Angiogenesis-associated pathways play critical roles in neonatal sepsis outcomes.

Neonatal sepsis is a major cause of childhood mortality. Limited diagnostic tools and mechanistic insights have hampered our abilities to develop prophylactic or therapeutic interventions. Biomarkers in human neonatal sepsis have been repeatedly identified as associated with dysregulation of angiopoietin signaling and altered arachidonic acid metabolism. We here provide the mechanistic evidence in support of the relevance for these observations. Angiopoetin-1 (Ang-1), which promotes vascular integrity, was decreased in blood plasma of human and murine septic newborns. In preclinical models, administration of Ang-1 provided prophylactic protection from septic death. Arachidonic acid metabolism appears to be functionally connected to Ang-1 via reactive oxygen species (ROS) with a direct role of nitric oxide (NO). Strengthening this intersection via oral administration of arachidonic acid and/or the NO donor L-arginine provided prophylactic as well as therapeutic protection from septic death while also increasing plasma Ang-1 levels among septic newborns. Our data highlight that targeting angiogenesis-associated pathways with interventions that increase Ang-1 activity directly or indirectly through ROS/eNOS provide promising avenues to prevent and/or treat severe neonatal sepsis.

Look Who's Talking: Host and Pathogen Drivers of Staphylococcus epidermidis Virulence in Neonatal Sepsis.

Preterm infants are at increased risk for invasive neonatal bacterial infections. S. epidermidis, a ubiquitous skin commensal, is a major cause of late-onset neonatal sepsis, particularly in high-resource settings. The vulnerability of preterm infants to serious bacterial infections is commonly attributed to their distinct and developing immune system. While developmentally immature immune defences play a large role in facilitating bacterial invasion, this fails to explain why only a subset of infants develop infections with low-virulence organisms when exposed to similar risk factors in the neonatal ICU. Experimental research has explored potential virulence mechanisms contributing to the pathogenic shift of commensal S. epidermidis strains. Furthermore, comparative genomics studies have yielded insights into the emergence and spread of nosocomial S. epidermidis strains, and their genetic and functional characteristics implicated in invasive disease in neonates. These studies have highlighted the multifactorial nature of S. epidermidis traits relating to pathogenicity and commensalism. In this review, we discuss the known host and pathogen drivers of S. epidermidis virulence in neonatal sepsis and provide future perspectives to close the gap in our understanding of S. epidermidis as a cause of neonatal morbidity and mortality.

Cyclic AMP in human preterm infant blood is associated with increased TLR-mediated production of acute-phase and anti-inflammatory cytokines in vitro.

BACKGROUND: Preterm infants are at high risk of infection and have distinct pathogen recognition responses. Suggested mechanisms include soluble mediators that enhance cellular levels of cAMP. The aim of this study was to assess the relationship between blood cAMP concentrations and TLR-mediated cytokine production in infants during the first month of life. METHODS: Cord and serial peripheral blood samples (days of life 1-28) were obtained from a cohort of very preterm (<30 weeks' gestational age) and term human infants. Whole-blood concentrations of cAMP and FSL-1 and LPS in vitro stimulated cytokine concentrations were measured by ELISA and multiplex bead assay. RESULTS: cAMP concentrations were higher in cord than in peripheral blood, higher in cord blood of female preterm infants, and lower at Days 1 and 7 in infants exposed to chorioamnionitis, even after adjusting for leukocyte counts. TLR2 and TLR4-mediated TNF-α, IL-1ß, IL-6, IL-12p70, and IL-10 production in vitro increased over the first month of life in preterm infants and were positively correlated with leukocyte-adjusted cAMP levels and reduced by exposure to chorioamnionitis. CONCLUSIONS: The ontogeny of blood cAMP concentrations and associations with chorioamnionitis and TLR-mediated production of cytokines suggest that this secondary messenger helps shape distinct neonatal pathogen responses in early life.

Exposure to the antimicrobial peptide LL-37 produces dendritic cells optimized for immunotherapy.

Immunization of patients with autologous, ex vivo matured dendritic cell (DC) preparations, in order to prime antitumor T-cell responses, is the focus of intense research. Despite progress and approval of clinical approaches, significant enhancement of these personalized immunotherapies is urgently needed to improve efficacy. We show that immunotherapeutic murine and human DC, generated in the presence of the antimicrobial host defense peptide LL-37, have dramatically enhanced expansion and differentiation of cells with key features of the critical CD103+/CD141+ DC subsets, including enhanced cross-presentation and co-stimulatory capacity, and upregulation of CCR7 with improved migratory capacity. These LL-37-DC enhanced proliferation, activation and cytokine production by CD8+ (but not CD4+) T cells in vitro and in vivo. Critically, tumor antigen-presenting LL-37-DC increased migration of primed, activated CD8+ T cells into established squamous cell carcinomas in mice, and resulted in tumor regression. This advance therefore has the potential to dramatically enhance DC immunotherapy protocols.

Effect of exercise on acute postprandial glucose concentrations and interleukin-6 responses in sedentary and overweight males.

This study examined the effect of 2 forms of exercise on glucose tolerance and the concurrent changes in markers associated with the interleukin (IL)-6 pathways. Fifteen sedentary, overweight males (29.0 ± 3.1 kg/m2) completed 2 separate, 3-day trials in randomised and counterbalanced order. An oral glucose tolerance test (OGTT; 75 g) was performed at the same time on each day of the trial. Day 2 of each trial consisted of a single 30-min workload-matched bout of either high-intensity intermittent exercise (HIIE; alternating 100% and 50% of peak oxygen uptake) or continuous moderate-intensity exercise (CME; 60 % of peak oxygen uptake) completed 1 h prior to the OGTT. Venous blood samples were collected before, immediately after, 1 h after, and 25 h after exercise for measurement of insulin, C-peptide, IL-6, and the soluble IL-6 receptors (sIL-6R; soluble glycoprotein 130 (sgp130)). Glucose area under the curve (AUC) was calculated from capillary blood samples collected throughout the OGTT. Exercise resulted in a modest (4.4%; p = 0.003) decrease in the glucose AUC when compared with the pre-exercise AUC; however, no differences were observed between exercise conditions (p = 0.65). IL-6 was elevated immediately after and 1 h after exercise, whilst sgp130 and sIL-6R concentrations were reduced immediately after exercise. In summary, exercise was effective in reducing glucose AUC, which was attributed to improvements that took place between 60 and 120 min into the OGTT, and was in parallel with an increased ratio of IL-6 to sIL-6R, which accords with an increased activation via the "classical" IL-6 signalling pathway. Our findings suggest that acute HIIE did not improve glycaemic response when compared with CME.

Evidence of functional cell-mediated immune responses to nontypeable Haemophilus influenzae in otitis-prone children.

Otitis media (OM) remains a common paediatric disease, despite advances in vaccinology. Susceptibility to recurrent acute OM (rAOM) has been postulated to involve defective cell-mediated immune responses to common otopathogenic bacteria. We compared the composition of peripheral blood mononuclear cells (PBMC) from 20 children with a history of rAOM (otitis-prone) and 20 healthy non-otitis-prone controls, and assessed innate and cell-mediated immune responses to the major otopathogen nontypeable Haemophilus influenzae (NTHi). NTHi was a potent stimulator of inflammatory cytokine secretion from PBMC within 4 hours, with no difference in cytokine levels produced between PBMC from cases or controls. In the absence of antigen stimulation, otitis-prone children had more circulating Natural Killer (NK) cells (p<0.01), particularly NKdim (CD56lo) cells (p<0.01), but fewer CD4+ T cells (p<0.01) than healthy controls. NTHi challenge significantly increased the proportion of activated (CD107a+) NK cells in otitis-prone and non-otitis-prone children (p<0.01), suggesting that NK cells from otitis-prone children are functional and respond to NTHi. CD8+ T cells and NK cells from both cases and controls produced IFNγ in response to polyclonal stimulus (Staphylococcal enterotoxin B; SEB), with more IFNγ+ CD8+ T cells present in cases than controls (p<0.05) but similar proportions of IFNγ+ NK cells. Otitis-prone children had more circulating IFNγ-producing NK cells (p<0.05) and more IFNγ-producing CD4+ (p<0.01) or CD8+ T-cells (p<0.05) than healthy controls. In response to SEB, more CD107a-expressing CD8+ T cells were present in cases than controls (p<0.01). Despite differences in PBMC composition, PBMC from otitis-prone children mounted innate and T cell-mediated responses to NTHi challenge that were comparable to healthy children. These data provide evidence that otitis-prone children do not have impaired functional cell mediated immunity.

Exposure to chorioamnionitis alters the monocyte transcriptional response to the neonatal pathogen Staphylococcus epidermidis.

Preterm infants are uniquely susceptible to late-onset sepsis that is frequently caused by the skin commensal Staphylococcus epidermidis. Innate immune responses, particularly from monocytes, are a key protective mechanism. Impaired cytokine production by preterm infant monocytes is well described, but few studies have comprehensively assessed the corresponding monocyte transcriptional response. Innate immune responses in preterm infants may be modulated by inflammation such as prenatal exposure to histologic chorioamnionitis which complicates 40-70% of preterm pregnancies. Chorioamnionitis alters the risk of late-onset sepsis, but its effect on monocyte function is largely unknown. Here, we aimed to determine the impact of exposure to chorioamnionitis on the proportions and phenotype of cord blood monocytes using flow cytometry, as well as their transcriptional response to live S. epidermidis. RNA-seq was performed on purified cord blood monocytes from very preterm infants (<32 weeks gestation, with and without chorioamnionitis-exposure) and term infants (37-40 weeks), pre- and postchallenge with live S. epidermidis. Preterm monocytes from infants without chorioamnionitis-exposure did not exhibit an intrinsically deficient transcriptional response to S. epidermidis compared to term infants. In contrast, chorioamnionitis-exposure was associated with hypo-responsive transcriptional phenotype regarding a subset of genes involved in antigen presentation and adaptive immunity. Overall, our findings suggest that prenatal exposure to inflammation may alter the risk of sepsis in preterm infants partly by modulation of monocyte responses to pathogens.

T-cell responses against rhinovirus species A and C in asthmatic and healthy children.

BACKGROUND: Infections by rhinovirus (RV) species A and C are the most common causes of exacerbations of asthma and a major cause of exacerbations of other acute and chronic respiratory diseases. Infections by both species are prevalent in pre-school and school-aged children and, particularly for RV-C, can cause severe symptoms and a need for hospitalization. While associations between RV infection and asthma are well established, the adaptive immune-mechanisms by which RV infections influence asthma exacerbations are yet to be defined. OBJECTIVE: The aim of this study was to characterize and compare T-cell responses between RV-A and RV-C and to test the hypothesis that T-cell responses would differ between asthmatic children and healthy controls. METHODS: A multi-parameter flow cytometry assay was used to characterize the in vitro recall T-cell response against RV-A and RV-C in PBMCs from children with acute asthma (n = 22) and controls (n = 26). The responses were induced by pools of peptides containing species-specific VP1 epitopes of RV-A and RV-C. RESULTS: Regardless of children's clinical status, all children that responded to the in vitro stimulation (>90%) had a similar magnitude of CD4+ T-cell responses to RV-A and RV-C. However, asthmatic children had a significantly lower number of circulating regulatory T cells (Tregs), and healthy controls had significantly more Tregs induced by RV-A than RV-C. CONCLUSIONS AND CLINICAL RELEVANCE: The comparable recall memory T-cell responses in asthmatic and control children to both RV-A and RV-C show that differences in the antibody and inflammatory responses previously described are likely to be due to regulation, with a demonstrated candidate being reduced regulatory T-cells. The reduced Treg numbers demonstrated here could explain the asthmatic's inability to appropriately control immunopathological responses to RV infections.

Identification of generic and pathogen-specific cord blood monocyte transcriptomes reveals a largely conserved response in preterm and term newborn infants.

Escherichia coli and Staphylococcus epidermidis are predominant causes of neonatal sepsis, particularly affecting preterm infants. Susceptibility to infection has been attributed to "immature" innate monocyte defences, but no studies have assessed global transcriptional responses of neonatal monocytes to these pathogens. Here, we aimed to identify and characterise the neonatal monocyte transcriptional responses to E. coli and S. epidermidis and the role of common modifiers such as gestational age (GA) and exposure to chorioamnionitis (a common complication of preterm birth) to better understand early life innate immune responses. RNA-sequencing was performed on purified cord blood monocytes from very preterm (< 32 weeks GA) and term infants (37-40 weeks GA) following standardised challenge with live S. epidermidis or E. coli. The major transcriptional changes induced by either pathogen were highly conserved between infant groups and stimuli, highlighting a common extant neonatal monocyte response to infection, largely mediated by TLR/NF-κB/TREM-1 signalling. In addition, we observed an activated interferon-centred immune response specific to stimulation with E. coli in both preterm and term infants. These data provide novel insights into the functionality of neonatal monocytes at birth and highlight potential pathways that could be targeted to reduce the harmful effects of bacterial-induced inflammation in sepsis. E. coli and S. epidermidis elicit common transcriptional changes in cord monocytes. The common transcriptional response is mediated by TLR/NF-κB/TREM-1 signalling. IFN genes are differentially regulated by E. coli and S. epidermidis in monocytes. These responses are largely unaffected by GA or exposure to chorioamnionitis. KEY MESSAGES: E. coli and S. epidermidis elicit common transcriptional changes in cord monocytes. The common transcriptional response is mediated by TLR/NF-κB/TREM-1 signalling. IFN-genes are differentially regulated by E. coli and S. epidermidis in monocytes. These responses are largely unaffected by GA or exposure to chorioamnionitis.

Human Infant Memory B Cell and CD4+ T Cell Responses to HibMenCY-TT Glyco-Conjugate Vaccine.

UNLABELLED: Carrier-specific T cell and polysaccharide-specific B cell memory responses are not well characterised in infants following glyco-conjugate vaccination. We aimed to determine if the number of Meningococcal (Men) C- and Y- specific memory B cells and; number and quality of Tetanus Toxoid (TT) carrier-specific memory CD4+ T cells are associated with polysaccharide-specific IgG post HibMenCY-TT vaccination. Healthy infants received HibMenCY-TT vaccine at 2, 4 and 6 months with a booster at 12 months. Peripheral blood mononuclear cells were isolated and polysaccharide-specific memory B cells enumerated using ELISpot. TT-specific memory CD4+ T cells were detected and phenotyped based on CD154 expression and intracellular TNF-α, IL-2 and IFN-γ expression following stimulation. Functional polysaccharide-specific IgG titres were measured using the serum bactericidal activity (SBA) assay. Polysaccharide-specific Men C- but not Men Y- specific memory B cell frequencies pre-boost (12 months) were significantly associated with post-boost (13 months) SBA titres. Regression analysis showed no association between memory B cell frequencies post-priming (at 6 or 7 months) and SBA at 12 months or 13 months. TT-specific CD4+ T cells were detected at frequencies between 0.001 and 0.112 as a percentage of CD3+ T cells, but their numbers were not associated with SBA titres. There were significant negative associations between SBA titres at M13 and cytokine expression at M7 and M12. CONCLUSION: Induction of persistent polysaccharide-specific memory B cells prior to boosting is an important determinant of secondary IgG responses in infants. However, polysaccharide-specific functional IgG responses appear to be independent of the number and quality of circulating carrier-specific CD4+ T cells after priming.

Restoration of defective cross-presentation in tumors by gemcitabine.

Tumor antigen cross-presentation by dendritic cells (DCs) to specific CD8+ T cells is central to antitumor immunity. Although highly efficient in draining lymph nodes, it is defective within the tumor site itself. Importantly, an immunogenic chemotherapy, gemcitabine, reverses this defect, allowing the potential re-stimulation of cytotoxic T lymphocytes within tumor sites.

Antimicrobial protein and Peptide concentrations and activity in human breast milk consumed by preterm infants at risk of late-onset neonatal sepsis.

OBJECTIVE: We investigated the levels and antimicrobial activity of antimicrobial proteins and peptides (AMPs) in breast milk consumed by preterm infants, and whether deficiencies of these factors were associated with late-onset neonatal sepsis (LOS), a bacterial infection that frequently occurs in preterm infants in the neonatal period. STUDY DESIGN: Breast milk from mothers of preterm infants (≤ 32 weeks gestation) was collected on days 7 (n = 88) and 21 (n = 77) postpartum. Concentrations of lactoferrin, LL-37, beta-defensins 1 and 2, and alpha-defensin 5 were measured by enzyme-linked immunosorbent assay. The antimicrobial activity of breast milk samples against Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, and Streptococcus agalactiae was compared to the activity of infant formula, alone or supplemented with physiological levels of AMPs. Samples of breast milk fed to infants with and without subsequent LOS were compared for levels of AMPs and inhibition of bacterial growth. RESULTS: Levels of most AMPs and antibacterial activity in preterm breast milk were higher at day 7 than at day 21. Lactoferrin was the only AMP that limited pathogen growth >50% when added to formula at a concentration equivalent to that present in breast milk. Levels of AMPs were similar in the breast milk fed to infants with and without LOS, however, infants who developed LOS consumed significantly less breast milk and lower doses of milk AMPs than those who were free from LOS. CONCLUSIONS: The concentrations of lactoferrin and defensins in preterm breast milk have antimicrobial activity against common neonatal pathogens.

Tumor-infiltrating dendritic cells exhibit defective cross-presentation of tumor antigens, but is reversed by chemotherapy.

Cross-presentation defines the unique capacity of an APC to present exogenous Ag via MHC class I molecules to CD8(+) T cells. DCs are specialized cross-presenting cells and as such have a critical role in antitumor immunity. DCs are routinely found within the tumor microenvironment, but their capacity for endogenous or therapeutically enhanced cross-presentation is not well characterized. In this study, we examined the tumor and lymph node DC cross-presentation of a nominal marker tumor Ag, HA, expressed by the murine mesothelioma tumor AB1-HA. We found that tumors were infiltrated by predominantly CD11b(+) DCs with a semimature phenotype that could not cross-present tumor Ag, and therefore, were unable to induce tumor-specific T-cell activation or proliferation. Although tumor-infiltrating DCs were able to take up, process, and cross-present exogenous cell-bound and soluble Ags, this was significantly impaired relative to lymph node DCs. Importantly, however, systemic chemotherapy using gemcitabine reversed the defect in Ag cross-presentation of tumor DCs. These data demonstrate that DC cross-presentation within the tumor microenvironment is defective, but can be reversed by chemotherapy. These results have important implications for anticancer therapy, particularly regarding the use of immunotherapy in conjunction with cytotoxic chemotherapy.

The efficacy of tumor debulking surgery is improved by adjuvant immunotherapy using imiquimod and anti-CD40.

BACKGROUND: Tumor debulking surgery followed by adjuvant chemotherapy or radiotherapy is a standard treatment for many solid malignancies. Although this approach can be effective, it often has limited success against recurrent or metastatic cancers and new multimodality approaches are needed. Adjuvant immunotherapy is another potentially effective approach. We therefore tested the efficacy of the TLR7 agonist imiquimod (IMQ) combined with agonistic anti-CD40 in an incomplete debulking model of malignant mesothelioma. METHODS: Established subcutaneous murine ABA-HA mesothelioma tumors in BALB/c mice were surgically debulked by 75% and treated with either: i) saline; ii) intratumoral IMQ; iii) systemic anti-CD40 antibody, or using a combination of IMQ and anti-CD40. Tumour growth and survival were monitored, and the role of anti-tumor CD4 and CD8 T cells in therapeutic responses was determined. RESULTS: The combination therapy of partial debulking surgery, IMQ and anti-CD40 significantly delayed tumor growth in a CD8 T cell dependent manner, and promoted tumor regression in 25% of animals with establishment of immunological memory. This response was associated with an increase in ICOS+ CD8 T cells and tumor-specific CTL activity in tumor draining lymph nodes along with an increase in ICOS+ CD8 T cells in responding tumours. CONCLUSIONS: We show that the post-surgical environment can be significantly altered by the co-administration of adjuvant IMQ and anti-CD40, resulting in strong, systemic anti-tumor activity. Both adjuvants are available for clinical use/trial, hence this treatment regimen has clear translational potential.

NOD1 and NOD2 expression and function in very preterm infant mononuclear cells.

AIM: To evaluate mononuclear cell expression and function of the cytosolic nucleotide-binding oligomerization domain-containing receptors, NOD1 and NOD2, in very preterm and full-term infants. METHODS: NOD1 and NOD2 gene and protein expression in very preterm infants, term infants and healthy adult, cord and peripheral blood mononuclear cells (C/PBMC) were quantified using qPCR and flow cytometry. Cytokine responses of purified infant and adult monocytes to NOD1- and NOD2-specific agonists were assessed using a multiplex immunoassay (Bioplex). RESULTS: NOD1 and NOD2 were expressed by a range of infant and adult mononuclear cell types, including T- and B cells, with highest expression in classical (CD14(++) CD16(-) ) and intermediate (CD14(++) CD16(+) ) monocytes. NOD1 and NOD2 expression levels by monocytes from very preterm infant were similar to those in term infants or adults. Monocyte production of TNFα, IL-6 and IL-1ß induced by activation of NOD1 and NOD2 was similar between very preterm infants, term infants and adults. CONCLUSION: Monocyte expression and function of NOD1 and NOD2 in very preterm infants are intact and comparable/equivalent to term infants and adults. Functional deficiencies in monocyte NOD signalling pathways are unlikely to contribute to the increased susceptibility to bacterial sepsis in preterm infants.

A practical method for preparation of pneumococcal and nontypeable Haemophilus influenzae inocula that preserves viability and immunostimulatory activity.

BACKGROUND: Convenience is a major reason for using killed preparations of bacteria to investigate host-pathogen interactions, however, host responses to such preparations can result in different outcomes when compared to live bacterial stimulation. We investigated whether cryopreservation of Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) permitted investigation of host responses to infection without the complications of working with freshly prepared live bacteria on the day of experimental challenge. FINDINGS: S. pneumoniae and NTHi retained >90% viability following cryopreservation in fetal calf serum for at least 8 weeks. Host responses to live, cryopreserved (1 week and 4 weeks), heat-killed or ethanol-killed S. pneumoniae and NTHi were assessed by measuring cytokine release from stimulated peripheral blood mononuclear cells (PBMCs). We found that cryopreserved bacteria, in contrast to heat-killed and ethanol-killed preparations, resulted in comparable levels of inflammatory cytokine release from PBMCs when compared with fresh live bacterial cultures. CONCLUSION: Cryopreservation of S. pneumoniae and NTHi does not alter the immunostimulatory properties of these species thereby enabling reproducible and biologically relevant analysis of host responses to infection. This method also facilitates the analysis of multiple strains on the same day and allows predetermination of culture purity and challenge dose.

Agonistic anti-CD40 antibody therapy is effective against postoperative cancer recurrence and metastasis in a murine tumor model.

Postresection recurrences of cancer arising from occult tumor deposits, either local or metastatic, represent major causes of death in patients with operable solid tumors. Thus, new therapies are required that complement existing treatments to eradicate these occult deposits. Agonistic anti-CD40 antibody is one of the most powerful new cancer immunotherapies, enhancing immune priming of effector CD8 T cells by dendritic cells, leading to increased antitumor activity. We investigated the use of anti-CD40 antibody for the treatment of postoperative recurrence and metastasis, with regional lymphadenectomy, in a murine model of cancer. Subcutaneous AB1-HA mesothelioma tumors were induced in BALB/c mice. Established tumors were surgically excised on day 16, with or without sentinel lymph node removal. On the day of surgery, animals were rechallenged with AB1-HA tumor cells at the surgical site (local recurrence) or the opposite flank (metastasis). Postoperative tumors were treated with anti-CD40 (FGK45) on emergence, delivered either intratumorally, peritumorally, or systemically. Local or systemic anti-CD40 treatment slowed postsurgical metastatic growth relative to untreated controls (P = 0.020) and improved survival from metastasis. Anti-CD40 also retarded the growth of local recurrences (P = 0.004) and improved survival from recurrence. Sentinel lymph node dissection did not impair efficacy (P > 0.05). This study demonstrates that anti-CD40 therapy, given either locally or systemically, may be a powerful and readily translatable adjuvant to cancer surgery, including in cases where regional lymphadenectomy is indicated.

Tumor cells, rather than dendritic cells, deliver antigen to the lymph node for cross-presentation.

It is widely accepted that generation of tumor specific CD8+ T-cell responses occur via cross-priming; however the source of tumor antigen for this event is unknown. We examined the source and form of tumor antigen required for cross-presentation in the local lymph node (LN) using a syngeneic mouse tumor model expressing a marker antigen. We found that cross-presentation of this model tumor antigen in the LN is dependent on continuous traffic of antigen from the tumor site, but without any detectable migration of tumor resident dendritic cells (DCs). Instead, small numbers of tumor cells metastasize to local LNs where they are exposed to a localized CTL attack, resulting in delivery of tumor antigen into the cross-presentation pathway.