Immunodeficient Hosts Research Articles

Interaction of acid ceramidase inhibitor LCL521 with tumor response to photodynamic therapy and photodynamic therapy-generated vaccine.

Acid ceramidase has been identified as a promising target for cancer therapy. One of its most effective inhibitors, LCL521, was examined as adjuvant to photodynamic therapy (PDT) using mouse squamous cell carcinoma SCCVII model of head and neck cancer. Lethal effects of PDT, assessed by colony forming ability of in vitro treated SCCVII cells, were greatly enhanced when combined with 10 µM LCL521 treatment particularly when preceding PDT. When PDT-treated SCCVII cells are used to vaccinate SCCVII tumor-bearing mice (PDT vaccine protocol), adjuvant LCL521 treatment (75 mg/kg) resulted in a marked retardation of tumor growth. This effect can be attributed to the capacity of LCL521 to effectively restrict the activity of two main immunoregulatory cell populations (Tregs and myeloid-derived suppressor cells, MDSCs) that are known to hinder the efficacy of PDT vaccines. The therapeutic benefit with adjuvant LCL521 was also achieved with SCCVII tumors treated with standard PDT when using immunocompetent mice but not with immunodeficient hosts. The interaction of LCL521 with PDT-based antitumor mechanisms is dominated by immune system contribution that includes overriding the effects of immunoregulatory cells, but could also include a tacit contribution from boosting direct tumor cell kill.

CD69 Deficiency Enhances the Host Response to Vaccinia Virus Infection through Altered NK Cell Homeostasis.

During the host response to viral infection, the transmembrane CD69 protein is highly upregulated in all immune cells. We have studied the role of CD69 in the murine immune response to vaccinia virus (VACV) infection, and we report that the absence of CD69 enhances protection against VACV at both short and long times postinfection in immunocompetent and immunodeficient mice. Natural killer (NK) cells were implicated in the increased infection control, since the differences were greatly diminished when NK cells were depleted. This role of NK cells was not based on an altered NK cell reactivity, since CD69 did not affect the NK cell activation threshold in response to major histocompatibility complex class I NK cell targets or protein kinase C activation. Instead, NK cell numbers were increased in the spleen and peritoneum of CD69-deficient infected mice. That was not just secondary to better infection control in CD69-deficient mice, since NK cell numbers in the spleens and the blood of uninfected CD69(-/-) mice were already augmented. CD69-deficient NK cells from infected mice did not have an altered proliferation capacity. However, a lower spontaneous cell death rate was observed for CD69(-/-) lymphocytes. Thus, our results suggest that CD69 limits the innate immune response to VACV infection at least in part through cell homeostatic survival. We show that increased natural killer (NK) cell numbers augment the host response and survival after infection with vaccinia virus. This phenotype is found in the absence of CD69 in immunocompetent and immunodeficient hosts. As part of the innate immune system, NK lymphocytes are activated and participate in the defense against infection. Several studies have focused on the contribution of NK cells to protection against infection with vaccinia virus. In this study, it was demonstrated that the augmented early NK cell response in the absence of CD69 is responsible for the increased protection seen during infection with vaccinia virus even at late times of infection. This work indicates that the CD69 molecule may be a target of therapy to augment the response to poxvirus infection.

NLRC5 elicits antitumor immunity by enhancing processing and presentation of tumor antigens to CD8+ T lymphocytes

ABSTRACTCancers can escape immunesurveillance by diminishing the expression of MHC class-I molecules (MHC-I) and components of the antigen-processing machinery (APM). Developing new approaches to reverse these defects could boost the efforts to restore antitumor immunity. Recent studies have shown that the expression of MHC-I and antigen-processing molecules is transcriptionally regulated by NOD-like receptor CARD domain containing 5 (NLRC5). To investigate whether NLRC5 could be used to improve tumor immunogenicity, we established stable lines of B16-F10 melanoma cells expressing NLRC5 (B16-5), the T cell co-stimulatory molecule CD80 (B16-CD80) or both (B16-5/80). Cells harboring NLRC5 constitutively expressed MHC-I and LMP2, LMP7 and TAP1 genes of the APM. The B16-5 cells efficiently presented the melanoma antigenic peptide gp10025–33 to Pmel-1 TCR transgenic CD8+ T cells and induced their proliferation. In the presence of CD80, B16-5 cells stimulated Pmel-1 cells even without the addition of gp100 peptide, indicating that NLRC5 facilitated the processing and presentation of endogenous tumor antigen. Upon subcutaneous implantation, B16-5 cells showed markedly reduced tumor growth in C57BL/6 hosts but not in immunodeficient hosts, indicating that the NLRC5-expressing tumor cells elicited antitumor immunity. Following intravenous injection, B16-5 and B16-5/80 cells formed fewer lung tumor foci compared to control cells. In mice depleted of CD8+ T cells, B16-5 cells formed large subcutaneous and lung tumors. Finally, immunization with irradiated B16-5 cells conferred protection against challenge by parental B16 cells. Collectively, our findings indicate that NLRC5 could be exploited to restore tumor immunogenicity and to stimulate protective antitumor immunity.

Abstract A152: The macrophage migration inhibitory factor promotes breast cancer metastasis through interaction with the host immune response

Abstract The Macrophage Migration Inhibitory Factor (MIF) is an inflammatory cytokine that has been well-characterized as a mediator of numerous inflammatory disease processes, including in the setting of breast cancer. Ongoing research has shown that increased levels of MIF in breast cancer patient serum and biopsy samples are a marker of poor prognosis. We have previously demonstrated that expression of MIF in the primary tumor is required for spontaneous pulmonary metastasis in the 4T1 murine model of breast cancer. We hypothesized that MIF, as a cytokine, would promote metastasis by controlling some aspect of the interaction between the tumor and the host immune system. This was supported by our observation that MIF expression was no longer required for metastasis in an immunodeficient host lacking T, B, and NK cells. Using flow cytometry, we discovered that MIF expression in the primary tumor increased the abundance of a subset of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. Furthermore, systemic depletion of MDSCs from tumor-bearing mice prevented spontaneous metastasis, suggesting that the MIF-induced increase in MDSCs is likely mediating the enhanced metastasis. These observations support a model in which MIF and MDSCs may be cooperating to protect the tumor cells from immune attack not only in the primary tumor, but also at sites of metastasis. Therefore, we hypothesize that MIF and MDSCs are functioning together to promote formation of a “metastatic niche” in the lung. Our ongoing studies aim to determine if MIF expression in the primary tumor drives matrix remodeling and/or establishment of an immunosuppressive microenvironment in the lungs of tumor-bearing mice, as well as whether inhibiting MIF pharmacologically can interrupt the process of niche formation. A more complete understanding of the role MIF plays in breast cancer development and metastasis will support deployment of MIF-targeted therapeutic interventions, as well as providing critical support for studies of how MIF inhibition might be combined with other immune-targeted therapies to enhance patient survival. Citation Format: Kristen N. Balogh, Janet V. Cross. The macrophage migration inhibitory factor promotes breast cancer metastasis through interaction with the host immune response. [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 A152.

Efficient Engraftment and Disease Replication of Myelodysplastic Syndromes Using a Novel Humanized Mice Model

Abstract Myelodysplastic Syndromes (MDS) are a heterogeneous disorder of the hematopoietic stem cell caused by a large number of genetic and epigenetic alterations. With the development of novel therapeutics a reliable model to test the drugs' efficacy in correlation with genetic and epigenetic alterations and disease phenotype is essential. Recent advances in the field of MDS xenotransplantation have been achieved by transgenic expression of human cytokines in the murine host as well as by co-transplantation of primary patient derived mesenchymal stromal cells (MSCs) concurrent with MDS stem cells. However, neither model to date affords efficient transplantation of MDS at a scale that allows in vivo mechanistic studies or provides a platform to develop and test novel therapeutics. We sought to establish a MDS xenotransplantation model in humanized immunodeficient mice amenable to mechanistic in vivo studies and therapeutic testing. Several murine cytokines essential for hematopoiesis are non-crossreactive with their human counterpart. "MISTRG" mice express several human, non-crossreactive cytokines, namely M -CSF, I L-3, GM-CSF, and T hrombopoietin from the respective murine loci, as well as human macrophage receptor signal regulatory protein-alpha (S IRPα) to prevent murine macrophage-mediated immune rejection in the Rag2-/- IL2rγ-/- background (Rongvaux et al. Nature Biotech 32(4): 364 - 372, 2014). To establish a reliable, efficient MDS xenotransplantation model we optimized the host irradiation dose, transplantation route, CD34+ cell number and cell preparation. Mice were allowed to engraft for >10 weeks. Peripheral blood (PB), bone marrow (BM), and spleen were analyzed for engraftment by flow cytometry. BM, spleen, and liver were also fixed and sectioned for histologic analysis. Human CD45+ cells were sorted from engrafted MISTRG bone marrow and genomic testing was performed by cytogenetics, FISH, and/or targeted exome sequencing. MISTRG mice consistently supported higher engraftment in peripheral blood and bone marrow than NSG mice for the majority of MDS samples assessed. Out of 25 different patient's BM samples, including 6 RCMD, 5 RAEB I, 12 RAEB II, and 2 CMML patient samples, 23 samples engrafted in MISTRG mice, while 19 of the samples were transplanted concurrently into NSG mice (6 RCMD, 3 RAEB I, and 11 RAEB II patient samples) out of which 12 samples engrafted. Mice were classified as engrafted when huCD45+ cells accounted for over 1% of all nucleated cells in BM. On average, huCD45 engraftment was 7.3-fold higher in MISTRG than in NSG mice (17.78% vs. 2.45%). 56.1% of all MISTRG mice compared with 26.7% of NSG mice transplanted with MDS were engrafted. The number of engrafted MISTRG mice per sample ranged from 2-10 mice could be further improved with optimal bone marrow sample collection. We verified engraftment of the MDS clone via cytogenetics, FISH, and/or targeted exome sequencing, also revealing preserved clonal distribution and mutant allele frequencies in engrafted mice. Flow cytometric analysis of lineage differentiation revealed robust myeloid engraftment in MISTRG mice as opposed to NSG mice. In addition terminal differentiation of myeloid cells was markedly improved in MISTRG over NSG mice, with immunophenotypic concordance between engrafted MISTRG mice and the patient's primary bone marrow. Histologic analysis showed striking similarities between engrafted MISTRG bone marrow and the concurrent patient's bone marrow, such as marked dysplasia and clustering of human CD61 positive megakarocytes with resultant myelofibrosis as evident by reticulin staining. MISTRG mice lack the DNA repair defect inherent to NSG mice and are thus tolerant of chemotherapeutic agents. Studies testing hypomethylating drugs and targeted agents are now underway to establish MISTRG as promising "co-clinical" model for MDS. In conclusion, physiologic expression of essential non-crossreactive human cytokines greatly facilitates long-term engraftment of MDS patient derived CD34+ HSPCs in the murine immunodeficient host. MISTRG mice engraft lower and higher grade MDS with replication of the disease genotypes and phenotypes, supporting higher engraftment in bone marrow and blood than NSG mice for all MDS samples assessed. MISTRG mice may provide a xenotransplantation model for MDS allowing us to study the biology of the disease and to test therapeutics in vivo. Disclosures No relevant conflicts of interest to declare.

Abstract 1449: The valuable experimental model system “Cancer Xenopatient” established in NOG mice

Abstract Viable and stable human cancer materials combined with adequate clinical information are required for advance in cancer research and patient care. Conventional in vitro cancer cell lines are commonly available without enough information on the patient including disease phenotypes and drug-sensitivity. Mice bearing Patient-derived xenografts (PDXs) with clinical information (so-called “cancer xenopatients”) could be essential and useful systems to accelerate cancer medicine. In this study, we investigated the establishment efficiency of PDX using NOG mice with clinical factors of xeno-transplantaion. The NOG mouse, the NOD/Shi-scid/IL-2Rγnull (NOG) mice derived from the NOD/SCID mouse with a common gamma chain, has multifunctional defects in natural killer cell activity, macrophage function, complement activity, and dendritic cell function in addition to lacking functional T and B lymphocytes. NOG mice were reported to be the best appropriate immunodeficient host animal for direct xenografting of fresh tumor tissue. We also discuss herein its contribution for not only reliable preclinical studies of new anticancer drugs but also personalized anti-cancer therapies. Sixty-one PDX lines were established from 116 surgically removed tumor tissues inoculated into NOG mice's subcutaneous (53%). PDX lines were established from various types of epithelial tumors and also from sarcomas including gastrointestinal stromal tumor and Ewing/PNET sarcoma. The metastatic tumors yielded PDX lines effectively (65%) than the primary tumors (27%, p<0.001). The group engrafted into NOG mice after 2 days or later from the surgical removal showed a higher establishment rate (61%) than that of the group engrafted at the day of surgery or the next day (51%), however non-significant (p = 0.49). Our PDX models were preserved well not only in morphological characteristics but also in gene expression and alteration patterns. In 8 cases (7%), the transplantable xenografts for several generations were composed of monotonous large non-epithelial cell growth of human origin, revealed to be the Epstein-Barr virus (EBV) infection associated lymphoprolipherative lesions prospected by chromogenic in situ hybridization for EBV-encoded RNA. We revealed efficient establishment rates both for primary tumors and metastatic tumors. PDXs linked with clinical information will contribute to reliable preclinical studies for new anticancer drugs. The fast and efficient establishment of individual PDXs may also contribute to future personalized anti-cancer therapies. Citation Format: Tsuyoshi Chijiwa, Kenji Kawai, Akira Noguchi, Hidemitsu Sato, Akimune Hayashi, Haruhiko Cho, Manabu Shiozawa, Takeshi Kishida, Soichiro Morinaga, Tomoyuki Yokose, Makoto Katayama, Nobuo Takenaka, Hiroshi Suemizu, Roppei Yamada, Yoshiyasu Nakamura, Yasuo Takano, Kohzoh Imai, Yohei Miyagi, Masato Nakamura. The valuable experimental model system “Cancer Xenopatient” established in NOG mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1449. doi:10.1158/1538-7445.AM2015-1449

Abstract 417: Endosome-Based Activation of Non-Canonical NF-kB Signaling by Complement Membrane Attack Complexes

Using sera from transplant patients with high-titer panel reactive antibody (PRA), we previously described a model for studying the effects of alloantibody binding and complement activation on human endothelial cells (EC). With this model we found that complement membrane attack complexes (MAC) induced rapid post-translational stabilization of NF-kB-inducing kinase (NIK), the activator of non-canonical NF-kB signaling, which was required for inflammatory changes in cultured human EC and for vasculopathic changes in human coronary artery segments implanted into immunodeficient mouse hosts. To elucidate the mechanism of NIK stabilization, we performed a genome-wide siRNA screen and discovered a novel, TRAF3-independent signaling mechanism in which MAC induces NIK stabilization on endosomes. MAC is internalized by EC in a clathrin-, AP2-, and dynamin-dependent mechanism into Rab5+ endosomes. Activated Akt is recruited in a Rab5-dependent manner to the same endosomes which then bind and stabilize NIK and subsequently recruit and activate IKK-alpha, the catalytic activator of p52/RelB complexes, the non-canonical form of NF-kB. Pharmacological inhibition of MAC endocytosis blocked this pathway both in cultured EC and in human coronary artery xenografts. These data reveal a novel endosome-based signaling cascade for activating non-canonical NF-kB and identify new targets for inhibiting complement-mediated tissue injury such as chronic antibody-mediated rejection and cardiac allograft vasculopathy.

Induction of anti-microbial immune responses in severely immunodeficient hosts by IFNγ-expressing Salmonella enterica serovar Typhimurium correlates with efficient activation of macrophage effectors (VAC8P.1050)

Abstract Susceptibility to infection by the intracellular bacterial pathogen, Salmonella enterica serovar Typhimurium (S. typhimurium), is controlled by many genes of innate and adaptive immunity. One of the most critical genes is IFN-γ and mice deficient in IFN-γ synthesis are highly susceptible to Salmonella. Previously, we also demonstrated that mice deficient in MyD88, an adaptor that regulates TLR signaling, are susceptible to Salmonella infection. In the current study, we compared immune responses in mice deficient in IFN-γ or MyD88 with wild-type controls following infection with an attenuated strain of S. typhimurium (designated BRD509) or a recombinant derivative engineered to express murine IFN-γ (GIDIFN). Infection studies with BRD509 or GIDIFN revealed that the latter strain was significantly less virulent in immunodeficient mice than BRD509 and correlated with decreased bacterial loads in systemic organs. Enhanced responsiveness was due to GIDIFN strain’s ability to activate effector macrophages, as shown by increased synthesis of inflammatory cytokines and anti-microbial effector molecules, including NO. Gene expression profiling by qPCR demonstrated stronger induction of key inflammatory modulators by GIDIFN in macrophages of immunodeficient animals. These findings suggest that immunotherapeutic approaches using attenuated bacterial strains expressing immunomodulatory genes is more efficacious and offers a superior safety profile even in severely immunodeficient hosts.

Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function.

Short bowel syndrome (SBS) is a devastating condition in which insufficient small intestinal surface area results in malnutrition and dependence on intravenous parenteral nutrition. There is an increasing incidence of SBS, particularly in premature babies and newborns with congenital intestinal anomalies. Tissue-engineered small intestine (TESI) offers a therapeutic alternative to the current standard treatment, intestinal transplantation, and has the potential to solve its biggest challenges, namely donor shortage and life-long immunosuppression. We have previously demonstrated that TESI can be generated from mouse and human small intestine and histologically replicates key components of native intestine. We hypothesized that TESI also recapitulates native small intestine function. Organoid units were generated from mouse or human donor intestine and implanted into genetically identical or immunodeficient host mice. After 4 wk, TESI was harvested and either fixed and paraffin embedded or immediately subjected to assays to illustrate function. We demonstrated that both mouse and human tissue-engineered small intestine grew into an appropriately polarized sphere of intact epithelium facing a lumen, contiguous with supporting mesenchyme, muscle, and stem/progenitor cells. The epithelium demonstrated major ultrastructural components, including tight junctions and microvilli, transporters, and functional brush-border and digestive enzymes. This study demonstrates that tissue-engineered small intestine possesses a well-differentiated epithelium with intact ion transporters/channels, functional brush-border enzymes, and similar ultrastructural components to native tissue, including progenitor cells, whether derived from mouse or human cells.

Probing Human NK Cell Biology Using Human Immune System (HIS) Mice.

Our incomplete understanding of the mechanisms that orchestrate human lymphocyte differentiation and condition human immune responses is in part due to the limited access to normal human tissue samples that can inform on these complex processes. In addition, in vitro culture conditions fail to recapitulate the three-dimensional microenvironments that influence cell-cell interactions and impact on immune outcomes. Small animals provide a preclinical model to dissect and probe immunity and over the past decades, development of immunodeficient hosts that can be engrafted with human hematopoietic precursors and mature cells have led to the development of new in vivo models to study human lymphocyte development and function. Natural killer (NK) cells are implicated in the recognition and elimination of pathogen-infected and transformed cells and belong to a family of diverse innate lymphoid cells (ILCs) that provide early immune defense against disease. Here, we summarize the use of humanized mouse models for the study of NK cell and group 1 ILCs and their respective roles in immunity and tissue homeostasis.

Abstract B50: A novel immunocompetent murine model for replicating oncolytic adenoviral therapy

Abstract Oncolytic adenoviruses are able to replicate selectively in cancerous but not normal human cells and are under investigation as potential cancer therapeutics. Unfortunately, mice are poor model systems for therapy with replication competent adenoviruses because murine cells are generally unable to produce infectious viral progeny. In order to model oncolytic adenovirus treatment, researchers have used human tumor xenografts that support viral replication in immunodeficient mice. Because xenografts require an immunodeficient host, xenograft model systems do not reflect a host's adaptive immune response against the virus and cannot model the potential for an immunostimulatory virus to induce an anti-tumor immune response. Using the tumor-selective oncolytic adenovirus TAV-255 previously developed in our laboratory, we find that the murine lung adenocarcinoma cell line LKR-13 supports adenoviral infection and generates infectious viral progeny. Western blotting showed that LKR-13 cells express the coxsackie and adenovirus receptor (CAR) to allow viral infection, and that infected cells express the viral protein E1A. MTT assays, crystal violet staining, and clonogenic assays showed that TAV-255 kills LKR-13 cells in a dose-dependent and time-dependent manner with similar potency and latency of infection as a panel of human cancer cell lines, with more than 50% cell death of murine LKR-13 cells and various human cancer cells (T1, 882 and A549) within 6 days of infection at a multiplicity of infection of 10. It produces infectious progeny from LKR-13 cells at titres similar to other human cancer cell lines. Other tested murine cancer cell lines do not share these features; for example, the CT-26 colon cancer cell line does not express CAR and is resistant TAV-255 infection, while the murine sarcoma cell line F244 is readily infected by adenovirus and produces the viral protein E1A but does not produce infectious progeny. Based on these in-vitro findings, we investigated LKR-13 cells as an in-vivo model system for replicating oncolytic adenoviruses. The LKR-13 cell line is derived from 129/Sv mice, and using fully immunocompetent 129/s4 mice, subcutaneous injection of 5x106 LKR-13 cells led to tumor formation in all injected mice. Intratumoral injection of 3x107 plaque forming units of TAV-255 in established tumors (greater than 200 mm3) caused a significant reduction in tumor growth: Eight days after treatment, tumors in mice treated with TAV-255 were 339 mm3 +/- 26 mm3 (n=3), and tumors in mice treated with PBS were 578 mm3 +/- 49 mm3 (n=5) (p=0.013). This model system represents the first fully immunocompetent mouse model for cancer treatment with replicating oncolytic adenoviruses, and will be useful to study the therapeutic effect of oncolytic adenoviruses in general and particularly immunostimulatory viruses designed to evoke an anti-tumor immune response. Citation Format: Lingzhi Zhang, Farah Hedjran, Christopher Larson, Min Yan, Tony Reid. A novel immunocompetent murine model for replicating oncolytic adenoviral therapy. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B50.

Wingless ligand Wnt5a promotes proliferation and inhibits apoptosis of first trimester trophoblasts

Wnts (wingless and int-related proteins) are a family of secreted cysteine-rich glycoproteins, expressed in a variety of tissues in developing embryos, thought to be involved in cell fate specification and stem cell commitment. To identify the specific Wnts involved in osteoblastic differentiation of human mesenchymal stem cells (hMSCs), we performed degenerative RT-PCR cloning method to amplify Wnt-encoding cDNAs expressed during osteoblastic differentiation of hMSCs in vitro and during hMSC-directed ectopic osteogenesis in the severe combined immunodeficient (SCID) mouse host. WNT5A was found to be the dominant Wnt expressed during osteoblastic differentiation of hMSCs both in vitro and in vivo. RT-PCR further revealed that hWNT5A and its receptor Frizzled family member 5 (hFZD5) was up-regulated during osteoblastic differentiation compared to uncommitted hMSCs. To evaluate the function of Wnt5a, calvarial cells were obtained from Wnt5a−/−, Wnt5a+/−, and wild type mice. Wnt5a−/− cells showed significantly slower growth when compared to Wnt5a+/− and wild type cells. Gene expression profiles of the Wnt5a−/− calvarial cells as compared to wild type cells were evaluated using microarray analysis. 255 genes exhibited at least 2-fold changes in expression. Clusters of genes regulating cell cycle, cell proliferation and cell growth, and gene transcription were altered with absence of Wnt5a expression. In addition, genes regulating osteoblastic differentiation including Runx2, osterix, and alkaline phosphatase (ALP) were shown to be down-regulated in Wnt5a−/− cells. In conclusion, Wnt5a is highly expressed during osteoblastic differentiation. Its function during mesenchymal stem cell differentiation as well as cell growth was suggested by comparing the gene expression profile of calvarial cells from the Wnt5a−/− and wild type mice.

CD40 ligand preferentially modulates immune response and enhances protection against influenza virus.

CD40L, a key regulator of the immune system, was studied as both a targeting ligand and a molecular adjuvant in nucleoprotein (NP)-based host defense against influenza in mouse models with different genetic backgrounds. Adenoviral vectors secreting NP-CD40L fusion protein (denoted as rAd-SNP40L) afforded full protection of immunocompetent and immunocompromised mice (CD40L(-/-) and CD4(-/-)) against lethal influenza infection. Mechanistically, rAd-SNP40L preferentially induced early and persistent B cell germinal center formation, and accelerated Ig isotype-switching and Th1-skewed, NP-specific Ab response. Moreover, it drastically augmented primary and memory NP-specific CTL activity and polyfunctional CD8(+) T cells. The markedly enhanced nonneutralizing Abs and CTLs significantly reduced viral burdens in the lungs of mice upon lethal virus challenge. Data generated from CD40L(-/-) and CD4(-/-) mice revealed that the protection was indeed CD40L mediated but CD4(+) T cell independent, demonstrating the viability of the fusion Ags in protecting immunodeficient hosts. Notably, a single dose of rAd-SNP40L completely protected mice from lethal viral challenge 4 mo after immunization, representing the first report, to our knowledge, on NP in conjunction with a molecular adjuvant inducing a robust and long-lasting memory immune response against influenza. This platform is characterized by an increased in vivo load of CD40-targeted Ag upon the secretion of the fusion protein from adenovirus-infected cells and may represent a promising strategy to enhance the breadth, durability, and potency of Ag-specific immune responses.

IL-15 enhances the anti-tumor effect of human tumor antigen-specific T cells by cellular senescence delay (TUM2P.880)

Abstract Optimal expansion protocol for adoptive T-cell therapy often include interleukin (IL)-15, however, the mechanism by which IL-15 improves the in vivo anti-tumor effect of T cells remains to be elucidated. Using T cells generated from HLA-A2+ normal donors against novel T-cell epitopes derived from the U266 myeloma Ig light chain V-region (idiotype) as a model, we confirmed that T cells cultured with IL-15 provide more potent anti-tumor effects than IL-2-cultured T cells, after adoptive transfer in vivo into immunodeficient hosts. This effect was associated with delayed/reversed senescence in IL-15 expanded idiotype-specific memory CD8 T cells. In vitro experiments revealed IL-15 mediated senescence delay/reversal through JAK3-STAT5 and IL-2 receptor βγ signaling pathway. Specifically, we found DNA damage molecules MDC1, 53BP1, and ATM are the direct targets of IL-15 signaling and STAT5 represses the promoter activity of these genes through the competition with STAT3 to bind to the STATs sites. Thus, our study provides the first evidence for IL-15-inducing the repression of transcription of DNA damage molecules and senescence delay in human memory CD8 T cells.

Elimination of Progressive Mammary Cancer by Repeated Administrations of Chimeric Antigen Receptor-Modified T Cells

Continuous oncogenic processes that generate cancer require an on-going treatment approach to eliminate the transformed cells, and prevent their further development. Here, we studied the ability of T cells expressing a chimeric antibody-based receptor (CAR) to offer a therapeutic benefit for breast cancer induced by erbB-2. We tested CAR-modified T cells (T-bodies) specific to erbB-2 for their antitumor potential in a mouse model overexpressing a human erbB-2 transgene that develops mammary tumors. Comparing the antitumor reactivity of CAR-modified T cells under various therapeutic settings, either prophylactic, prior to tumor development, or therapeutically. We found that repeated administration of CAR-modified T cells is required to eliminate spontaneously developing mammary cancer. Systemic, as well as intratumoral administered CAR-modified T cells accumulated at tumor sites and eventually eliminated the malignant cells. Interestingly, within a few weeks after a single CAR T cells' administration, and rejection of primary lesion, tumors usually relapsed both in treated mammary gland and at remote sites; however, repeated injections of CAR-modified T cells were able to control the secondary tumors. Since spontaneous tumors can arise repeatedly, especially in the case of syndromes characterized by specific susceptibility to cancer, multiple administrations of CAR-modified T cells can serve to control relapsing disease.

Multifunctional CD26hi Th17 cells eradicate large human tumors (TUM2P.902)

Abstract Human CD4+ T cells differentiate into multiple effector subsets, but their distinct roles in anti-tumor immunity remain elusive. CD4+ T cell subsets enriched from bulk human CD4+ T cells [Th1 (CXCR3+), Th2 (CCR4+), and Th17 (CCR6+ or CD26hi)] were stimulated with anti-CD3 beads bearing agonists to either CD28 or ICOS and were then engineered with a chimeric antigen receptor that recognizes human mesothelioma. In vitro, ICOS costimulation proved superior to CD28 for augmenting the function of human Th1, Th2, CCR6+ Th17 and CD26hi Th17 cells, as indicated by elevated production of IFN-γ, IL-4 and IL-17A, respectively. Moreover, CD26hi Th17 cells possessed strikingly enhanced polyfunctionality compared to Th1, Th2 or CCR6+ Th17 cells, as demonstrated by their heightened capacity to co-secrete IL-17A, IFN-γ, IL-22, IL-2, and TNF-α simultaneously. Compared to other enriched T cell subsets, a greater percentage of CD26hi Th17 cells exhibit an effector memory phenotype. In vivo, CD26hi Th17 cells more efficiently reconstituted immunodeficient hosts and persisted long-term. Furthermore, CD26hi Th17 cells possessed a superior ability to kill large human tumors (>150mm2) when infused into mice compared to Th1, Th2 or CCR6+ Th17 cells. These results suggest that the generation of multifunctional, long-lived human Th17 populations could be instrumental to the design of novel, effective T cell-based cancer therapies.

Human Skeletal Muscle–derived CD133+ Cells Form Functional Satellite Cells After Intramuscular Transplantation in Immunodeficient Host Mice

Stem cell therapy is a promising strategy for treatment of muscular dystrophies. In addition to muscle fiber formation, reconstitution of functional stem cell pool by donor cells is vital for long-term treatment. We show here that some CD133(+) cells within human muscle are located underneath the basal lamina of muscle fibers, in the position of the muscle satellite cell. Cultured hCD133(+) cells are heterogeneous and multipotent, capable of forming myotubes and reserve satellite cells in vitro. They contribute to extensive muscle regeneration and satellite cell formation following intramuscular transplantation into irradiated and cryodamaged tibialis anterior muscles of immunodeficient Rag2-/γ chain-/C5-mice. Some donor-derived satellite cells expressed the myogenic regulatory factor MyoD, indicating that they were activated. In addition, when transplanted host muscles were reinjured, there was significantly more newly-regenerated muscle fibers of donor origin in treated than in control, nonreinjured muscles, indicating that hCD133(+) cells had given rise to functional muscle stem cells, which were able to activate in response to injury and contribute to a further round of muscle regeneration. Our findings provide new evidence for the location and characterization of hCD133(+) cells, and highlight that these cells are highly suitable for future clinical application.

Multiresistant Elizabethkingia meningoseptica infections in tertiary care

Elizabethkingia meningoseptica is an emerging pathogen known to cause meningitis, pneumonia, endocarditis, bacteremia, sepsis, wound, soft tissue, abdominal, respiratory and ocular infections, dialysis associated peritonitis and prosthesis associated septic arthritis; especially in immunodeficient hosts.1,2 It is ubiquitous Gram-negative nonfermenter found in soil, plants, fresh and marine water, food products, hospital environments, chlorinated water and may colonize sinks and taps causing both community and nosocomial infections, though does not constitute human microflora.1 It has many attributes of Gram-positive bacteria which confuse its characterization in routine diagnostic settings. Elizabethkingia is an eponym from its discoverer Elizabeth O. King. Earlier classified under CDC group IIa, Flavobacterium (1959) and Chryseobacterium (1994), it was assigned the genus Elizabethkingia under Flavobacteriaceae family in 2005 based on 16s rRNA phylogenetic studies.3 Few infections of E. meningoseptica have been reported from India. We report four cases of Elizabethkingia meningoseptica from an apex tertiary care setup.

Tumor-derived inducible heat-shock protein 70 (HSP70) is an essential component of anti-tumor immunity

The anti-apoptotic function and tumor-associated expression of heat-shock protein 70 (HSP70) is consistent with HSP70 functioning as a survival factor to promote tumorigenesis. However, its immunomodulatory activities to induce anti-tumor immunity predict the suppression of tumor growth. Using the Hsp70.1/3(-/-)(Hsp70(-/-)) mouse model, we observed that tumor-derived HSP70 was neither required for cellular transformation nor for in vivo tumor growth. Hsp70(-/-)murine embryonic fibroblasts (MEFs) were transformed by E1A/Ras and generated tumors in immunodeficient hosts as efficiently as wild-type (WT) transformants. Comparison of Bcr-Abl-mediated transformation of WT and Hsp70(-/-)bone marrow and progression of B-cell leukemogenesis in vivo revealed no differences in disease onset or survival rates, and Eμ-Myc-driven lymphoma in Hsp70(-/-)mice was phenotypically indistinguishable from that in WT Eμ-Myc mice. However, Hsp70(-/-)E1A/Ras MEFs generated significantly larger tumors than their WT counterparts in C57BL/6 J immune-competent hosts. Concurrent with this was a reduction in intra-tumoral infiltration of innate and adaptive immune cells, including macrophages and CD8(+) T cells. Evaluation of several potential mechanisms revealed an HSP70-chemokine-like activity to promote cellular migration. These observations support a role for tumor-derived HSP70 in facilitating anti-tumor immunity to limit tumor growth and highlight the potential consequences of anti-HSP70 therapy as an efficacious anti-cancer strategy.

Contribution of acidic melanoma cells undergoing epithelial-to-mesenchymal transition to aggressiveness of non-acidic melanoma cells

Tumor cell plasticity largely depends on epithelial-to-mesenchymal transition (EMT) and its reversion. It was ascertained that EMT characterizes disease progression, including melanoma malignancy. As most solid tumors, melanoma shows extracellular acidosis, we analyse the impact of acidic environment on the EMT development in human melanoma cells. Melanoma cells were exposed to an acidic extracellular environment (pH 6.7) and tested for EMT markers. We found that acidic cells express a significant up-regulation of mesenchymal markers (N-cadherin, Vimentin), transcription factors (Twist, NF-κB) and a significant, although modest, reduction of E-cadherin expression. Acidic cell also express an increased invasiveness through Matrigel associated with an up-regulation of MMP-9 activity. When we injected acidic cells intravenously into immunodeficient animals, we found a number of lung micrometastases not different from non-acidic cells. Indeed, they show a partial G1 cell cycle arrest, which might interfere with the growth of lung colonies. When we investigated the in vitro invasiveness and lung colonization of a mixed population of acidic and non acidic melanoma cells, we found that acidic cells promote in vitro invasiveness of non-acidic cells and this cooperation leads to an higher migration rate than acidic cells. Moreover, acidic cells cooperate for a better lung colonization of non-acidic cells, that represent the greater part of cells participating to lung micrometastases. We found evidence that acidity triggers in melanoma cells an EMT program, which although "incomplete", potentiates migration rate and development of lung colonies into immunodeficient host of cells grown in standard pH.