Rationale for the clinical use of adipose-derived mesenchymal stem cells for COVID-19 patients.

In late 2019, a novel coronavirus (SARS-CoV-2) emerged in Wuhan, capital city of Hubei province in China. Cases of SARS-CoV-2 infection quickly grew by several thousand per day. Less than 100 days later, the World Health Organization declared that the rapidly spreading viral outbreak had become a global pandemic. Coronavirus disease 2019 (COVID-19) is typically associated with fever and respiratory symptoms. It often progresses to severe respiratory distress and multi-organ failure which carry a high mortality rate. Older patients or those with medical comorbidities are at greater risk for severe disease. Inflammation, pulmonary edema and an over-reactive immune response can lead to hypoxia, respiratory distress and lung damage. Mesenchymal stromal/stem cells (MSCs) possess potent and broad-ranging immunomodulatory activities. Multiple in vivo studies in animal models and ex vivo human lung models have demonstrated the MSC's impressive capacity to inhibit lung damage, reduce inflammation, dampen immune responses and aid with alveolar fluid clearance. Additionally, MSCs produce molecules that are antimicrobial and reduce pain. Upon administration by the intravenous route, the cells travel directly to the lungs where the majority are sequestered, a great benefit for the treatment of pulmonary disease. The in vivo safety of local and intravenous administration of MSCs has been demonstrated in multiple human clinical trials, including studies of acute respiratory distress syndrome (ARDS). Recently, the application of MSCs in the context of ongoing COVID-19 disease and other viral respiratory illnesses has demonstrated reduced patient mortality and, in some cases, improved long-term pulmonary function. Adipose-derived stem cells (ASC), an abundant type of MSC, are proposed as a therapeutic option for the treatment of COVID-19 in order to reduce morbidity and mortality. Additionally, when proven to be safe and effective, ASC treatments may reduce the demand on critical hospital resources. The ongoing COVID-19 outbreak has resulted in significant healthcare and socioeconomic burdens across the globe. There is a desperate need for safe and effective treatments. Cellular based therapies hold great promise for the treatment of COVID-19. This literature summary reviews the scientific rationale and need for clinical studies of adipose-derived stem cells and other types of mesenchymal stem cells in the treatment of patients who suffer with COVID-19.

Delivery of oncolytic vaccinia virus by matched allogeneic stem cells overcomes critical innate and adaptive immune barriers.

BACKGROUND: Previous studies have identified IFNγ as an important early barrier to oncolytic viruses including vaccinia. The existing innate and adaptive immune barriers restricting oncolytic virotherapy, however, can be overcome using autologous or allogeneic mesenchymal stem cells as carrier cells with unique immunosuppressive properties. METHODS: To test the ability of mesenchymal stem cells to overcome innate and adaptive immune barriers and to successfully deliver oncolytic vaccinia virus to tumor cells, we performed flow cytometry and virus plaque assay analysis of ex vivo co-cultures of stem cells infected with vaccinia virus in the presence of peripheral blood mononuclear cells from healthy donors. Comparative analysis was performed to establish statistically significant correlations and to evaluate the effect of stem cells on the activity of key immune cell populations. RESULTS: Here, we demonstrate that adipose-derived stem cells (ADSCs) have the potential to eradicate resistant tumor cells through a combination of potent virus amplification and sensitization of the tumor cells to virus infection. Moreover, the ADSCs demonstrate ability to function as a virus-amplifying Trojan horse in the presence of both autologous and allogeneic human PBMCs, which can be linked to the intrinsic immunosuppressive properties of stem cells and their unique potential to overcome innate and adaptive immune barriers. The clinical application of ready-to-use ex vivo expanded allogeneic stem cell lines, however, appears significantly restricted by patient-specific allogeneic differences associated with the induction of potent anti-stem cell cytotoxic and IFNγ responses. These allogeneic responses originate from both innate (NK)- and adaptive (T)- immune cells and might compromise therapeutic efficacy through direct elimination of the stem cells or the induction of an anti-viral state, which can block the potential of the Trojan horse to amplify and deliver vaccinia virus to the tumor. CONCLUSIONS: Overall, our findings and data indicate the feasibility to establish simple and informative assays that capture critically important patient-specific differences in the immune responses to the virus and stem cells, which allows for proper patient-stem cell matching and enables the effective use of off-the-shelf allogeneic cell-based delivery platforms, thus providing a more practical and commercially viable alternative to the autologous stem cell approach.

First-in-human study of TK-positive oncolytic vaccinia virus delivered by adipose stromal vascular fraction cells.

BACKGROUND: ACAM2000, a thymidine kinase (TK)-positive strain of vaccinia virus, is the current smallpox vaccine in the US. Preclinical testing demonstrated potent oncolytic activity of ACAM2000 against several tumor types. This Phase I clinical trial of ACAM2000 delivered by autologous adipose stromal vascular fraction (SVF) cells was conducted to determine the safety and feasibility of such a treatment in patients with advanced solid tumors or acute myeloid leukemia (AML). METHODS: Twenty-four patients with solid tumors and two patients with AML participated in this open-label, non-randomized dose-escalation trial. All patients were treated with SVF derived from autologous fat and incubated for 15 min to 1 h with ACAM2000 before application. Six patients received systemic intravenous application only, one patient received intra-tumoral application only, 15 patients received combination intravenous with intra-tumoral deployment, 3 patients received intravenous and intra-peritoneal injection and 1 patient received intravenous, intra-tumoral and intra-peritoneal injections. Safety at each dose level of ACAM2000 (1.4 × 106 plaque-forming units (PFU) to 1.8 × 107 PFU) was evaluated. Blood samples for PK assessments, flow cytometry and cytokine analysis were collected at baseline and 1 min, 1 h, 1 day, 1 week, 1 month, 3 months and 6 months following treatment. RESULTS: No serious toxicities (> grade 2) were reported. Seven patients reported an adverse event (AE) in this study: self-limiting skin rashes, lasting 7 to 18 days-an expected adverse reaction to ACAM2000. No AEs leading to study discontinuation were reported. Viral DNA was detected in all patients' blood samples immediately following treatment. Interestingly, in 8 patients viral DNA disappeared 1 day and re-appeared 1 week post treatment, suggesting active viral replication at tumor sites, and correlating with longer survival of these patients. No major increase in cytokine levels or correlation between cytokine levels and skin rashes was noted. We were able to assess some initial efficacy signals, especially when the ACAM2000/SVF treatment was combined with checkpoint inhibition. CONCLUSIONS: Treatment with ACAM2000/SVF in patients with advanced solid tumors or AML is safe and well tolerated, and several patients had signals of an anticancer effect. These promising initial clinical results merit further investigation of therapeutic utility. Trial registration Retrospectively registered (ISRCTN#10201650) on October 22, 2018.

Artificial human antigen-presenting cells are superior to dendritic cells at inducing cytotoxic T-cell responses.

Peptide recognition through the MHC class I molecule by cytotoxic T lymphocytes (CTLs) leads to the killing of cancer cells. A potential challenge for T-cell immunotherapy is that dendritic cells (DCs) are exposed to the MHC class I-peptide complex for an insufficient amount of time. To improve tumour antigen presentation to T cells and thereby initiate a more effective T-cell response, we generated artificial antigen-presenting cells (aAPCs) by incubating human immature DCs (imDCs) with poly(lactic-co-glycolic) acid nanoparticles (PLGA-NPs) encapsulating tumour antigenic peptides, followed by maturation with lipopolysaccharide. Tumour antigen-specific CTLs were then induced using either peptide-loaded mature DCs (mDCs) or aAPCs, and their activities were analysed using both ELISpot and cytotoxicity assays. We found that the aAPCs induced significantly stronger tumour antigen-specific CTL responses than the controls, which included both mDCs and aAPCs loaded with empty nanoparticles. Moreover, frozen CTLs that were generated by exposure to aAPCs retained the capability to eradicate HLA-A2-positive tumour antigen-bearing cancer cells. These results indicated that aAPCs are superior to DCs when inducing the CTL response because the former are capable of continuously presenting tumour antigens to T cells in a sustained manner. The development of aAPCs with PLGA-NPs encapsulating tumour antigenic peptides is a promising approach for the generation of effective CTL responses in vitro and warrants further assessments in clinical trials.

Colonization of xenograft tumors by oncolytic vaccinia virus (VACV) results in enhanced tumor killing due to the involvement of myeloid cells.

BACKGROUND: The mechanisms by which vaccinia virus (VACV) interacts with the innate immune components are complex and involve different mechanisms. iNOS-mediated NO production by myeloid cells is one of the central antiviral mechanisms and this study aims to investigate specifically whether iNOS-mediated NO production by myeloid cells, is involved in tumor eradication following the virus treatment. METHODS: Human colon adenocarcinoma (HCT-116) xenograft tumors were infected by VACV. Infiltration of iNOS+ myeloid cell population into the tumor, and virus titer was monitored following the treatment. Single-cell suspensions were stained for qualitative and quantitative flow analysis. The effect of different myeloid cell subsets on tumor growth and colonization were investigated by depletion studies. Finally, in vitro culture experiments were carried out to study NO production and tumor cell killing. Student's t test was used for comparison between groups in all of the experiments. RESULTS: Infection of human colon adenocarcinoma (HCT-116) xenograft tumors by VACV has led to recruitment of many CD11b+ ly6G+ myeloid-derived suppressor cells (MDSCs), with enhanced iNOS expression in the tumors, and to an increased intratumoral virus titer between days 7 and 10 post-VACV therapy. In parallel, both single and multiple rounds of iNOS-producing cell depletions caused very rapid tumor growth within the same period after virus injection, indicating that VACV-induced iNOS+ MDSCs could be an important antitumor effector component. A continuous blockade of iNOS by its specific inhibitor, L-NIL, showed similar tumor growth enhancement 7-10 days post-infection. Finally, spleen-derived iNOS+ MDSCs isolated from virus-injected tumor bearing mice produced higher amounts of NO and effectively killed HCT-116 cells in in vitro transwell experiments. CONCLUSIONS: We initially hypothesized that NO could be one of the factors that limits active spreading of the virus in the cancerous tissue. In contrast to our initial hypothesis, we observed that PMN-MDSCs were the main producer of NO through iNOS and NO provided a beneficial antitumor effect, The results strongly support an important novel role for VACV infection in the tumor microenvironment. VACV convert tumor-promoting MDSCs into tumor-killing cells by inducing higher NO production.

Overcoming tumor immune evasion with an unique arbovirus.

Combining dendritic cell vaccination with the adjuvant effect of a strain of dengue virus may be a way to overcome known tumor immune evasion mechanisms. Dengue is unique among viruses as primary infections carry lower mortality than the common cold, but secondary infections carry significant risk of hypovolemic shock. While current immuno-therapies rely on a single axis of attack, this approach combines physiological (hyperthermic reduction of tumor perfusion), immunological (activation of effector cells of the adaptive and innate immune system), and apoptosis-inducing pathways (sTRAIL) to destroy tumor cells. The premise of using multiple mechanisms of action in synergy with a decline in the ability of the tumor cells to employ resistance methods suggests the potential of this combination approach in cancer immunotherapy.

TLR4-dependent activation of dendritic cells by an HMGB1-derived peptide adjuvant.

High mobility group box protein 1 (HMGB1) acts as an endogenous danger molecule that is released from necrotic cells and activated macrophages. We have previously shown that peptide Hp91, whose sequence corresponds to an area within the B-Box domain of HMGB1, activates dendritic cells (DCs) and acts as an adjuvant in vivo. Here we investigated the underlying mechanisms of Hp91-mediated DC activation. Hp91-induced secretion of IL-6 was dependent on clathrin- and dynamin-driven endocytosis of Hp91 and mediated through a MyD88- and TLR4-dependent pathway involving p38 MAPK and NFκB. Endosomal TLR4 has been shown to activate the MyD88-independent interferon pathway. Hp91-induced activation of pIRF3 and IL-6 secretion was reduced in IFNαßR knockout DCs, suggesting an amplification loop via the IFNαßR. These findings elucidate the mechanisms by which Hp91 acts as immunostimulatory peptide and may serve as a guide for the future development of synthetic Th1-type peptide adjuvants for vaccines.

Intravenous ascorbic acid as an adjuvant to interleukin-2 immunotherapy.

Interleukin-2 (IL-2) therapy has been demonstrated to induce responses in 10-20% of advanced melanoma and renal cell carcinoma patients, which translates into durable remissions in up to half of the responsers. Unfortunately the use of IL-2 has been associated with severe toxicity and death. It has been previously observed and reported that IL-2 therapy causes a major drop in circulating levels of ascorbic acid (AA). The IL-2 induced toxicity shares many features with sepsis such as capillary leakage, systemic complement activation, and a relatively non-specific rise in inflammatory mediators such as TNF-alpha, C-reactive protein, and in advanced cases organ failure. Animal models and clinical studies have shown rapid depletion of AA in conditions of sepsis and amelioration associated with administration of AA (JTM 9:1-7, 2011). In contrast to other approaches to dealing with IL-2 toxicity, which may also interfere with therapeutic effects, AA possesses the added advantage of having direct antitumor activity through cytotoxic mechanisms and suppression of angiogenesis. Here we present a scientific rationale to support the assessment of intravenous AA as an adjuvant to decrease IL-2 mediated toxicity and possibly increase treatment efficacy.

Oncolytic vaccinia virus GLV-1h68 strain shows enhanced replication in human breast cancer stem-like cells in comparison to breast cancer cells.

BACKGROUND: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. METHODS: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44(+)CD24(+)ESA(+) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. RESULTS: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44(+)CD24(+)ESA(+) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44(+)CD24(-)ESA(+) cells. CONCLUSIONS: Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors.

Characterization of iNOS(+) Neutrophil-like ring cell in tumor-bearing mice.

BACKGROUND: Myeloid-derived Suppressor Cells (MDSC) have been identified as tumor-induced immature myeloid cells (IMC) with potent immune suppressive activity in cancer. Whereas strict phenotypic classification of MDSC has been challenging due to the highly heterogeneous nature of cell surface marker expression, use of functional markers such as Arginase and inducible nitric oxide synthase (iNOS) may represent a better categorization strategy. In this study we investigated whether iNOS could be utilized as a specific marker for the identification of a more informative homogenous MDSC subset. METHODS: Single-cell suspensions from tumors and other organs were prepared essentially by enzymatic digestion. Flow cytometric analysis was performed on a four-color flow cytometer. Morphology, intracellular structure and localization of iNOS(+) ring cells in the tumor were determined by cytospin analysis, immunofluorescence microscopy and immunohistochemistry, respectively. For functional analysis, iNOS(+) ring subset were sorted and tested in vitro cell culture experiments. Pharmacologic inhibition of iNOS was performed both in vivo and in vitro. RESULTS: The results showed that intracellular iNOS staining distinguished a granular iNOS(+) SSC(hi) CD11b(+) Gr-1(dim) F4/80(+) subset with ring-shaped nuclei (ring cells) among the CD11b(+) Gr-1(+) cell populations found in tumors. The intensity of the ring cell infiltrate correlated with tumor size and these cells constituted the second major tumor-infiltrating leukocyte subset found in established tumors. Although phenotypic analysis demonstrated that ring cells shared characteristics with tumor-associated macrophages (TAM), morphological analysis revealed a neutrophil-like appearance as detected by cytospin and immunofluorescence microscopy analysis. The presence of distinct iNOS filled granule-like structures located next to the cell membrane suggested that iNOS was stored in pre-formed vesicles and available for rapid release upon activation. Tumor biopsies showed large areas with infiltrating ring cells primarily surrounding necrotic areas. Importantly, these cells significantly impaired CD8(+) T-cell proliferation and induced apoptotic death. The intratumoral accumulation and suppressive activity of ring cells could be blocked through pharmacologic inhibition of iNOS, demonstrating the critical role of this enzyme in mediating both the differentiation and the activity of these cells. CONCLUSIONS: In this study, iNOS expression was linked to a homogeneous subset; ring cells with a particular phenotype and immune suppressive function, in a common and well-established murine tumor model; 4T-1. Since the absence of a Gr-1 homolog in humans has made the identification of MDSC much more challenging, use of iNOS as a functional marker of MDSC may also have clinical importance.

Development of Allogeneic Stem Cell-Based Platform for Delivery and Potentiation of Oncolytic Virotherapy.

We describe the repurposing and optimization of the TK-positive (thymidine kinase) vaccinia virus strain ACAM1000/ACAM2000™ as an oncolytic virus. This virus strain has been widely used as a smallpox vaccine and was also used safely in our recent clinical trial in patients with advanced solid tumors and Acute Myeloid Leukemia (AML). The vaccinia virus was amplified in CV1 cells and named CAL1. CAL1 induced remarkable oncolysis in various human and mouse cancer cells and preferentially amplified in cancer cells, supporting the use of this strain as an oncolytic virus. However, the therapeutic potential of CAL1, as demonstrated with other oncolytic viruses, is severely restricted by the patients' immune system. Thus, to develop a clinically relevant oncolytic virotherapy agent, we generated a new off-the-shelf therapeutic called Supernova1 (SNV1) by loading CAL1 virus into allogeneic adipose-derived mesenchymal stem cells (AD-MSC). Culturing the CAL1-infected stem cells allows the expression of virally encoded proteins and viral amplification prior to cryopreservation. We found that the CAL1 virus loaded into AD-MSC was resistant to humoral inactivation. Importantly, the virus-loaded stem cells (SNV1) released larger number of infectious viral particles and virally encoded proteins, leading to augmented therapeutic efficacy in vitro and in animal tumor models.

Enhanced presentation of MHC class Ia, Ib and class II-restricted peptides encapsulated in biodegradable nanoparticles: a promising strategy for tumor immunotherapy.

BACKGROUND: Many peptide-based cancer vaccines have been tested in clinical trials with a limited success, mostly due to difficulties associated with peptide stability and delivery, resulting in inefficient antigen presentation. Therefore, the development of suitable and efficient vaccine carrier systems remains a major challenge. METHODS: To address this issue, we have engineered polylactic-co-glycolic acid (PLGA) nanoparticles incorporating: (i) two MHC class I-restricted clinically-relevant peptides, (ii) a MHC class II-binding peptide, and (iii) a non-classical MHC class I-binding peptide. We formulated the nanoparticles utilizing a double emulsion-solvent evaporation technique and characterized their surface morphology, size, zeta potential and peptide content. We also loaded human and murine dendritic cells (DC) with the peptide-containing nanoparticles and determined their ability to present the encapsulated peptide antigens and to induce tumor-specific cytotoxic T lymphocytes (CTL) in vitro. RESULTS: We confirmed that the nanoparticles are not toxic to either mouse or human dendritic cells, and do not have any effect on the DC maturation. We also demonstrated a significantly enhanced presentation of the encapsulated peptides upon internalization of the nanoparticles by DC, and confirmed that the improved peptide presentation is actually associated with more efficient generation of peptide-specific CTL and T helper cell responses. CONCLUSION: Encapsulating antigens in PLGA nanoparticles offers unique advantages such as higher efficiency of antigen loading, prolonged presentation of the antigens, prevention of peptide degradation, specific targeting of antigens to antigen presenting cells, improved shelf life of the antigens, and easy scale up for pharmaceutical production. Therefore, these findings are highly significant to the development of synthetic vaccines, and the induction of CTL for adoptive immunotherapy.

Intravenous ascorbic acid to prevent and treat cancer-associated sepsis?

The history of ascorbic acid (AA) and cancer has been marked with controversy. Clinical studies evaluating AA in cancer outcome continue to the present day. However, the wealth of data suggesting that AA may be highly beneficial in addressing cancer-associated inflammation, particularly progression to systemic inflammatory response syndrome (SIRS) and multi organ failure (MOF), has been largely overlooked. Patients with advanced cancer are generally deficient in AA. Once these patients develop septic symptoms, a further decrease in ascorbic acid levels occurs. Given the known role of ascorbate in: a) maintaining endothelial and suppression of inflammatory markers; b) protection from sepsis in animal models; and c) direct antineoplastic effects, we propose the use of ascorbate as an adjuvant to existing modalities in the treatment and prevention of cancer-associated sepsis.

Safety evaluation of allogeneic umbilical cord blood mononuclear cell therapy for degenerative conditions.

BACKGROUND: The current paradigm for cord blood transplantation is that HLA matching and immune suppression are strictly required to prevent graft versus host disease (GVHD). Immunological arguments and historical examples have been made that the use of cord blood for non-hematopoietic activities such as growth factor production, stimulation of angiogenesis, and immune modulation may not require matching or immune suppression. METHODS: 114 patients suffering from non-hematopoietic degenerative conditions were treated with non-matched, allogeneic cord blood. Doses of 1-3 x 10(7) cord blood mononuclear cells per treatment, with 4-5 treatments both intrathecal and intravenously were performed. Adverse events and hematological, immunological, and biochemical parameters were analyzed for safety evaluation. RESULTS: No serious adverse effects were reported. Hematological, immunological, and biochemical parameters did not deviate from normal ranges as a result of therapy. CONCLUSION: The current hematology-based paradigm of need for matching and immune suppression needs to be revisited when cord blood is used for non-hematopoietic regenerative purposes in immune competent recipients.

Nutraceutical augmentation of circulating endothelial progenitor cells and hematopoietic stem cells in human subjects.

The medical significance of circulating endothelial or hematopoietic progenitors is becoming increasing recognized. While therapeutic augmentation of circulating progenitor cells using G-CSF has resulted in promising preclinical and early clinical data for several degenerative conditions, this approach is limited by cost and inability to perform chronic administration. Stem-Kine is a food supplement that was previously reported to augment circulating EPC in a pilot study. Here we report a trial in 18 healthy volunteers administered Stem-Kine twice daily for a 2 week period. Significant increases in circulating CD133 and CD34 cells were observed at days 1, 2, 7, and 14 subsequent to initiation of administration, which correlated with increased hematopoietic progenitors as detected by the HALO assay. Augmentation of EPC numbers in circulation was detected by KDR-1/CD34 staining and colony forming assays. These data suggest Stem-Kine supplementation may be useful as a stimulator of reparative processes associated with mobilization of hematopoietic and endothelial progenitors.

Autologous stromal vascular fraction cells: a tool for facilitating tolerance in rheumatic disease.

Since the days of Medawar, the goal of therapeutic tolerogenesis has been a "Holy Grail" for immunologists. While knowledge of cellular and molecular mechanisms of this process has been increasing at an exponential rate, clinical progress has been minimal. To provide a mechanistic background of tolerogenesis, we overview common processes in the naturally occurring examples of: pregnancy, cancer, oral tolerance and anterior chamber associated immune deviation. The case is made that an easily accessible byproduct of plastic surgery, the adipose stromal vascular fraction, contains elements directly capable of promoting tolerogenesis such as T regulatory cells and inhibitory macrophages. The high content of mesenchymal and hematopoietic stem cells from this source provides the possibility of trophic/regenerative potential, which would augment tolerogenic processes by decreasing ongoing inflammation. We discuss the application of this autologous cell source in the context of rheumatoid arthritis, concluding with some practical examples of its applications.

Delivery of a peptide via poly(D,L-lactic-co-glycolic) acid nanoparticles enhances its dendritic cell-stimulatory capacity.

Nanoparticles (NPs) are attractive carriers for vaccines. We have previously shown that a short peptide (Hp91) activates dendritic cells (DCs), which are critical for initiation of immune responses. In an effort to develop Hp91 as a vaccine adjuvant with NP carriers, we evaluated its activity when encapsulated in or conjugated to the surface of poly(d,l-lactic-co-glycolic) acid (PLGA) NPs. We found that Hp91, when encapsulated in or conjugated to the surface of PLGA-NPs, not only activates both human and mouse DCs, but is in fact more potent than free Hp91. Hp91 packaged within NPs was about fivefold more potent than the free peptide, and Hp91 conjugated to the surface of NPs was ∼20-fold more potent than free Hp91. Because of their capacity to activate DCs, such NP-Hp91 systems are promising as delivery vehicles for subunit vaccines against infectious disease or cancer. FROM THE CLINICAL EDITOR: In this paper, nanoparticle-based dendritic cell activating vaccines are described and discussed. The authors report that the presented PLGA NP based vaccine constructs increase the potency of the studied vaccine by up to 20-fold, making them promising as delivery vehicles for subunit vaccines against infectious diseases or cancer.

Novel Survivin Peptides Screened With Computer Algorithm Induce Cytotoxic T Lymphocytes With Higher Cytotoxic Efficiency to Cancer Cells.

The identification of novel biomarkers and therapeutic targets in advanced cancer is critical for improving cancer diagnosis and therapeutics. Survivin (SV) is highly expressed predominantly in most cancer cells and tissues but is absent or undetectable in terminally differentiated normal adult tissues. Therefore, it functions as an almost universal tumor antigen. Peptides are short chains of amino acids linked by peptide bonds. To obtain novel SV decamers that are able to induce SV-specific cytotoxic T lymphocytes (CTLs) with a higher cytotoxic efficiency against cancer cells, major histocompatibility complex (MHC) peptide binding algorithms were conducted to predict nine modified SV95 decamers (from SV95-2 to SV95-10) based on the natural SV95-104 peptide sequence of ELTLGEFLKL (here defined as SV95-1). The fluorescent density of each SV95 peptide was determined by a MHC stability assay, followed by the generation of SV95-specific CTLs with each SV95 peptide (from SV95-1 to SV95-10) and human dendritic cells (DCs) loaded with Poly(lactic-co-glycolic) acid (PLGA) nanoparticles encapsulated with SV95 peptide. Finally, IFN-γ ELISpot and CytoTox 96® Non-Radioactive Cytotoxicity Assays were employed to verify their cytotoxic efficiency of the SV95-specific CTLs generated with the corresponding artificial antigen presenting cells (aAPCs) containing SV95 (SV95-1 to SV95-10) peptide. Furthermore, the cytotoxicity of the SV95 specific CTLs generated with nine mutated SV95 peptides was compared to the one generated with natural SV95-1 peptide and TIL2080 cells. The results indicated that the HLA-A2-restricted mutated SV95 epitope decamers (SV95-6 and SV95-7) showed significant higher binding ability compared to natural peptide SV95-1 in MHC stability assay. More importantly, SV95-specific CTLs with higher cytotoxicity were successfully induced with both SV95-6 and SV95-7 peptides, which significantly eliminated target cells (not only SV95-1 peptide pulsed T2 cells, but also both HLA-A2 and SV positive cancer cells) when compared to those generated with natural SV95-1 peptide and TIL2080 cells. These findings suggest that the SV95-6 and SV95-7 peptides are two novel HLA-A2-restricted CTL epitopes and may be useful for the immunotherapy for patients with survivin expressing cancer.

Human embryonic stem cells hemangioblast express HLA-antigens.

BACKGROUND: It has been suggested that the initial differentiation of endothelial and hematopoietic cells during embryogenesis occurs from a common progenitor, called hemangioblast (hB). We hypothesized that these cells with dual hematopoietic/endothelial potential could be used in future regenerative medicine. METHODS: We used the two-step differentiation technology to generate bipotential blast cells from human embryonic stem cells (hES). This involved short differentiation in our in vitro EB system followed by differentiation in semisolid culture medium supplemented with mixture of cytokines. RESULTS: The occurrence of blast-colony-forming cells (BL-CFC) during EB differentiation (day 0-6) was transient and peaked on day 3. The emergence of this event was associated with expression of mesoderm gene T, and inversely correlated with expression of endoderm gene FoxA2. Similarly, the highest BL-CFC number was associated with increase in expression of early hematopoietic/endothelial genes: CD34, CD31 and KDR. The derived colonies were composed of 30-50 blast cells on day 6 in culture. These cells had homogenous appearance in Wright-Giemsa stain, but to a different extent expressed markers of immature hematopoietic and endothelial cells (CD31, CD34, VE-cadherin, Flt-1) and mature differentiated cells (CD45, CD33, CD146). We found that some of them expressed fetal and embryonic globin genes. Interestingly, these cells expressed also HLA class I molecules, however at very low levels compared to endothelial and hematopoietic cells. The blast cells could be successfully differentiated to hematopoietic cells in a CFU assay. In these conditions, blast cells formed CFU-M colonies (63.4 +/- 0.8%) containing macrophages, BFU-E colonies (19.5 +/- 3.5%) containing nucleated red blood cells, and CFU-EM colonies (17.1 +/- 2.7%) composed of macrophages and nucleated erythrocytes. Cells of CFU-EM and BFU-E colonies expressed both epsilon - and gamma- globin genes, but not adult-type gamma-globin. When in endothelial cell culture conditions, blast cells differentiated to endothelial cells which had the ability to take up Dil-Ac-LDL and to form complex vascular networks in Matrigel. CONCLUSION: 1) Hematoendothelial precursors exist transiently in early embryonic development and form single cell-derived colonies; 2) their differentiation can be tracked by the use of chosen molecular markers; 3) blast colonies consist of cells having properties of endothelial and hematopoietic precursors, however the issue of their ability to maintain dual properties over time needs to be further explored; 4) blast cells can potentially be used in regenerative medicine due to their low expression of HLA molecules.