis underlying this disabling disorder. Club cells are local progenitors critical for repairing the airway epithelium after exposure to various airborne toxins, and a prior study using an inducible transgenic murine model reported that 10 days of sustained targeted club cell injury causes constrictive bronchiolitis. To further understand the mechanisms leading to small airway fibrosis, a murine model was employed to show that sustained club cell injury elicited acute weight loss, caused increased local production of proinflammatory cytokines, and promoted accumulation of numerous myeloid cell subsets in the lung. Transition to a chronic phase was characterized by up-regulated expression of oxidative stress-associated genes, increased activation of transforming growth factor-ß, accumulation of alternatively activated macrophages, and enhanced peribronchiolar collagen deposition. Comparative histopathologic analysis demonstrated that sustained club cell injury was sufficient to induce epithelial metaplasia, airway wall thickening, peribronchiolar infiltrates, and clusters of intraluminal airway macrophages that recapitulated key abnormalities observed in DRCB. Depletion of alveolar macrophages in mice decreased activation of transforming growth factor-ß and ameliorated constrictive bronchiolitis. Collectively, these findings implicate sustained club cell injury in the development of DRCB and delineate pathways that may yield biomarkers and treatment targets for this disorder.

CD36/Lyn kinase interactions within macrophages promotes pulmonary fibrosis in response to oxidized phospholipid.

Recent data from human studies and animal models have established roles for type II alveolar epithelial cell (AEC2) injury/apoptosis and monocyte/macrophage accumulation and activation in progressive lung fibrosis. Although the link between these processes is not well defined, we have previously shown that CD36-mediated uptake of apoptotic AEC2s by lung macrophages is sufficient to drive fibrosis. Importantly, apoptotic AEC2s are rich in oxidized phospholipids (oxPL), and amongst its multiple functions, CD36 serves as a scavenger receptor for oxPL. Recent studies have established a role for oxPLs in alveolar scarring, and we hypothesized that uptake and accrual of oxPL by CD36 would cause a macrophage phenotypic change that promotes fibrosis. To test this hypothesis, we treated wild-type and CD36-null mice with the oxPL derivative oxidized phosphocholine (POVPC) and found that CD36-null mice were protected from oxPL-induced scarring. Compared to WT mice, fewer macrophages accumulated in the lungs of CD36-null animals, and the macrophages exhibited a decreased accumulation of intracellular oxidized lipid. Importantly, the attenuated accrual of oxPL in CD36-null macrophages was associated with diminished expression of the profibrotic mediator, TGFß. Finally, the pathway linking oxPL uptake and TGFß expression was found to require CD36-mediated activation of Lyn kinase. Together, these observations elucidate a causal pathway that connects AEC2 injury with lung macrophage activation via CD36-mediated uptake of oxPL and suggest several potential therapeutic targets.

Diverse Injury Pathways Induce Alveolar Epithelial Cell CCL2/12, Which Promotes Lung Fibrosis.

Accumulating evidence suggests that fibrosis is a multicellular process with contributions from alveolar epithelial cells (AECs), recruited monocytes/macrophages, and fibroblasts. We have previously shown that AEC injury is sufficient to induce fibrosis, but the precise mechanism remains unclear. Several cell types, including AECs, can produce CCL2 and CCL12, which can promote fibrosis through CCR2 activation. CCR2 signaling is critical for the initiation and progression of pulmonary fibrosis, in part through recruitment of profibrotic bone marrow-derived monocytes. Attempts at inhibiting CCL2 in patients with fibrosis demonstrated a marked upregulation of CCL2 production and no therapeutic response. To better understand the mechanisms involved in CCL2/CCR2 signaling, we generated mice with conditional deletion of CCL12, a murine homolog of human CCL2. Surprisingly, we found that mice with complete deletion of CCL12 had markedly increased concentrations of other CCR2 ligands and were not protected from fibrosis after bleomycin injury. In contrast, mice with lung epithelial cell-specific deletion of CCL12 were protected from bleomycin-induced fibrosis and had expression of CCL2 and CCL7 similar to that of control mice treated with bleomycin. Deletion of CCL12 within AECs led to decreased recruitment of exudate macrophages. Finally, injury to murine and human primary AECs resulted in increased production of CCL2 and CCL12, in part through activation of the mTOR pathway. In conclusion, these data suggest that targeting CCL2 may be a viable antifibrotic strategy once the pathways involved in the production and function of CCL2 and other CCR2 ligands are better defined.

Autocrine IL-10 Signaling Promotes Dendritic Cell Type-2 Activation and Persistence of Murine Cryptococcal Lung Infection.

The substantial morbidity and mortality caused by invasive fungal pathogens, including Cryptococcus neoformans, necessitates increased understanding of protective immune responses against these infections. Our previous work using murine models of cryptococcal lung infection demonstrated that dendritic cells (DCs) orchestrate critical transitions from innate to adaptive immunity and that IL-10 signaling blockade improves fungal clearance. To further understand interrelationships among IL-10 production, fungal clearance, and the effect of IL-10 on lung DCs, we performed a comparative temporal analysis of cryptococcal lung infection in wild type C57BL/6J mice (designated IL-10+/+) and IL-10-/- mice inoculated intratracheally with C. neoformans (strain 52D). Early and sustained IL-10 production by lung leukocytes was associated with persistent infection in IL-10+/+ mice, whereas fungal clearance was improved in IL-10-/- mice during the late adaptive phase of infection. Numbers of monocyte-derived DCs, T cells, and alveolar and exudate macrophages were increased in lungs of IL-10-/- versus IL-10+/+ mice concurrent with evidence of enhanced DC type-1, Th1/Th17 CD4 cell, and classical macrophage activation. Bone marrow-derived DCs stimulated with cryptococcal mannoproteins, a component of the fungal capsule, upregulated expression of IL-10 and IL-10R, which promoted DC type-2 activation in an autocrine manner. Thus, our findings implicate fungus-triggered autocrine IL-10 signaling and DC type-2 activation as important contributors to the development of nonprotective immune effector responses, which characterize persistent cryptococcal lung infection. Collectively, this study informs and strengthens the rationale for IL-10 signaling blockade as a novel treatment for fungal infections.

Anti-PD-1 Antibody Treatment Promotes Clearance of Persistent Cryptococcal Lung Infection in Mice.

Activation of immunomodulatory pathways in response to invasive fungi can impair clearance and promote persistent infections. The programmed cell death protein-1 (PD-1) signaling pathway inhibits immune effector responses against tumors, and immune checkpoint inhibitors that block this pathway are being increasingly used as cancer therapy. The objective of this study was to investigate whether this pathway contributes to persistent fungal infection and to determine whether anti-PD-1 Ab treatment improves fungal clearance. Studies were performed using C57BL/6 mice infected with a moderately virulent strain of Cryptococcus neoformans (52D), which resulted in prolonged elevations in fungal burden and histopathologic evidence of chronic lung inflammation. Persistent infection was associated with increased and sustained expression of PD-1 on lung lymphocytes, including a mixed population of CD4+ T cells. In parallel, expression of the PD-1 ligands, PD-1 ligands 1 and 2, was similarly upregulated on specific subsets of resident and recruited lung dendritic cells and macrophages. Treatment of persistently infected mice for 4 wk by repetitive administration of neutralizing anti-PD-1 Ab significantly improved pulmonary fungal clearance. Treatment was well tolerated without evidence of morbidity. Immunophenotyping revealed that anti-PD-1 Ab treatment did not alter immune effector cell numbers or myeloid cell activation. Treatment did reduce gene expression of IL-5 and IL-10 by lung leukocytes and promoted sustained upregulation of OX40 by Th1 and Th17 cells. Collectively, this study demonstrates that PD-1 signaling promotes persistent cryptococcal lung infection and identifies this pathway as a potential target for novel immune-based treatments of chronic fungal disease.

Local GM-CSF-Dependent Differentiation and Activation of Pulmonary Dendritic Cells and Macrophages Protect against Progressive Cryptococcal Lung Infection in Mice.

Patients with acquired deficiency in GM-CSF are susceptible to infections with Cryptococcus neoformans and other opportunistic fungi. We previously showed that GM-CSF protects against progressive fungal disease using a murine model of cryptococcal lung infection. To better understand the cellular and molecular mechanisms through which GM-CSF enhances antifungal host defenses, we investigated temporal and spatial relationships between myeloid and lymphoid immune responses in wild-type C57BL/6 mice capable of producing GM-CSF and GM-CSF-deficient mice infected with a moderately virulent encapsulated strain of C. neoformans (strain 52D). Our data demonstrate that GM-CSF deficiency led to a reduction in: 1) total lung leukocyte recruitment; 2) Th2 and Th17 responses; 3) total numbers of CD11b(+) dendritic cells (DC) and CD11b(-) and CD11b(+) macrophages (Mϕ); 4) DC and Mϕ activation; and 5) localization of DC and Mϕ to the microanatomic sites of alveolar infection. In contrast, GM-CSF deficiency resulted in increased accumulation of DC and Mϕ precursors, namely Ly-6C(high) monocytes, in the blood and lungs of infected mice. Collectively, these results show that GM-CSF promotes the local differentiation, accumulation, activation, and alveolar localization of lung DC and Mϕ in mice with cryptococcal lung infection. These findings identify GM-CSF as central to the protective immune response that prevents progressive fungal disease and thus shed new light on the increased susceptibility to these infections observed in patients with acquired GM-CSF deficiency.

Resident alveolar macrophages suppress, whereas recruited monocytes promote, allergic lung inflammation in murine models of asthma.

The role and origin of alveolar macrophages (AMs) in asthma are incompletely defined. We sought to clarify these issues in the context of acute allergic lung inflammation using house dust mite and OVA murine models. Use of liposomal clodronate to deplete resident AMs (rAMs) resulted in increased levels of inflammatory cytokines and eosinophil numbers in lavage fluid and augmented the histopathologic evidence of lung inflammation, suggesting a suppressive role for rAMs. Lung digests of asthmatic mice revealed an increased percentage of Ly6C(high)/CD11b(pos) inflammatory monocytes. Clodronate depletion of circulating monocytes, by contrast, resulted in an attenuation of allergic inflammation. A CD45.1/CD45.2 chimera model demonstrated that recruitment at least partially contributes to the AM pool in irradiated nonasthmatic mice, but its contribution was no greater in asthma. Ki-67 staining of AMs supported a role for local proliferation, which was increased in asthma. Our data demonstrate that rAMs dampen, whereas circulating monocytes promote, early events in allergic lung inflammation. Moreover, maintenance of the AM pool in the early stages of asthmatic inflammation depends on local proliferation, but not recruitment.

Early or late IL-10 blockade enhances Th1 and Th17 effector responses and promotes fungal clearance in mice with cryptococcal lung infection.

The potent immunoregulatory properties of IL-10 can counteract protective immune responses and, thereby, promote persistent infections, as evidenced by studies of cryptococcal lung infection in IL-10-deficient mice. To further investigate how IL-10 impairs fungal clearance, the current study used an established murine model of C57BL/6J mice infected with Cryptococcus neoformans strain 52D. Our results demonstrate that fungal persistence is associated with an early and sustained expression of IL-10 by lung leukocytes. To examine whether IL-10-mediated immune modulation occurs during the early or late phase of infection, assessments of fungal burden and immunophenotyping were performed on mice treated with anti-IL-10R-blocking Ab at 3, 6, and 9 d postinfection (dpi) (early phase) or at 15, 18, and 21 dpi (late phase). We found that both early and late IL-10 blockade significantly improved fungal clearance within the lung compared with isotype control treatment when assessed 35 dpi. Immunophenotyping identified that IL-10 blockade enhanced several critical effector mechanisms, including increased accumulation of CD4(+) T cells and B cells, but not CD8(+) T cells; specific increases in the total numbers of Th1 and Th17 cells; and increased accumulation and activation of CD11b(+) dendritic cells and exudate macrophages. Importantly, IL-10 blockade effectively abrogated dissemination of C. neoformans to the brain. Collectively, this study identifies early and late cellular and molecular mechanisms through which IL-10 impairs fungal clearance and highlights the therapeutic potential of IL-10 blockade in the treatment of fungal lung infections.

DEK in the synovium of patients with juvenile idiopathic arthritis: characterization of DEK antibodies and posttranslational modification of the DEK autoantigen.

OBJECTIVE: DEK is a nuclear phosphoprotein and autoantigen in a subset of children with juvenile idiopathic arthritis (JIA). Autoantibodies to DEK are also found in a broad spectrum of disorders associated with abnormal immune activation. We previously demonstrated that DEK is secreted by macrophages, is released by apoptotic T cells, and attracts leukocytes. Since DEK has been identified in the synovial fluid (SF) of patients with JIA, this study was undertaken to investigate how DEK protein and/or autoantibodies may contribute to the pathogenesis of JIA. METHODS: DEK autoantibodies, immune complexes (ICs), and synovial macrophages were purified from the SF of patients with JIA. DEK autoantibodies and ICs were purified by affinity-column chromatography and analyzed by 2-dimensional gel electrophoresis, immunoblotting, and enzyme-linked immunosorbent assay. DEK in supernatants and exosomes was purified by serial centrifugation and immunoprecipitation with magnetic beads, and posttranslational modifications of DEK were identified by nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS). RESULTS: DEK autoantibodies and protein were found in the SF of patients with JIA. Secretion of DEK by synovial macrophages was observed both in a free form and via exosomes. DEK autoantibodies (IgG2) may activate the complement cascade, primarily recognize the C-terminal portion of DEK protein, and exhibit higher affinity for acetylated DEK. Consistent with these observations, DEK underwent acetylation on an unprecedented number of lysine residues, as demonstrated by nano-LC-MS/MS. CONCLUSION: These results indicate that DEK can contribute directly to joint inflammation in JIA by generating ICs through high-affinity interaction between DEK and DEK autoantibodies, a process enhanced by acetylation of DEK in the inflamed joint.

In vivo sensitized and in vitro activated B cells mediate tumor regression in cancer adoptive immunotherapy.

Adoptive cellular immunotherapy utilizing tumor-reactive T cells has proven to be a promising strategy for cancer treatment. However, we hypothesize that successful treatment strategies will have to appropriately stimulate not only cellular immunity, but also humoral immunity. We previously reported that B cells in tumor-draining lymph nodes (TDLNs) may function as APCs. In this study, we identified TDLN B cells as effector cells in an adoptive immunotherapy model. In vivo primed and in vitro activated TDLN B cells alone mediated effective (p < 0.05) tumor regression after adoptive transfer into two histologically distinct murine pulmonary metastatic tumor models. Prior lymphodepletion of the host with either chemotherapy or whole-body irradiation augmented the therapeutic efficacy of the adoptively transferred TDLN B cells in the treatment of s.c. tumors as well as metastatic pulmonary tumors. Furthermore, B cell plus T cell transfers resulted in substantially more efficient antitumor responses than B cells or T cells alone (p < 0.05). Activated TDLN B cells conferred strong humoral responses to tumor. This was evident by the production of IgM, IgG, and IgG2b, which bound specifically to tumor cells and led to specific tumor cell lysis in the presence of complement. Collectively, these data indicate that in vivo primed and in vitro activated B cells can be employed as effector cells for cancer therapy. The synergistic antitumor efficacy of cotransferred activated B effector cells and T effector cells represents a novel approach for cancer adoptive immunotherapy.

Serum autoantibody profiling using a natural glycoprotein microarray for the prognosis of early melanoma.

The poor prognosis of melanoma and the high cost of lymph node biopsy for melanoma patients have led to an urgent need for the discovery of convenient and accurate prognostic indicators. Here, we have developed a natural glycoprotein microarray to discover serum autoantibodies to distinguish between patients with node negative melanoma and node positive melanoma. Dual-lectin affinity chromatography was used to extract glycoproteins from a melanoma cell line. Liquid-based reverse phase separation and microarray platforms were then applied to separate and spot these natural proteins on nitrocellulose slides. The serum autoantibodies were investigated by exposing these proteins to sera from 43 patients that have already been diagnosed to have different stages of early melanoma. The combination of 9 fractions provides a 55% sensitivity with 100% specificity for the detection of node positive against node negative and a 62% sensitivity with 100% specificity for the detection of node negative against node positive. Recombinant proteins were used to confirm the results using a sample set with 79 patients with diagnosed melanoma. The response of sera against recombinant 94 kD glucose-regulated protein (GRP94), acid ceramidase (ASAH1), cathepsin D (CTSD), and lactate dehydrogenase B (LDHB) shared a similar pattern to the fractions where they were identified. The glycoarray platform provides a convenient and highly reproducible method to profile autoantibodies that could be used as serum biomarkers for prognosis of melanoma.

Urokinase Plasminogen Activator Overexpression Reverses Established Lung Fibrosis.

INTRODUCTION: Impaired plasminogen activation (PA) is causally related to the development of lung fibrosis. Prior studies demonstrate that enhanced PA in the lung limits the severity of scarring following injury and in vitro studies indicate that PA promotes matrix degradation and fibroblast apoptosis. These findings led us to hypothesize that increased PA in an in vivo model would enhance the resolution of established lung fibrosis in conjunction with increased myofibroblast apoptosis. METHODS: Transgenic C57BL/6 mice with doxycycline inducible lung-specific urokinase plasminogen activator (uPA) expression or littermate controls were treated (day 0) with bleomycin or saline. Doxycycline was initiated on days 1, 9, 14, or 21. Lung fibrosis, stiffness, apoptosis, epithelial barrier integrity, and inflammation were assessed. RESULTS: Protection from fibrosis with uPA upregulation from day 1 through day 28 was associated with reduced parenchymal stiffness as determined by atomic force microscopy. Initiation of uPA expression beginning in the late inflammatory or the early fibrotic phase reduced stiffness and fibrosis at day 28. Induction of uPA activity in mice with established fibrosis decreased lung collagen and lung stiffness while increasing myofibroblast apoptosis. Upregulation of uPA did not alter lung inflammation but was associated with improved epithelial cell homeostasis. CONCLUSION: Restoring intrapulmonary PA activity diminishes lung fibrogenesis and enhances the resolution of established lung fibrosis. This PA-mediated resolution is associated with increased myofibroblast apoptosis and improved epithelial cell homeostasis. These studies support the potential capacity of the lung to resolve existing scar in murine models.

Dendritic cells pulsed with keyhole limpet hemocyanin and cryopreserved maintain anti-tumor activity in a murine melanoma model.

We compared viability, phenotype, in vitro function and therapeutic efficacy of murine unpulsed-dendritic cells (-DC), DC pulsed with keyhole limpet hemocyanin (KLH-DC) and cryopreserved KLH-DC (C-KLH-DC). Mean viability (%+/-SE) of unpulsed-DC, KLH-DC and C-KLH-DC was 93.6+/-0.9, 93.9+/-0.8 and 87.4+/-1.6, respectively. Pulsing DC with KLH did not induce maturation or affect in vitro function. Cryopreservation of KLH-DC reduced MHC I, CD80 and CD86 expression, endocytic capacity and allogeneic splenocyte stimulatory capacity. Intratumoral (i.t.) vaccination of mice bearing s.c. D5 melanoma with unpulsed-DC, KLH-DC or C-KLH-DC elicited comparable anti-tumor immune responses and inhibited tumor growth to the same extent. Combining radiotherapy with i.t. unpulsed-DC, KLH-DC or C-KLH-DC administration enhanced induction of anti-tumor immune responses and inhibition of tumor growth to a similar degree. Cryopreservation of KLH-DC slightly reduces viability, expression of co-stimulatory cell surface markers and in vitro function; however, in vivo anti-tumor activity is fully maintained with or without radiotherapy.

Polarization effects of 4-1BB during CD28 costimulation in generating tumor-reactive T cells for cancer immunotherapy.

Using murine tumor-draining lymph node (TDLN) cells, we investigated the polarization effect of 4-1BB (CD137) during CD28 costimulation in generating antitumor T cells. Costimulation of TDLN cells through the newly induced 4-1BB molecules, CD3, and CD28 using monoclonal antibodies significantly enhanced cell proliferation. The greater cell yield with 4-1BB signaling appeared to be related to the inhibition of activation-induced cell death. Activation of TDLN cells through 4-1BB in addition to CD3/CD28 signaling shifted T-cell responses toward a type 1 cytokine pattern because 4-1BB ligation plus CD3/CD28 stimulation significantly augmented type 1 cytokine (e.g., IFN-gamma) and granulocyte macrophage colony-stimulating factor secretion. By contrast, type 2 cytokine (e.g., interleukin 10) secretion by the activated TDLN cells was significantly reduced. The in vivo antitumor reactivity of TDLN cells activated through 4-1BB in conjunction with CD3/CD28 pathways was examined using an adoptive immunotherapy model. The number of pulmonary metastases was significantly reduced and survival was prolonged after the transfer of anti-CD3/anti-CD28/anti-4-1BB-activated TDLN cells compared with an equivalent number of cells activated without anti-4-1BB. The antitumor effect through 4-1BB involvement during CD28 costimulation was dependent on IFN-gamma production and abrogated after IFN-gamma neutralization. By contrast, interleukin 10 neutralization resulted in significantly enhanced tumor regression. These results indicate that costimulation of TDLN cells through newly induced 4-1BB and CD3/CD28 signaling can significantly increase antitumor reactivity by shifting T-cell responses toward a type 1 cytokine pattern while concomitantly decreasing type 2 responses.

Anti-CD137 monoclonal antibody administration augments the antitumor efficacy of dendritic cell-based vaccines.

In weakly and poorly immunogenic tumor models, we examined the effects of stimulating CD137 (4-1BB) in vivo by administering anti-CD137 monoclonal antibody after tumor lysate-pulsed dendritic cell (TP-DC) vaccination. TP-DC subcutaneous vaccination induced a transient up-regulation of CD137 on T cells and natural killer (NK) cells within vaccine-primed lymph nodes (VPLNs). In established pulmonary and subcutaneous tumor models, anti-CD137 synergistically enhanced tumor regression after TP-DC vaccination. In the subcutaneous tumor model, the combined therapy resulted in improved survival. Combined therapy also resulted in improved local control of subcutaneous tumor after surgical resection. Anti-CD137 polarized the cytokine release of VPLNs and spleen cells in response to tumor antigen toward a type 1 (interferon-gamma) versus a type 2 (interleukin-4) profile. Cell depletion and the use of knockout animals identified that CD8(+), CD4(+), and NK cells were involved in the tumor rejection response and that CD8(+) cells had the major effector role. Anti-CD137 administration resulted in increased proliferation of adoptively transferred OT-1 CD8(+) T cells in the VPLNs of mice inoculated with B16-OVA TP-DCs. Polarization toward type 1 (interferon-gamma) versus type 2 (interleukin-4) was also observed with the OT-1 cells from VPLNs and spleen cells after anti-CD137 injections. This polarization effect was abrogated by the in vivo depletion of NK cells. These findings indicate that the adjuvant effect of anti-CD137 given in conjunction with TP-DC vaccination is associated with the polarization of T effector cells toward a type 1 response to tumor antigen and is mediated via NK cells.

Radiotherapy potentiates the therapeutic efficacy of intratumoral dendritic cell administration.

We examined whether radiotherapy (RT) could enhance the efficacy of dendritic cell (DC)-based immunotherapy of cancer. Mice bearing s.c. D5 melanoma or MCA 205 sarcoma tumors were treated with intratumoral (i.t.) injections of bone marrow-derived unpulsed DCs in combination with local fractionated tumor irradiation. DC administration alone slightly inhibited D5 tumor growth and had no effect on MCA 205. RT alone caused a modest inhibition of both tumors. DC administration combined with RT inhibited D5 and MCA 205 tumor growth in an additive and synergistic manner, respectively. In both tumor models, RT intensified the antitumor efficacy of DC administration independent of apoptosis or necrosis within the tumor mass. Combination treatment of i.t. DCs plus RT was superior to s.c. injections of tumor lysate-pulsed DCs plus interleukin 2 in inhibiting D5 tumor growth and prolonging survival of mice. Splenocytes from mice treated with i.t. DCs plus RT contained significantly more tumor-specific, IFN-gamma-secreting T cells compared with control groups. Moreover, adoptive transfer of these splenocytes mediated significant tumor regression in mice bearing established pulmonary metastases. Combined treatment followed by resection of residual s.c. tumor conferred protective immunity against a subsequent i.v. tumor challenge. Furthermore, i.t. DC plus RT treatment of s.c. tumor in mice bearing concomitant pulmonary metastases resulted in a significant reduction of lung tumors. i.t. DC administration combined with RT induces a potent local and systemic antitumor response in tumor-bearing mice. This novel regimen may be beneficial in the treatment of human cancers.

Generation of a novel dendritic-cell vaccine using melanoma and squamous cancer stem cells.

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDH(high) CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c. tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFNγ and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Effects of tumor irradiation on host T-regulatory cells and systemic immunity in the context of adoptive T-cell therapy in mice.

In this study, we used a murine D5 melanoma model to study the effects of local tumor irradiation on the therapeutic efficacy of adoptive T-cell therapy. Tumor irradiation was delivered in 5 daily fractions (8.5 Gy) to subcutaneous tumors on days 7-11 after tumor inoculation. After the last radiation dose, activated tumor-draining lymph node cells were transferred intravenously followed by intraperitoneal IL-2 administration. Tumor irradiation alone had no significant effect on tumor growth; however, it synergistically enhanced the therapeutic efficacy of T-cell therapy. For 2 days after tumor irradiation there was a significant reduction in T cells, B cells, and CD11c(+) dendritic cells in both the tumor microenvironment and the systemic lymphoid compartments. By days 4-6 after irradiation, the relative reduction in the number of Treg cells within the tumor and the systemic compartments was greater than the reduction in conventional T cells. Furthermore, the suppressive function of the Tregs was significantly impaired in irradiated versus untreated mice. Using effector T cells derived from congenic mice, we found that local tumor irradiation resulted in increased proliferation of donor T cells within the tumor and the systemic lymphoid compartments. Radiation was associated with increased expression of the effector cytokines IFN-γ and TNF-α by donor and host CD4(+) and CD8(+) T cells. Altogether, our data indicate that local tumor irradiation has a distinct modulatory effect on Tregs and can enhance systemic antitumor immunity associated with adoptive T-cell therapy.

Murine colitis is mediated by vimentin.

Vimentin, an abundant intermediate filament protein, presumably has an important role in stabilizing intracellular architecture, but its function is otherwise poorly understood. In a vimentin knockout (Vim KO) mouse model, we note that Vim KO mice challenged with intraperitoneal Escherichia coli control bacterial infection better than do wild-type (WT) mice. In vitro, Vim KO phagocytes show significantly increased capacity to mediate bacterial killing by abundant production of reactive oxygen species (ROS) and nitric oxides, likely due to interactions with the p47phox active subunit of NADPH oxidase. In acute colitis induced by dextran sodium sulfate (DSS), Vim KO mice develop significantly less gut inflammation than do WT mice. Further, Vim KO mice have markedly decreased bacterial extravasation in the setting of DSS-induced acute colitis, consistent with decreased intestinal disease. Our results suggest that vimentin impedes bacterial killing and production of ROS, thereby contributing to the pathogenesis of acute colitis.

Targeting cancer stem cells via dendritic-cell vaccination.

Using syngeneic murine tumor models established in immunocompetent hosts, we showed that cancer stem cells are immunogenic and can be selectively targeted by dendritic cell-based vaccines. This new approach induced both humoral and cellular immune responses and conferred significantly superior antitumor immunity as compared with conventional vaccines.