Targeting the CD47/thrombospondin-1 signaling axis regulates immune cell bioenergetics in the tumor microenvironment to potentiate antitumor immune response.

BACKGROUND: CD47 is an integral membrane protein that alters adaptive immunosurveillance when bound to the matricellular glycoprotein thrombospondin-1 (TSP1). We examined the impact of the CD47/TSP1 signaling axis on melanoma patient response to anti-PD-1 therapy due to alterations in T cell activation, proliferation, effector function, and bioenergetics. METHODS: A syngeneic B16 mouse melanoma model was performed to determine if targeting CD47 as monotherapy or in combination with anti-PD-1 impacted tumor burden. Cytotoxic (CD8+) T cells from Pmel-1 transgenic mice were used for T cell activation, cytotoxic T lymphocyte, and cellular bioenergetic assays. Single-cell RNA-sequencing, ELISA, and flow cytometry was performed on peripheral blood mononuclear cells and plasma of melanoma patients receiving anti-PD-1 therapy to examine CD47/TSP1 expression. RESULTS: Human malignant melanoma tissue had increased CD47 and TSP1 expression within the tumor microenvironment compared with benign tissue. Due to the negative implications CD47/TSP1 can have on antitumor immune responses, we targeted CD47 in a melanoma model and observed a decrease in tumor burden due to increased tumor oxygen saturation and granzyme B secreting CD8+ T cells compared with wild-type tumors. Additionally, Pmel-1 CD8+ T cells exposed to TSP1 had reduced activation, proliferation, and effector function against B16 melanoma cells. Targeting CD47 allowed CD8+ T cells to overcome this TSP1 interaction to sustain these functions. TSP1 exposed CD8+ T cells have a decreased rate of glycolysis; however, targeting CD47 restored glycolysis when CD8+ T cells were exposed to TSP1, suggesting CD47 mediated metabolic reprogramming of T cells. Additionally, non-responding patients to anti-PD-1 therapy had increased T cells expressing CD47 and circulating levels of TSP1 compared with responding patients. Since CD47/TSP1 signaling axis negatively impacts CD8+ T cells and non-responding patients to anti-PD-1 therapy have increased CD47/TSP1 expression, we targeted CD47 in combination with anti-PD-1 in a melanoma model. Targeting CD47 in combination with anti-PD-1 treatment further decreased tumor burden compared with monotherapy and control. CONCLUSION: CD47/TSP1 expression could serve as a marker to predict patient response to immune checkpoint blockade treatment, and targeting this pathway may preserve T cell activation, proliferation, effector function, and bioenergetics to reduce tumor burden as a monotherapy or in combination with anti-PD-1.

Circulating Immune Bioenergetic, Metabolic, and Genetic Signatures Predict Melanoma Patients' Response to Anti-PD-1 Immune Checkpoint Blockade.

PURPOSE: Immunotherapy with checkpoint inhibitors is improving the outcomes of several cancers. However, only a subset of patients respond. Therefore, predictive biomarkers are critically needed to guide treatment decisions and develop approaches to the treatment of therapeutic resistance. EXPERIMENTAL DESIGN: We compared bioenergetics of circulating immune cells and metabolomic profiles of plasma obtained at baseline from patients with melanoma treated with anti-PD-1 therapy. We also performed single-cell RNA sequencing (scRNAseq) to correlate transcriptional changes associated with metabolic changes observed in peripheral blood mononuclear cells (PBMC) and patient plasma. RESULTS: Pretreatment PBMC from responders had a higher reserve respiratory capacity and higher basal glycolytic activity compared with nonresponders. Metabolomic analysis revealed that responder and nonresponder patient samples cluster differently, suggesting differences in metabolic signatures at baseline. Differential levels of specific lipid, amino acid, and glycolytic pathway metabolites were observed by response. Further, scRNAseq analysis revealed upregulation of T-cell genes regulating glycolysis. Our analysis showed that SLC2A14 (Glut-14; a glucose transporter) was the most significant gene upregulated in responder patients' T-cell population. Flow cytometry analysis confirmed significantly elevated cell surface expression of the Glut-14 in CD3+, CD8+, and CD4+ circulating populations in responder patients. Moreover, LDHC was also upregulated in the responder population. CONCLUSIONS: Our results suggest a glycolytic signature characterizes checkpoint inhibitor responders; consistently, both ECAR and lactate-to-pyruvate ratio were significantly associated with overall survival. Together, these findings support the use of blood bioenergetics and metabolomics as predictive biomarkers of patient response to immune checkpoint inhibitor therapy.

SMARCA4 mutations in KRAS-mutant lung adenocarcinoma: a multi-cohort analysis.

KRAS is a key oncogenic driver in lung adenocarcinoma (LUAD). Chromatin-remodeling gene SMARCA4 is comutated with KRAS in LUAD; however, the impact of SMARCA4 mutations on clinical outcome has not been adequately established. This study sought to shed light on the clinical significance of SMARCA4 mutations in LUAD. The association of SMARCA4 mutations with survival outcomes was interrogated in four independent cohorts totaling 564 patients: KRAS-mutant patients with LUAD who received nonimmunotherapy treatment from (a) The Cancer Genome Atlas (TCGA) and (b) the MSK-IMPACT Clinical Sequencing (MSK-CT) cohorts; and KRAS-mutant patients with LUAD who received immune checkpoint inhibitor-based immunotherapy treatment from (c) the MSK-IMPACT (MSK-IO) and (d) the Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) immunotherapy cohorts. Of the patients receiving nonimmunotherapy treatment, in the TCGA cohort (n = 155), KRAS-mutant patients harboring SMARCA4 mutations (KS) showed poorer clinical outcome [P = 6e-04 for disease-free survival (DFS) and 0.031 for overall survival (OS), respectively], compared to KRAS-TP53 comutant (KP) and KRAS-only mutant (K) patients; in the MSK-CT cohort (n = 314), KS patients also exhibited shorter OS than KP (P = 0.03) or K (P = 0.022) patients. Of patients receiving immunotherapy, KS patients consistently exhibited the shortest progression-free survival (PFS; P = 0.0091) in the MSK-IO (n = 77), and the shortest PFS (P = 0.0026) and OS (P = 0.0014) in the WFBCCC (n = 18) cohorts, respectively. Therefore, mutations of SMARCA4 represent a genetic factor leading to adverse clinical outcome in lung adenocarcinoma treated by either nonimmunotherapy or immunotherapy.

Higher Frequency and Increased Expression of Molecules Associated with Suppression on T Regulatory Cells from Newborn Compared with Adult Nonhuman Primates.

T regulatory cells (Tregs) play a critical role in controlling the immune response, often limiting pathogen-specific cells to curb immune-mediated damage. Studies in human infants have reported an increased representation of Tregs in these individuals. However, how these cells differ from those in adults at various sites and how they respond to activation signals is relatively unknown. In this study, we used a newborn nonhuman primate model to assess Treg populations present at multiple sites with regard to frequency and phenotype in comparison with those present in adult animals. We found that Foxp3+ cells were more highly represented in the T cell compartment of newborn nonhuman primates for all sites examined (i.e., the spleen, lung, and circulation). In the spleen and circulation, newborn-derived Tregs expressed significantly higher levels of Foxp3 and CD25 compared with adults, consistent with an effector phenotype. Strikingly, the phenotype of Tregs in the lungs of adult and infant animals was relatively similar, with both adult and newborn Tregs exhibiting a more uniform PD-1+CD39+ phenotype. Finally, in vitro, newborn Tregs exhibited an increased requirement for TCR engagement for survival. Further, these cells upregulated CD39 more robustly than their adult counterpart. Together, these data provide new insights into the quantity of Tregs in newborns, their activation state, and their potential to respond to activation signals.

Neuroinflammation after Intracerebral Hemorrhage and Potential Therapeutic Targets.

Spontaneous intracerebral hemorrhage (ICH) is a catastrophic illness causing significant morbidity and mortality. Despite advances in surgical technique addressing primary brain injury caused by ICH, little progress has been made treating the subsequent inflammatory cascade. Pre-clinical studies have made advancements identifying components of neuroinflammation, including microglia, astrocytes, and T lymphocytes. After cerebral insult, inflammation is initially driven by the M1 microglia, secreting cytokines (e.g., interleukin-1ß [IL-1ß] and tumor necrosis factor-α) that are involved in the breakdown of the extracellular matrix, cellular integrity, and the blood brain barrier. Additionally, inflammatory factors recruit and induce differentiation of A1 reactive astrocytes and T helper 1 (Th1) cells, which contribute to the secretion of inflammatory cytokines, augmenting M1 polarization and potentiating inflammation. Within 7 days of ICH ictus, the M1 phenotype coverts to a M2 phenotype, key for hematoma removal, tissue healing, and overall resolution of inflammation. The secretion of anti-inflammatory cytokines (e.g., IL-4, IL-10) can drive Th2 cell differentiation. M2 polarization is maintained by the secretion of additional anti-inflammatory cytokines by the Th2 cells, suppressing M1 and Th1 phenotypes. Elucidating the timing and trigger of the anti-inflammatory phenotype may be integral in improving clinical outcomes. A challenge in current translational research is the absence of an equivalent disease animal model mirroring the patient population and comorbid pathophysiologic state. We review existing data and describe potential therapeutic targets around which we are creating a bench to bedside translational research model that better reflects the pathophysiology of ICH patients.

Dissecting intratumoral myeloid cell plasticity by single cell RNA-seq.

Tumor-infiltrating myeloid cells are the most abundant leukocyte population within tumors. Molecular cues from the tumor microenvironment promote the differentiation of immature myeloid cells toward an immunosuppressive phenotype. However, the in situ dynamics of the transcriptional reprogramming underlying this process are poorly understood. Therefore, we applied single cell RNA-seq (scRNA-seq) to computationally investigate the cellular composition and transcriptional dynamics of tumor and adjacent normal tissues from 4 early-stage non-small cell lung cancer (NSCLC) patients. Our scRNA-seq analyses identified 11 485 cells that varied in identity and gene expression traits between normal and tumor tissues. Among these, myeloid cell populations exhibited the most diverse changes between tumor and normal tissues, consistent with tumor-mediated reprogramming. Through trajectory analysis, we identified a differentiation path from CD14+ monocytes to M2 macrophages (monocyte-to-M2). This differentiation path was reproducible across patients, accompanied by increased expression of genes (eg, MRC1/CD206, MSR1/CD204, PPARG, TREM2) with significantly enriched functions (Oxidative phosphorylation and P53 pathway) and decreased expression of genes (eg, CXCL2, IL1B) with significantly enriched functions (TNF-α signaling via NF-κB and inflammatory response). Our analysis further identified a co-regulatory network implicating upstream transcription factors (JUN, NFKBIA) in monocyte-to-M2 differentiation, and activated ligand-receptor interactions (eg, SFTPA1-TLR2, ICAM1-ITGAM) suggesting intratumoral mechanisms whereby epithelial cells stimulate monocyte-to-M2 differentiation. Overall, our study identified the prevalent monocyte-to-M2 differentiation in NSCLC, accompanied by an intricate transcriptional reprogramming mediated by specific transcriptional activators and intercellular crosstalk involving ligand-receptor interactions.

Immunogenicity of high-dose influenza vaccination in patients with primary central nervous system malignancy.

BACKGROUND: For cancer patients, rates of influenza-associated hospitalization and death are 4 times greater than that of the general population. Previously, we reported reduced immunogenicity to the standard-dose influenza vaccine in patients with central nervous system malignancy. In other poorly responding populations (eg, elderly patients), high-dose vaccination has improved efficacy and immunogenicity. METHODS: A prospective cohort study was designed to evaluate the immunogenicity of the Fluzone® high-dose influenza vaccine in brain tumor patients. Data on diagnosis, active oncologic treatment, and immunologic status (eg, CD4 count, CD8 count, CD4:CD8 ratio) were collected. All patients received the high-dose vaccine (180 µg). Hemagglutination inhibition titers were measured at baseline, day 28, and 3 months following vaccination to determine seroconversion (≥4-fold rise) and seroprotection (titer ≥1:40), which were compared to our prior results. RESULTS: Twenty-seven patients enrolled. Diagnoses included high-grade glioma (85%), CNS lymphoma (11%), and meningioma (4%). Treatment at enrollment included glucocorticoids (n = 8, 30%), radiation (n = 2, 7%), and chemotherapy (n = 9, 33%). Posttreatment lymphopenia (PTL, CD4 ≤ 200) was observed in 4 patients (15%). High-dose vaccination was well tolerated with no grade III-IV toxicity. Overall, seroconversion rates for the A/H1N1, A/H3N2, and B vaccine strains were significantly higher than in our prior study: 65% vs 37%, 69% vs 23%, and 50% vs 23%, respectively (all P < .04). Seroconversion was universally poor in patients with PTL. While seroprotection at 3 months declined in our prior study, no drop was observed following high-dose vaccination in this cohort. CONCLUSIONS: The immunologic response to HD influenza vaccination was higher in this cohort than standard-dose influenza vaccination in our prior report. These findings mirror those in elderly patients where high-dose vaccination is the standard of care and raise the possibility of an immunosenescence phenotype.

A Novel Function for the Streptococcus pneumoniae Aminopeptidase N: Inhibition of T Cell Effector Function through Regulation of TCR Signaling.

Streptococcus pneumoniae (Spn) causes a variety of disease states including fatal bacterial pneumonia. Our previous finding that introduction of Spn into an animal with ongoing influenza virus infection resulted in a CD8+ T cell population with reduced effector function gave rise to the possibility of direct regulation by pneumococcal components. Here, we show that treatment of effector T cells with lysate derived from Spn resulted in inhibition of IFNγ and tumor necrosis factor α production as well as of cytolytic granule release. Spn aminopeptidase N (PepN) was identified as the inhibitory bacterial component and surprisingly, this property was independent of the peptidase activity found in this family of proteins. Inhibitory activity was associated with reduced activation of ZAP-70, ERK1/2, c-Jun N-terminal kinase, and p38, demonstrating the ability of PepN to negatively regulate TCR signaling at multiple points in the cascade. These results reveal a novel immune regulatory function for a bacterial aminopeptidase.

Coinfection with Streptococcus pneumoniae negatively modulates the size and composition of the ongoing influenza-specific CD8⁺ T cell response.

Infection with influenza A virus can lead to increased susceptibility to subsequent bacterial infection, often with Streptococcus pneumoniae. Given the substantial modification of the lung environment that occurs following pathogen infection, there is significant potential for modulation of immune responses. In this study, we show that infection of mice with influenza virus, followed by the noninvasive EF3030 strain of Streptococcus pneumoniae, leads to a significant decrease in the virus-specific CD8(+) T cell response in the lung. Adoptive-transfer studies suggest that this reduction contributes to disease in coinfected animals. The reduced number of lung effector cells in coinfected animals was associated with increased death, as well as a reduction in cytokine production in surviving cells. Further, cells that retained the ability to produce IFN-γ exhibited a decreased potential for coproduction of TNF-α. Reduced cytokine production was directly correlated with a decrease in the level of mRNA. Negative regulation of cells in the mediastinal lymph node was minimal compared with that present in the lung, supporting a model of selective regulation in the tissue harboring high pathogen burden. These results show that entry of a coinfecting pathogen can have profound immunoregulatory effects on an ongoing immune response. Together, these findings reveal a novel dynamic interplay between concurrently infecting pathogens and the adaptive immune system.

CD4+ T cell subset differentiation and avidity setpoint are dictated by the interplay of cytokine and antigen mediated signals.

CD4(+) T cell differentiation has been shown to be regulated by the cytokine milieu present during activation as well as peptide MHC levels. However, the extent to which these two important regulatory signals work in concert to shape CD4(+) T cell function has not been investigated. Using a murine OT-II transgenic TCR model of in vitro differentiation, we demonstrate that the ability of CD4(+) T cells to commit to a distinct lineage, i.e. Th1 vs. Th2 vs. Th17, is restricted by the amount of peptide antigen present in the stimulating environment. In addition, whether cells succumb to inhibitory effects associated with high dose antigen is dependent on the array of cytokine signals encountered. Specifically, stimulation with high dose antigen in Th1 or Th17 conditions promoted efficient generation of functional cells, while Th2 polarizing conditions did not. Finally, we found that the peptide sensitivity of an effector cell was determined by the combined actions of cytokine and peptide level, with Th1 cells exhibiting the highest avidity, followed by Th17 and Th2 cells. Together, these data show that the interplay of antigen and cytokine signals shape both the differentiation fate and avidity setpoint of CD4(+) T cells.

In vivo modulation of avidity in highly sensitive CD8(+) effector T cells following viral infection.

Numerous studies have demonstrated a critical role for T cell avidity in predicting in vivo efficacy. Even though the measurement of avidity is now a routine assessment for the analysis of effector and memory T cell populations, our understanding of how this property is controlled in vivo at both the population and individual cell levels is limited. Our previous studies have identified high avidity as a property of the initial effector population generated in mice following respiratory virus infection. As the response progresses, lower avidity cells appear in the effector pool. The studies described here investigate the mechanistic basis of this in vivo regulation of avidity. We present data supporting in vivo avidity modulation within the early high avidity responders that results in a population of lower avidity effector cells. Changes in avidity were correlated with decreased lck expression and increased sensitivity to lck inhibitors in effector cells present at late versus early times postinfection. The possibility of tuning within select individual effectors is a previously unappreciated mechanism for the control of avidity in vivo.

High viral burden restricts short-lived effector cell number at late times postinfection through increased natural regulatory T cell expansion.

Generating and maintaining a robust CD8(+) T cell response in the face of high viral burden is vital for host survival. Further, balancing the differentiation of effectors along the memory precursor effector cell pathway versus the short-lived effector cell (SLEC) pathway may be critical in controlling the outcome of virus infection with regard to clearance and establishing protection. Although recent studies have identified several factors that have the capacity to regulate effector CD8(+) T cell differentiation-for example, inflammatory cytokines-we are far from a complete understanding of how cells choose the memory precursor effector cell versus SLEC fate following infection. In this study, we have modulated the infectious dose of the poxvirus vaccinia virus as an approach to modulate the environment present during activation and expansion of virus-specific effector cells. Surprisingly, in the face of a high virus burden, the number of SLECs was decreased. This decrease was the result of increased natural regulatory T cells (Tregs) generated by high viral burden, as depletion of these cells restored SLECs. Our data suggest Treg modulation of differentiation occurs via competition for IL-2 during the late expansion period, as opposed to the time of T cell priming. These findings support a novel model wherein modulation of the Treg response as a result of high viral burden regulates late-stage SLEC number.

Omega-3 fatty acids ameliorate atherosclerosis by favorably altering monocyte subsets and limiting monocyte recruitment to aortic lesions.

OBJECTIVE: Fish oil, containing omega-3 fatty acids, attenuates atherosclerosis. We hypothesized that omega-3 fatty acid-enriched oils are atheroprotective through alteration of monocyte subsets and their trafficking into atherosclerotic lesions. METHODS AND RESULTS: Low-density lipoprotein receptor knockout and apolipoprotein E(-/-) mice were fed diets containing 10% (calories) palm oil and 0.2% cholesterol, supplemented with an additional 10% palm oil, echium oil (containing 18:4 n-3), or fish oil. Compared with palm oil-fed low-density lipoprotein receptor knockout mice, echium oil and fish oil significantly reduced plasma cholesterol, splenic Ly6C(hi) monocytosis by ≈50%, atherosclerosis by 40% to 70%, monocyte trafficking into the aortic root by ≈50%, and atherosclerotic lesion macrophage content by 30% to 44%. In contrast, atherosclerosis and monocyte trafficking into the artery wall was not altered by omega-3 fatty acids in apolipoprotein E(-/-) mice; however, Ly6C(hi) splenic monocytes positively correlated with aortic root intimal area across all diet groups. In apolipoprotein E(-/-) mice, fish oil reduced the percentage of blood Ly6C(hi) monocytes, despite an average 2-fold higher plasma cholesterol relative to palm oil. CONCLUSIONS: The presence of splenic Ly6C(hi) monocytes parallels the appearance of atherosclerotic disease in both low-density lipoprotein receptor knockout and apolipoprotein E(-/-) mice. Furthermore, omega-3 fatty acids favorably alter monocyte subsets independently from effects on plasma cholesterol and reduce monocyte recruitment into atherosclerotic lesions.

Increased sensitivity to antigen in high avidity CD8(+) T cells results from augmented membrane proximal T-cell receptor signal transduction.

The functional avidity of a cytotoxic T lymphocyte (CTL) is known to be a critical determinant of the efficacy with which it clears pathogens. High avidity cells, which are by definition highly sensitive to peptide antigen, are superior for elimination of viruses and tumours. Our studies have established the ability of T cells to undergo avidity modulation as a result of antigen encounter. High and low avidity cells established in this manner exhibit significant differences in the amount of peptide required to elicit effector function. However, how signalling is regulated in these cells as it relates to the control of peptide sensitivity remains to be defined. To address this question, we compared T-cell receptor (TCR) signal transduction events in high and low avidity CTL generated from OT-I(rag2-) TCR transgenic mice. Our data suggest that divergent signalling is initiated at the TCR-associated CD3ζ, with low avidity CTL requiring higher amounts of pMHC to achieve threshold levels of phosphorylated CD3ζ compared with high avidity CTL. Further, this difference is transduced further downstream to mitogen-activated protein kinase and Ca(2+) signalling pathways. These results suggest that regulated control of the initiation of TCR signalling in high versus low avidity cells determines the amount of peptide required for T-cell activation.

Engineered expression of the TLR5 ligand flagellin enhances paramyxovirus activation of human dendritic cell function.

The paramyxovirus simian virus 5 (SV5) is a poor activator of human dendritic cell (DC) maturation pathways in vitro, and infected DC do not upregulate cell surface costimulatory proteins or secretion of immunomodulatory cytokines. We evaluated the hypothesis that activation of SV5-infected DC would be enhanced by engineering SV5 to express a Toll-like-receptor (TLR) ligand. To test this hypothesis, a novel virus was engineered such that the gene encoding an intracellular form of the TLR5 ligand flagellin was expressed from the genome of wild-type (WT) SV5 (SV5-flagellin). Cells infected in vitro with the flagellin-expressing virus released low levels of biologically active flagellin, which was capable of stimulating TLR5 signaling. Infection of human peripheral blood mononuclear cell-derived immature DC with SV5-flagellin resulted in enhanced levels of interleukin-6 (IL-6) and IL-12 compared to infection with DC with the parental virus, WT SV5. In contrast to cytokine induction, the flagellin-expressing virus did not appreciably increase DC surface expression of the costimulatory molecule CD80 or CD86 above the level seen with WT SV5 alone. In mixed-culture assays, DC infected with the flagellin-expressing virus were more effective at activating gamma interferon secretion from both CD8(+) and CD4(+) allogeneic T cells than DC infected with WT SV5. Our results with SV5-directed intracellular expression of flagellin may be applicable to other vectors or pathogenic viruses where overcoming impairment of DC activation could contribute to the development of safer and more effective vaccines.

Cutting edge: Dendritic cells prime a high avidity CTL response independent of the level of presented antigen.

CTL that possess a high functional avidity are known to be optimal for the clearance of pathogens in vivo. We have shown that the amount of peptide encountered by a CD8+ CTL determines its functional avidity. Notably, in these studies nonprofessional APC were used. However, it is mature dendritic cells (DC) that are predominantly responsible for the activation of naive T cells in vivo. Whether DC also direct dose dependent-differences in avidity is unknown. In this work we examined the ability of mature DC presenting a high vs low level of peptide to generate CTL of distinct avidities. In contrast to what was observed with nonprofessional APC, CTL generated by stimulation with mature DC were of high avidity regardless of the amount of peptide presented. This DC property may promote generation of highly effective CTL that retain plasticity, which would allow the tuning of avidity in the periphery to promote optimal pathogen recognition and clearance.

Distinct pathways for signaling maturation in macrophages and dendritic cells after infection with paramyxovirus simian virus 5.

Professional antigen-presenting cells are critical components of both the innate and adaptive immune responses. Although dendritic cells (DCs) are generally thought to be the primary activators of naive T cells, macrophages have also been shown to fulfill this role. As with DCs, the capacity to induce optimal activation of T cells requires that macrophages undergo a process that results in the increased expression of costimulatory molecules, such as CD40, CD80, and CD86, and the production of cytokines. In this study we analyzed the effect of infection of macrophages generated from BALB/c mice with the paramyxovirus simian virus 5 (SV5). Here we have shown that bone marrow-derived macrophages (BMMs) are not productively infected at any multiplicity of infection tested. Analysis of activation markers revealed that SV5-infected BMMs robustly upregulated CD40 and modestly upregulated CD86, but did not upregulate the expression of CD80. Further, SV5-infected BMMs secreted low levels of interferon-beta and interleukin (IL)-12p40, but high levels of tumor necrosis factor-alpha and IL-6. Intriguingly, upregulation of these molecules on BMMs, unlike our previous results using bone marrow-derived dendritic cells, was not dependent on live virus. These findings provide evidence that different professional antigen-presenting cells can detect and respond to virus via distinct mechanisms.

Vaccinia virus infection of mature dendritic cells results in activation of virus-specific naïve CD8+ T cells: a potential mechanism for direct presentation.

Understanding how vaccinia virus (VV) generates immunity necessitates an appreciation for how this virus interacts with dendritic cells (DC), which are the most potent activators of naïve CD8(+) T cells. In order to optimally activate naïve CD8(+) T cells, DC must undergo maturation, during which costimulatory molecules are upregulated and cytokines are produced. In this report, we show that VV infection of immature murine bone marrow-derived DC (BMDC) failed to induce maturation. Similar results were obtained when CD8(+) DC were analyzed, a subset shown previously to be important in vivo in the generation of a vaccinia-specific response. The finding that VV infection of DC resulted in APC that were incapable of initiating T-cell activation was surprising given the previously reported role for direct presentation in the generation of anti-VV CD8(+) T-cell responses in mice. To address the potential mechanism responsible for direct presentation, we tested the hypothesis that previously matured DC were susceptible to vaccinia virus infection and could present newly synthesized VV-derived epitopes for CD8(+) T-cell activation. Our results show, that during VV infection of mature DC, threshold levels of viral protein are produced that promote T-cell activation. These results suggest that, even though VV cannot mature DC, previously matured DC exposed to VV can generate a VV-specific CD8(+) T-cell response providing a potential mechanism by which direct infection results in T-cell activation in vivo.

Exchange of P/V genes between two non-cytopathic simian virus 5 variants results in a recombinant virus that kills cells through death pathways that are sensitive to caspase inhibitors.

The paramyxovirus Simian virus 5 (SV5) is largely non-cytopathic in human epithelial and fibroblast cells. WF-PIV has been described previously as a naturally occurring SV5 variant that encodes P and V proteins differing from the wild-type (WT) SV5 proteins in eight and five amino acid positions, respectively. In this study, it is shown that WF-PIV is like WT SV5 by being largely non-cytopathic in A549 lung epithelial cells. However, substitution of the WF-PIV P/V gene into the background of WT SV5 resulted in a hybrid virus (P/V-WF) that induced apoptotic cell death not seen with either of the parental viruses. The kinetics of HeLa cell killing and induction of apoptosis by the P/V-WF chimera differed from those of the previously described P/V-CPI- chimera by being slower and less extensive. HeLa cell killing by the P/V-WF chimera was effectively reduced by inhibitors of caspase-9, but not of caspase-8. These results demonstrate that an exchange of P/V genes from two non-cytopathic SV5 variants can produce apoptosis-inducing chimeras, and that the role of the SV5 P/V gene products in limiting apoptosis can be dependent on expression in the context of a native viral genome.

Transferable anticancer innate immunity in spontaneous regression/complete resistance mice.

Spontaneous regression/complete resistance (SR/CR) mice resist very high doses of cancer cells that are lethal to WT mice even at low doses. In this study, we show that this resistance is mediated by rapid infiltration of leukocytes, mostly of innate immunity, in both primary and repeated challenges. Formation of rosettes with infiltrating natural killer cells, neutrophils, and macrophages was required for the subsequent destruction of cancer cells through rapid cytolysis. Highly purified natural killer cells, macrophages, and neutrophils from the SR/CR mice independently killed cancer cells in vitro. The independent killing activity by each subset of effector cells is consistent with the observation that the resistance was abolished by depleting total infiltrating leukocytes but not by depleting only one or two subsets of leukocytes. The resistance was completely transferable to WT recipient mice through SR/CR splenocytes, bone marrow cells, or enriched peritoneal macrophages, either for prevention against subsequent cancer challenges or eradication of established malignancy at distant sites.