Amy Prosser

Tumor eradication after cyclophosphamide depends on concurrent depletion of regulatory T cells: a role for cycling TNFR2-expressing effector-suppressor T cells in limiting effective chemotherapy

Tumor cell death potentially engages with the immune system. However, the efficacy of anti-tumor chemotherapy may be limited by tumor-driven immunosuppression, e.g., through CD25+ regulatory T cells. We addressed this question in a mouse model of mesothelioma by depleting or reconstituting CD25+ regulatory T cells in combination with two different chemotherapeutic drugs. We found that the efficacy of cyclophosphamide to eradicate established tumors, which has been linked to regulatory T cell depletion, was negated by adoptive transfer of CD25+ regulatory T cells. Analysis of post-chemotherapy regulatory T cell populations revealed that cyclophosphamide depleted cycling (Ki-67hi) T cells, including foxp3+ regulatory CD4+ T cells. Ki-67hi CD4+ T cells expressed increased levels of two markers, TNFR2 and ICOS, that have been associated with a maximally suppressive phenotype according to recently published studies. This suggest that cyclophosphamide depletes a population of maximally suppressive regulatory T cells, which may explain its superior anti-tumor efficacy in our model. Our data suggest that regulatory T cell depletion could be used to improve the efficacy of anti-cancer chemotherapy regimens. Indeed, we observed that the drug gemcitabine, which does not deplete cycling regulatory T cells, eradicates established tumors in mice only when CD25+ CD4+ T cells are concurrently depleted. Cyclophosphamide could be used to achieve regulatory T cell depletion in combination with chemotherapy.

Locally Administered TLR7 Agonists Drive Systemic Antitumor Immune Responses That Are Enhanced by Anti-CD40 Immunotherapy

Topical application of tumors with the TLR7 agonist imiquimod is an effective adjunct treatment for a range of primary dermatological cancers. However, for therapy to be effective against a broad range of solid tumor types, it must promote a strong systemic antitumor response that targets metastases in addition to primary tumor. We therefore investigated the potential of locally delivered imiquimod to stimulate an effective systemic antitumor response in a murine model of malignant mesothelioma (AB1-HA) with primary and distal tumors (dual tumor). Persistent delivery of imiquimod into primary tumor significantly retarded tumor growth in all treated mice compared with vehicle control. This local antitumor immune response required both CD8 T cells and NK cells, but not CD4 T cells, and was reliant on type I IFN induction. In vivo CTL studies and Ly6A/E staining of lymphocytes suggested that local imiquimod treatment had indeed induced a systemic, Ag-specific CD8 response. However, notably this response was not sufficient to retard the growth of an untreated distal tumor. Because local imiquimod treatment did not induce significant CD4 T cell responses, we investigated the efficacy of combining imiquimod with agonistic CD40 Ab (as a surrogate for CD4 T cell help). Combination of locally delivered imiquimod with systemic anti-CD40 immunotherapy not only significantly enhanced the local antitumor response, with 30% complete resolution, but it was also effective at significantly retarding growth of distal tumor. These results demonstrate that antitumor responses induced by locally delivered TLR7 agonists can be harnessed systemically for treating distal tumor.

Cyclophosphamide Chemotherapy Sensitizes Tumor Cells to TRAIL-Dependent CD8 T Cell-Mediated Immune Attack Resulting in Suppression of Tumor Growth

Background Anti-cancer chemotherapy can be simultaneously lymphodepleting and immunostimulatory. Pre-clinical models clearly demonstrate that chemotherapy can synergize with immunotherapy, raising the question how the immune system can be mobilized to generate anti-tumor immune responses in the context of chemotherapy. Methods and Findings We used a mouse model of malignant mesothelioma, AB1-HA, to investigate T cell-dependent tumor resolution after chemotherapy. Established AB1-HA tumors were cured by a single dose of cyclophosphamide in a CD8 T cell- and NK cell-dependent manner. This treatment was associated with an IFN-α/β response and a profound negative impact on the anti-tumor and total CD8 T cell responses. Despite this negative effect, CD8 T cells were essential for curative responses. The important effector molecules used by the anti-tumor immune response included IFN-γ and TRAIL. The importance of TRAIL was supported by experiments in nude mice where the lack of functional T cells could be compensated by agonistic anti-TRAIL-receptor (DR5) antibodies. Conclusion The data support a model in which chemotherapy sensitizes tumor cells for T cell-, and possibly NK cell-, mediated apoptosis. A key role of tumor cell sensitization to immune attack is supported by the role of TRAIL in tumor resolution and explains the paradox of successful CD8 T cell-dependent anti-tumor responses in the absence of CD8 T cell expansion.

Targeting the Effector Site with IFN-αβ-Inducing TLR Ligands Reactivates Tumor-Resident CD8 T Cell Responses to Eradicate Established Solid Tumors

Effective antitumor CD8 T cell responses may be activated by directly targeting the innate immune system within tumors. We investigated this response by injecting a range of TLR agonists into established tumors using a mouse model of malignant mesothelioma stably transduced with the hemagglutinin (HA) gene as a marker Ag (AB1-HA). Persistent delivery of the dsRNA mimetic poly(I:C) into established AB1-HA tumors resulted in complete tumor resolution in 40% of mice, with the remaining mice also showing a significant delay in tumor progression. Experiments in athymic nude mice along with CD8 depletion and IFN-αβ blocking studies revealed that tumor resolution required both CD8 T cells and type I IFN induction, and was associated with local changes in MHC class I expression. Surprisingly, however, tumor resolution was not associated with systemic dissemination or tumor infiltration of effector CD8 T cells. Instead, the antitumor response was critically dependent on the reactivation of tumor-resident CD8 T cell responses. These studies suggest that, once reactivated, pre-existing local CD8 T cell responses are sufficient to resolve established tumors and that in situ type I IFN is a determining factor.

Dual control of antitumor CD8 T cells through the programmed death-1/programmed death-ligand 1 pathway and immunosuppressive CD4 T cells: regulation and counterregulation

Tumors have evolved multiple mechanisms to evade immune destruction. One of these is expression of T cell inhibitory ligands such as programmed death-ligand 1 (PD-L1; B7-H1). In this study, we show that PD-L1 is highly expressed on mesothelioma tumor cells and within the tumor stroma. However, PD-L1 blockade only marginally affected tumor growth and was associated with the emergence of activated programmed death-1+ ICOS+ CD4 T cells in tumor-draining lymph nodes, whereas few activated CD8 T cells were present. Full activation of antitumor CD8 T cells, characterized as programmed death-1+ ICOS+ Ki-67+ and displaying CTL activity, was only observed when CD4 T cells were depleted, suggesting that a population of suppressive CD4 T cells exists. ICOS+ foxp3+ regulatory T cells were found to be regulated through PD-L1, identifying one potentially suppressive CD4 T cell population. Thus, PD-L1 blockade activates antitumor CD8 T cell most potently in the absence of CD4 T cells. These findings have implications for the development of PD-L1-based therapies.

CD8α+ DC are not the sole subset cross-presenting cell-associated tumor antigens from a solid tumor

One of the clear paradoxes in tumor immunology is the fact that cross‐presentation of cell‐associated tumor antigens to CD8+ T cells is efficient, yet CTL generation is weak, and tumors continue to grow. We examined, for the first time whether this may be due to alterations in the phenotype or function of cross‐presenting DC using a solid tumor model expressing a membrane bound neo‐antigen (hemagglutinin, HA). Tumor antigen was constitutively cross‐presented in the tumor‐draining LN throughout tumor progression by CD11c+ DC. Further analysis revealed that both CD8α+ and CD8α− DC subsets, but not plasmacytoid DC, were effective at cross‐presenting HA tumor antigen. The proportions of DC subsets in the tumor‐draining LN were equivalent to those seen in the LN of naïve mice; however, a significant increase in the expression of the potential inhibitory B7 molecule, B7‐DC, was noted and appeared to be restricted to the CD8α– DC subset. Therefore LN resident CD8α+ DC are not the sole DC subset capable of cross‐presenting cell‐associated tumor antigens. Migratory tumor DC subsets with altered co‐stimulatory receptor expression may contribute to induction and regulation of tumor‐specific responses.

Responsiveness of human monocytes to the commensal bacterium Staphylococcus epidermidis develops late in gestation

Introduction:Staphylococcus epidermidis (SE) rarely causes infection in term infants but is a leading cause of late-onset sepsis in preterm infants. We hypothesized that the innate immune responses to SE in preterm infants are impaired in a gestational age (GA)-dependent manner.Methods:Cord and peripheral blood mononuclear cells (MNCs) were stimulated with SE bacteria, and a range of innate immune responses were assessed, including phagocytosis, intracellular killing, Toll-like receptor (TLR) pathway transcriptional activation, cytokine production, TLR2 and TLR4 expression, and cell signaling.Results:Phagocytosis and intracellular killing of SE bacteria were similar in neonatal and adult monocytes. Cytokine gene expression and protein synthesis increased in a GA-dependent manner, which was confirmed at the single-cell level. These GA-related effects were not associated with differences in expression of TLR2 or TLR4, nor with downstream activation of nuclear factor-κB or mitogen-activated protein kinase pathways.Discussion:The expression of TLRs, phagocytic capacity, and intracellular killing by monocytes develops early in fetal development, whereas the ability to mount a bacteria-induced cytokine response requires further maturation. The functional immaturity of monocyte activation pathways in the preterm infant may underpin their particular susceptibility to sepsis with commensal bacteria.

Phagocytosis of neonatal pathogens by peripheral blood neutrophils and monocytes from newborn preterm and term infants

Background:Deficiencies in phagocytosis may contribute to the increased susceptibility of infants to early life infections. Data on phagocytosis of the major neonatal pathogens Staphylococcus epidermidis (SE), Staphylococcus aureus (SA), and Escherichia coli (EC) by preterm infant leukocytes are inconsistent.Methods:Cord and <24-h peripheral blood were collected from very preterm (<30.1 wks gestational age (GA)) and term (37–42 wks GA) infants. Monocyte and neutrophil phagocytosis of pHrodo-labeled SE, SA, and EC were analyzed using a small-volume flow cytometry assay, with simultaneous characterization of surface activation marker expression.Results:Preterm infants had lower proportions of monocytes and neutrophils capable of phagocytosis than term infants, but preterm infant phagocytes had higher phagocytic capacity. Phagocytosis was strongly correlated between cord and <24-h peripheral blood. Supplementation with exogenous complement significantly increased phagocytosis of EC but not of SE or SA. Monocyte human leukocyte antigen (HLA)-DR expression was lower in preterm infants but did not correlate with phagocytosis.Conclusion:There is no defect in phagocytosis by monocytes and neutrophils from preterm compared with term infants, although preterm infants possess fewer phagocytes, possibly contributing to susceptibility to bacterial infection. Further investigation into the development of postnatal phagocytic competence is warranted.

Method of bacterial killing differentially affects the human innate immune response to Staphylococcus epidermidis

Background: In vitro investigations of human innate immune responses to extracellular bacteria commonly utilise killed preparations in preference to live organisms. The effects of the bacterial preparation method on the activation of innate signalling pathways by the common opportunistic pathogen Staphylococcus epidermidis (SE) are unknown. Materials and methods: Mononuclear cell cytokine expression patterns induced by live (LSE), heat-killed (HKSE) and ethanol-killed SE (EKSE) were characterized at the transcriptional and translational level. Toll-like receptor (TLR)-activating capacity of the preparations was analysed using TLR-transfected human embryonic kidney cells. Results: Live SE activated NF-κB, STAT1, type I interferon, and inflammasome pathways. Killed preparations engaged the NF-κB pathway, but had significantly lower capacity to activate other innate immune pathways. Conclusions: Killing of extracellular bacteria has significant qualitative and quantitative effects on key aspects of innate responses in vitro. Interpretation of in vitro data and extrapolation of findings should take into account the potential effects of bacterial preparation and should not assume that responses to killed bacteria are predictive of responses to live organisms.

Haemophilus haemolyticus Interaction with Host Cells Is Different to Nontypeable Haemophilus influenzae and Prevents NTHi Association with Epithelial Cells

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that resides in the upper respiratory tract and contributes to a significant burden of respiratory related diseases in children and adults. Haemophilus haemolyticus is a respiratory tract commensal that can be misidentified as NTHi due to high levels of genetic relatedness. There are reports of invasive disease from H. haemolyticus, which further blurs the species boundary with NTHi. To investigate differences in pathogenicity between these species, we optimized an in vitro epithelial cell model to compare the interaction of 10 H. haemolyticus strains with 4 NTHi and 4 H. influenzae-like haemophili. There was inter- and intra-species variability but overall, H. haemolyticus had reduced capacity to attach to and invade nasopharyngeal and bronchoalveolar epithelial cell lines (D562 and A549) within 3 h when compared with NTHi. H. haemolyticus was cytotoxic to both cell lines at 24 h, whereas NTHi was not. Nasopharyngeal epithelium challenged with some H. haemolyticus strains released high levels of inflammatory mediators IL-6 and IL-8, whereas NTHi did not elicit an inflammatory response despite higher levels of cell association and invasion. Furthermore, peripheral blood mononuclear cells stimulated with H. haemolyticus or NTHi released similar and high levels of IL-6, IL-8, IL-10, IL-1β, and TNFα when compared with unstimulated cells but only NTHi elicited an IFNγ response. Due to the relatedness of H. haemolyticus and NTHi, we hypothesized that H. haemolyticus may compete with NTHi for colonization of the respiratory tract. We observed that in vitro pre-treatment of epithelial cells with H. haemolyticus significantly reduced NTHi attachment, suggesting interference or competition between the two species is possible and warrants further investigation. In conclusion, H. haemolyticus interacts differently with host cells compared to NTHi, with different immunostimulatory and cytotoxic properties. This study provides an in vitro model for further investigation into the pathogenesis of Haemophilus species and the foundation for exploring whether H. haemolyticus can be used to prevent NTHi disease.