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Mutations in DNA mismatch repair (MMR) genes are causative in Lynch syndrome and a significant proportion of sporadic colorectal cancers (CRCs). MMR-deficient (dMMR) CRCs display increased mutation rates, with mutations frequently accumulating at short repetitive DNA sequences throughout the genome (microsatellite instability). The TGFBR2 gene is one of the most frequently mutated genes in dMMR CRCs. Therefore, we generated an animal model to study how the loss of both TGFBR2 signaling impacts dMMR-driven intestinal tumorigenesis in vivo and explore the impact of the gut microbiota.We generated VCMsh2/Tgfbr2 mice in which Msh2VCMsh2/Tgfbr2 mice developed small intestinal adenocarcinomas and CRCs with histopathological features highly similar to CRCs in Lynch syndrome patients. The CRCs in VCMsh2/Tgfbr2 mice were associated with the presence of colitis and displayed genetic and histological features that resembled inflammation-associated CRCs in human patients. The development of CRCs in VCMsh2/Tgfbr2 mice was strongly modulated by the gut microbiota composition, which in turn was impacted by the TGFBR2 status of the tumors.Our results demonstrate a synergistic interaction between MMR and TGFBR2 inactivation in inflammation-associated colon tumorigenesis and highlight the crucial impact of the gut microbiota on modulating the incidence of inflammation-associated CRCs.

Summary Tumor-associated macrophages (TAMs) promote metastasis and inhibit T cells, but macrophages can be polarized to kill cancer cells. Macrophage polarization could thus be a strategy for controlling cancer. We show that macrophages from metastatic pleural effusions of breast cancer patients can be polarized to kill cancer cells with monophosphoryl lipid A (MPLA) and interferon (IFN) γ. MPLA + IFNγ injected intratumorally or intraperitoneally reduces primary tumor growth and metastasis in breast cancer mouse models, suppresses metastasis, and enhances chemotherapy response in an ovarian cancer model. Both macrophages and T cells are critical for the treatment's anti-metastatic effects. MPLA + IFNγ stimulates type I IFN signaling, reprograms CD206+ TAMs to inducible NO synthase (iNOS)+ macrophages, and activates cytotoxic T cells through macrophage-secreted interleukin-12 (IL-12) and tumor necrosis factor alpha (TNFα). MPLA and IFNγ are used individually in clinical practice and together represent a previously unexplored approach for engaging a systemic anti-tumor immune response.

Protein glutathionylation is a posttranslational process that regulates protein function in response to redox cellular changes. Furthermore, carbon monoxide-induced cellular pathways involve reactive oxygen species (ROS) signaling and mitochondrial protein glutathionylation. Herein, it is described as a technique to assess mitochondrial glutathionylation due to low concentrations of CO exposure. Mitochondria are isolated from cell culture or tissue, followed by an immunoprecipitation assay, which allows the capture of any glutathionylated mitochondrial protein using a specific antibody coupled to a solid matrix that binds to glutathione antigen. The precipitated protein is further identified and quantified by immunoblotting analysis.

Patients with colorectal cancer (CRC) harbor gut microbiomes that differ in structure and function from those of healthy individuals, suggesting this altered microbiome could contribute to tumorigenesis. Despite increasing evidence implicating the gut microbiome in CRC, the collective role of different microbial consortia in CRC carcinogenesis is unclear. We have previously described these consortia as co-abundance groups that co-exist at different abundance levels in the same patient. Here, we report that tumor biopsy tissue from patients with a “high-risk” Pathogen-type microbiome had a different immune transcriptome and immune cell infiltrate from those with a “low-risk” Lachnospiraceae-type microbiome. Transplantation from patients of the two fecal microbiome types into mice with an orthotopic tumor differentially affected tumor growth and the systemic anti-tumor immune response. The differences in tumor volume and immunophenotype between mice receiving the high-risk and the low-risk microbiome correlated with differences in the engrafted human microbial species and predicted microbiome-encoded metabolites in the two groups. Of twelve taxa whose abundance in recipient mice led to increased tumor onset, seven corresponded with differentially abundant taxa in a global dataset of 325 CRC patients versus 310 healthy controls. These data suggest that the enrichment for a Lachnospiraceae-type configuration of the gut microbiome may influence colon cancer progression and disease outcome by modulating the local and systemic anti-tumor immune response.Graphical AbstractProposed model of how the high-risk Pathogen and low-risk Lachnospiraceae CAGs differentially modulate the tumor immune response.

Protein glutathionylation is a posttranslational process that regulates protein function in response to redox cellular changes. Furthermore, carbon monoxide-induced cellular pathways involve reactive oxygen species (ROS) signaling and mitochondrial protein glutathionylation. Herein, it is described as a technique to assess mitochondrial glutathionylation due to low concentrations of CO exposure. Mitochondria are isolated from cell culture or tissue, followed by an immunoprecipitation assay, which allows the capture of any glutathionylated mitochondrial protein using a specific antibody coupled to a solid matrix that binds to glutathione antigen. The precipitated protein is further identified and quantified by immunoblotting analysis.

Flow cytometry is a popular laser-based technology that allows the phenotypic and functional characterization of individual cells in a high-throughput manner. Here, we describe a detailed procedure for preparing a single-cell suspension from mammary tumors of the mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) and analyzing these cells by multi-color flow cytometry. This protocol can be used to study the following tumor-infiltrating immune cell populations, defined by the expression of cell surface molecules: total leukocytes, tumor-associated macrophages (TAMs), conventional dendritic cells (DCs), CD103-expressing DCs, tumor-associated neutrophils, inflammatory monocytes, natural killer (NK) cells, CD4(+ )T cells, CD8(+) T cells, γδT cells, and regulatory T cells.

Many cancers recruit monocytes/macrophages and polarize them into tumor-associated macrophages (TAMs). TAMs promote tumor growth and metastasis and inhibit cytotoxic T cells. Yet, macrophages can also kill cancer cells after polarization by e.g., lipopolysaccharide (LPS, a bacteria-derived toll-like receptor 4 [TLR4] agonist) and interferon gamma (IFNγ). They do so via nitric oxide (NO), generated by inducible NO synthase (iNOS). Altering the polarization of macrophages could therefore be a strategy for controlling cancer. Here, we show that monophosphoryl lipid A (MPLA, a derivative of LPS) with IFNγ activated macrophages isolated from metastatic pleural effusions of breast cancer patients to kill the corresponding patients’ cancer cells in vitro. Importantly, intratumoral injection of MPLA with IFNγ not only controlled local tumor growth but also reduced metastasis in mouse models of luminal and triple negative breast cancers. Furthermore, intraperitoneal administration of MPLA with IFNγ reprogrammed peritoneal macrophages, suppressed metastasis, and enhanced the response to chemotherapy in the ID8-p53−/− ovarian carcinoma mouse model. The combined MPLA+IFNγ treatment reprogrammed the immunosuppressive microenvironment to be immunostimulatory by recruiting leukocytes, stimulating type I interferon signaling, decreasing tumor-associated (CD206+) macrophages, increasing tumoricidal (iNOS+) macrophages, and activating cytotoxic T cells through macrophage-secreted interleukin 12 (IL-12) and tumor necrosis factor α (TNFα). Both macrophages and T cells were critical for the anti-metastatic effects of MPLA+IFNγ. MPLA and IFNγ are already used individually in clinical practice, so our strategy to engage the anti-tumor immune response, which requires no knowledge of unique tumor antigens, may be ready for near-future clinical testing.

Plasmodium parasites undergo a dramatic transformation during the liver stage of their life cycle, amplifying over 10,000-fold inside infected hepatocytes within a few days. Such a rapid growth requires large-scale interactions with, and manipulations of, host cell functions. Whereas hepatocyte polarity is well-known to be critical for liver function, little is presently known about its involvement during the liver stage of Plasmodium development. Apical domains of hepatocytes are critical components of their polarity machinery and constitute the bile canalicular network, which is central to liver function. Here, we employed high resolution 3-D imaging and advanced image analysis of Plasmodium-infected liver tissues to show that the parasite associates preferentially with the apical domain of hepatocytes and induces alterations in the organization of these regions, resulting in localized changes in the bile canalicular architecture in the liver tissue. Pharmacological perturbation of the bile canalicular network by modulation of AMPK activity reduces the parasite's association with bile canaliculi and arrests the parasite development. Our findings using Plasmodium-infected liver tissues reveal a host-Plasmodium interaction at the level of liver tissue organization. We demonstrate for the first time a role for bile canaliculi, a central component of the hepatocyte polarity machinery, during the liver stage of Plasmodium development.

Öhlund et al. develop a three-dimensional co-culture platform of neoplastic pancreatic ductal organoids and pancreatic stellate cells (PSCs) to characterize the dynamic crosstalk between cancer cells and stromal cells, and to address stromal heterogeneity. The co-cultures reveal the co-existence of two phenotypically distinct populations of PSCs, providing insights into PDA biology and prompting a reconsideration of interventional strategies., Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated αSMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development.

Several studies have highlighted the role of the gut microbiota in colorectal cancer (CRC) progression. Patients with CRC harbor gut microbiomes that differ in structure and function from those of healthy individuals, suggesting this altered microbiome could contribute to carcinogenesis. Despite the increasing evidence implicating the gut microbiome in CRC, the collective role of different microbial consortia in CRC carcinogenesis is unclear. Using a combination of 16S rRNA amplicon sequencing, transcriptomics, and metagenomics, we assessed the capacity of the gut microbiota to shape the tumor microbiome, modulate the immune system and, ultimately, affect tumor growth. Here, we found that tumor biopsy tissue from patients with a “high-risk” Pathogen-type microbiome had a different immune transcriptome from those with a “low-risk” Lachnospiraceae-type microbiome. Transplantation from patients of the two fecal microbiome types into mice with an orthotopic tumor differentially affected tumor growth and the systemic anti-tumor immune response. The differences in tumor volume and immunophenotype between mice receiving the high-risk and the low-risk microbiome correlated with differences in the engrafted human microbial species and predicted microbiome-encoded metabolites in the two groups. These data suggest that the configuration of the gut microbiome may influence colon cancer progression and disease outcome by modulating the anti-tumor immune response.

C-C chemokine receptor type 2 (CCR2) expressed on monocytes facilitates their recruitment to tumors. Here, Fein et al. show that CCR2 signaling in cancer cells suppresses immune control of tumors, in part by reducing CD103+ dendritic cell recruitment., C-C chemokine receptor type 2 (CCR2) is expressed on monocytes and facilitates their recruitment to tumors. Though breast cancer cells also express CCR2, its functions in these cells are unclear. We found that Ccr2 deletion in cancer cells led to reduced tumor growth and approximately twofold longer survival in an orthotopic, isograft breast cancer mouse model. Deletion of Ccr2 in cancer cells resulted in multiple alterations associated with better immune control: increased infiltration and activation of cytotoxic T lymphocytes (CTLs) and CD103+ cross-presenting dendritic cells (DCs), as well as up-regulation of MHC class I and down-regulation of checkpoint regulator PD-L1 on the cancer cells. Pharmacological or genetic targeting of CCR2 increased cancer cell sensitivity to CTLs and enabled the cancer cells to induce DC maturation toward the CD103+ subtype. Consistently, Ccr2−/− cancer cells did not induce immune suppression in Batf3−/− mice lacking CD103+ DCs. Our results establish that CCR2 signaling in cancer cells can orchestrate suppression of the immune response., Graphical Abstract

Neutrophils, the most abundant type of leukocytes in blood, can form neutrophil extracellular traps (NETs). These are pathogen-trapping structures generated by expulsion of the neutrophil's DNA with associated proteolytic enzymes. NETs produced by infection can promote cancer metastasis. We show that metastatic breast cancer cells can induce neutrophils to form metastasis-supporting NETs in the absence of infection. Using intravital imaging, we observed NET-like structures around metastatic 4T1 cancer cells that had reached the lungs of mice. We also found NETs in clinical samples of triple-negative human breast cancer. The formation of NETs stimulated the invasion and migration of breast cancer cells in vitro. Inhibiting NET formation or digesting NETs with deoxyribonuclease I (DNase I) blocked these processes. Treatment with NET-digesting, DNase I-coated nanoparticles markedly reduced lung metastases in mice. Our data suggest that induction of NETs by cancer cells is a previously unidentified metastasis-promoting tumor-host interaction and a potential therapeutic target.

High expression of the C-C chemokine receptor type 2 (CCR2) in tumor samples and serum correlates with poor prognosis in human breast cancer, which has sparked interest in targeting the CCR2 pathway for therapeutic benefit. Importantly, a human CCR2 inhibitor (PF-04136309) has been investigated in clinical trials, and is currently being studied in a phase 1b/2 trial for pancreatic cancer (NCT02732938). Previous studies have focused on the protumor effects of CCR2 signaling in inflammatory monocytes. However, CCR2 is also expressed in breast cancer cells, but its potential function in cancer cells is poorly understood. To determine the specific roles of CCR2 in cancer and host cells, respectively, we established an orthotopic breast cancer mouse model by injecting primary cancer cells from MMTV-PyMT; Ccr2+/+ or MMTV-PyMT; Ccr2-/- mice into the mammary glands of wide-type or Ccr2 null hosts. In this cancer model, deleting Ccr2 in host cells, including monocytes, did not alter tumor growth. However, when tumor cells had lost Ccr2, immune surveillance was much more efficient, with greater infiltration and expansion of cytotoxic CD8+ T cell lymphocytes (CTLs) that efficiently recognized and destroyed the cancer cells, resulting in significant inhibition of tumor growth. Consistently, the delayed tumor growth of Ccr2 null cancer cells was fully reversed when these same cancer cells were transplanted into athymic (immunodeficient) mice. The reduced growth of tumors derived from Ccr2-/- cancer cells was associated with upregulation of IFN-γ response genes and genes involved in MHC class I presentation, as well as decreased expression of checkpoint inhibitor PD-L1 in cancer cells. Finally, greater infiltration of CD103+ dendritic cells (DCs) in Ccr2-/- tumor microenvironment also contributed to increased CTL function by cross presentation. Taken together, our results show that CCR2-expressing breast cancer cells orchestrated global changes in the infiltration of CTLs and DCs, leading to effective immune suppression. This suggests that CCR2 may be an immunotherapeutic target in breast cancer. Citation Format: Miriam R. Fein, Xue-Yan He, Ana S. Almeida, Arnaud Pommier, Emilis Bružas, Anaïs Eberhardt, John Erby Wilkinson, Camila dos Santos, Mikala Egeblad. Targeting cancer cell CCR2 enhances synergistic immune surveillance in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B42.

Rhinoscleroma is a human specific chronic granulomatous infection of the nose and upper airways caused by the Gram-negative bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. Although considered a rare disease, it is endemic in low-income countries where hygienic conditions are poor. A hallmark of this pathology is the appearance of atypical foamy monocytes called Mikulicz cells. However, the pathogenesis of rhinoscleroma remains poorly investigated. Capsule polysaccharide (CPS) is a prominent virulence factor in bacteria. All K. rhinoscleromatis strains are of K3 serotype, suggesting that CPS can be an important driver of rhinoscleroma disease. In this study, we describe the creation of the first mutant of K. rhinoscleromatis, inactivated in its capsule export machinery. Using a murine model recapitulating the formation of Mikulicz cells in lungs, we observed that a K. rhinoscleromatis CPS mutant (KR cps-) is strongly attenuated and that mice infected with a high dose of KR cps- are still able to induce Mikulicz cells formation, unlike a K. pneumoniae capsule mutant, and to partially recapitulate the characteristic strong production of IL-10. Altogether, the results of this study show that CPS is a virulence factor of K. rhinoscleromatis not involved in the specific appearance of Mikulicz cells., Author summary Rhinoscleroma is a human specific chronic infection characterized by the formation of granuloma in the nose and upper airways. It is a rare disease endemic in low-income countries where hygienic conditions are poor and caused by the Gram-negative bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. A hallmark of this pathology is the appearance of atypical foamy monocytes called Mikulicz cells. Very little is known about the cellular and molecular mechanisms underlying this disease and the bacterial virulence factors of K. rhinoscleromatis are unknown. In this study, we created the first mutant made in K. rhinoscleromatis and inactivated the production of capsule, an outer-membrane-anchored polysaccharide. Using a murine model recapitulating the formation of Mikulicz cells and this bacterial capsule mutant, we observed that capsule is a virulence factor for K. rhinoscleromatis which is not required for the formation of Mikulicz cells, indicating that other specific virulence factors are present in the genome of the bacterium. This works opens the way to further genetic analysis of K. rhinoscleromatis and identification of new specific virulence factors.

Protein glutathionylation is a posttranslational process that regulates protein function in response to redox cellular changes. Furthermore, carbon monoxide-induced cellular pathways involve reactive oxygen species (ROS) signaling and mitochondrial protein glutathionylation. Herein, it is described a technique to assess mitochondrial glutathionylation due to low concentrations of CO exposure. Mitochondria are isolated from cell culture or tissue, followed by an immunoprecipitation assay, which allows the capture of any glutathionylated mitochondrial protein using a specific antibody coupled to a solid matrix that binds to glutathione antigen. The precipitated protein is further identified and quantified by immunoblotting analysis.

Background: Klebsiella pneumoniae strains are pathogenic to animals and humans, in which they are both a frequent cause of nosocomial infections and a re-emerging cause of severe community-acquired infections. K. pneumoniae isolates of the capsular serotype K2 are among the most virulent. In order to identify novel putative virulence factors that may account for the severity of K2 infections, the genome sequence of the K2 reference strain Kp52.145 was determined and compared to two K1 and K2 strains of low virulence and to the reference strains MGH 78578 and NTUH-K2044. Results: In addition to diverse functions related to host colonization and virulence encoded in genomic regions common to the four strains, four genomic islands specific for Kp52.145 were identified. These regions encoded genes for the synthesis of colibactin toxin, a putative cytotoxin outer membrane protein, secretion systems, nucleases and eukaryotic-like proteins. In addition, an insertion within a type VI secretion system locus included sel1 domain containing proteins and a phospholipase D family protein (PLD1). The pld1 mutant was avirulent in a pneumonia model in mouse. The pld1 mRNA was expressed in vivo and the pld1 gene was associated with K. pneumoniae isolates from severe infections. Analysis of lipid composition of a defective E. coli strain complemented with pld1 suggests an involvement of PLD1 in cardiolipin metabolism. Conclusions: Determination of the complete genome of the K2 reference strain identified several genomic islands comprising putative elements of pathogenicity. The role of PLD1 in pathogenesis was demonstrated for the first time and suggests that lipid metabolism is a novel virulence mechanism of K. pneumoniae., LMSL, AT and this work were supported by an ERA-Net Pathogenomic 2008 grant allocated to both P. Sansonetti and J. Bengoechea. ASA is a recipient of a fellowship from the Fundacao para a Ciencia e a Tecnologia from Portugal. The authors thank Stephane Salamone and David Touboul for their help with lipid analysis.

International audience; Rhinoscleroma is a human specific chronic disease characterized by the formation of granuloma in the airways, caused by the bacterium Klebsiella pneumoniae subspecies rhinoscleromatis, a species very closely related to K. pneumoniae subspecies pneumoniae. It is characterized by the appearance of specific foamy macrophages called Mikulicz cells. However, very little is known about the pathophysiological processes underlying rhinoscleroma. Herein, we characterized a murine model recapitulating the formation of Mikulicz cells in lungs and identified them as atypical inflammatory monocytes specifically recruited from the bone marrow upon K. rhinoscleromatis infection in a CCR2-independent manner. While K. pneumoniae and K. rhinoscleromatis infections induced a classical inflammatory reaction, K. rhinoscleromatis infection was characterized by a strong production of IL-10 concomitant to the appearance of Mikulicz cells. Strikingly, in the absence of IL-10, very few Mikulicz cells were observed, confirming a crucial role of IL-10 in the establishment of a proper environment leading to the maturation of these atypical monocytes. This is the first characterization of the environment leading to Mikulicz cells maturation and their identification as inflammatory monocytes.

C-C chemokine receptor type 2 (CCR2) is highly expressed in human breast cancer tissue by both cancer cells and infiltrating immune cells. High expression of CCR2 is correlated with advanced tumor stage, metastasis, and early relapse. Studies in mice have suggested that CCR2 expression on infiltrating inflammatory monocytes promotes primary tumor growth and metastasis. However, the role of CCR2 expression on cancer cells has not been well studied. To determine how CCR2 directly impacts tumor growth and metastasis, we used a murine model of luminal breast cancer, the mouse mammary tumor virus (MMTV) promoter polyoma middle T antigen (PyMT) model. We crossed MMTV-PyMT mice to Ccr2-/- mice and found that loss of CCR2 did not alter tumor onset. However, primary tumors of MMTV-PyMT;Ccr2+/+ mice grew significantly faster than tumors of MMTV-PyMT;Ccr2-/- mice. Lung metastases were also larger in MMTV-PyMT;Ccr2+/+ mice than in MMTV-PyMT;Ccr2-/- mice. We determined by RNA in situ hybridization that CCR2 was indeed expressed by both host cells and cancer cells in MMTV-PyMT tumors. To differentiate between the contributions of CCR2 expression in the tumor and host, we transplanted primary cancer cells derived from MMTV-PyMT;Ccr2+/+ and MMTV-PyMT;Ccr2-/- mice to syngeneic Ccr2+/+ and Ccr2-/- hosts. Surprisingly, CCR2 expression in the host had no significant effect on tumor growth. However, deletion of CCR2 from cancer cells resulted in delayed tumor growth and increased survival. Furthermore, when Ccr2+/+ and Ccr2-/- cancer cells were transplanted to athymic nude mice (lacking T cells), the resulting tumors no longer showed a delayed growth. Consistent with this finding, flow cytometry of infiltrating leukocytes in tumors transplanted to syngeneic mice revealed increased levels of CD8+ cytotoxic T cells and antigen-presenting CD103+ dendritic cells into Ccr2-/- tumors compared to wild type tumors. These data suggest that CCR2 expression on cancer cells regulates an immune response to the tumors. Supporting this conclusion, expression of programmed cell death ligand 1 (PD-L1) on Ccr2-/- cancer cells was reduced to just 30% of the level on wild type cancer cells, indicating that CCR2 expression on cancer cells suppresses the anti-tumor immune response. Together, our results establish a novel role for cancer cell CCR2 expression in breast cancer progression by suppression of cytotoxic T cells through an immune checkpoint. Citation Format: Miriam R. Fein, Ana S. Almeida, Anaïs Eberhardt, Mikala Egeblad. Cancer cell expression of CCR2 regulates the PD-L1/ PD-1 immune checkpoint in breast cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A063.

Rhinoscleroma is a chronic granulomatous infection of the upper airways caused by the bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. The disease is endemic in tropical and subtropical areas, but its diagnosis remains difficult. As a consequence, and despite available antibiotherapy, some patients evolve advanced stages that can lead to disfiguration, severe respiratory impairment and death by anoxia. Because identification of the etiologic agent is crucial for the definitive diagnosis of the disease, the aim of this study was to develop two simple PCR assays. We took advantage of the fact that all Klebsiella pneumoniae subsp. rhinoscleromatis isolates are (i) of capsular serotype K3; and (ii) belong to a single clone with diagnostic single nucleotide polymorphisms (SNP). The complete sequence of the genomic region comprising the capsular polysaccharide synthesis (cps) gene cluster was determined. Putative functions of the 21 genes identified were consistent with the structure of the K3 antigen. The K3-specific sequence of gene Kr11509 (wzy) was exploited to set up a PCR test, which was positive for 40 K3 strains but negative when assayed on the 76 other Klebsiella capsular types. Further, to discriminate Klebsiella pneumoniae subsp. rhinoscleromatis from other K3 Klebsiella strains, a specific PCR assay was developed based on diagnostic SNPs in the phosphate porin gene phoE. This work provides rapid and simple molecular tools to confirm the diagnostic of rhinoscleroma, which should improve patient care as well as knowledge on the prevalence and epidemiology of rhinoscleroma., Author Summary In humans, the bacterium Klebsiella pneumoniae subsp. rhinoscleromatis (also called clone Rhinoscleromatis, as it evolved from a single Klebsiella pneumoniae ancestral strain) causes rhinoscleroma, a chronic infection of the nose and throat. Identification of the bacterium from biopsies or nasal secretions is essential for diagnosis, and currently relies on the biochemical characteristics of clone Rhinoscleromatis and on detection of its capsule of antigenic type K3. Our aim was to develop two identification tests based on amplification by polymerase chain reaction (PCR) of specific portions of the genome of clone Rhinoscleromatis. We established the sequence of the capsular polysaccharide synthesis cluster and identified one gene sequence that was unique to K3 strains. A PCR test that targets this gene was shown to be specific for K3 strains. We also exploited unique DNA signatures of clone Rhinoscleromatis to develop a second PCR test, which is specific for this clone, thus allowing distinction from other K. pneumoniae K3 strains. These novel and simple identification tests should help to diagnose rhinoscleroma and to understand the epidemiology of this disease.

A variety of immune cells, the most abundant being macrophages and T cells, typically infiltrate tumors. These immune cells can recognize and kill cancer cells. For example, macrophages activated with interferon-gamma and lipopolysaccharide can kill cancer cells both directly and indirectly, by enhancing the cytotoxic T cells' killing activities. Cancer cells, however, evade the attacking immune cells and even re-instruct macrophages and cytotoxic T cells to facilitate tumor growth, rather than halt it. It is now clear that in a clinical setting T cell deactivation can be overcome using anti-CTLA4 or anti-PDL1 antibodies, leading to tumor regression. Although exciting recoveries are experienced by many patients, the majority of patients do not have long-term benefits of such treatments. Therefore, ways are urgently needed to modify these immune activating approaches so they become beneficial to an expanded patient population. One underexplored strategy, which we are undertaking, is to re-activate the macrophages so they can kill cancer cells and further enhance the anti-tumor T cell responses. To test different strategies for re-activating macrophages, we are developing co-culture models with cancer cells, T cells and macrophages. In addition, we study interactions between these three cell types in vivo, using intravital imaging of mouse models of breast and pancreatic cancer. Our findings will offer new insights into the mechanisms by immune suppressive signals can be overcome and generate new methods for studying immune responses to cancer in real-time. Citation Format: Ana S. Almeida, Tim Kees, Mikala Egeblad. Modulating innate-adaptive immune cell interactions to achieve tumor rejection. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B62.