Generation Of Cytotoxic T Lymphocytes Research Articles

Unprecedented Approach of Fabrication and Analysis of a Bioactive PDMS/Hydroxyapatite/Graphene Nanocomposite Scaffold with a Vascular Channel to Combat Carcinogenesis.

In the present investigation, natural bone-derived hydroxyapatite (HA, 2 wt %) and/or exfoliated graphene (Gr, 0.1 wt %)-embedded polydimethylsiloxane (PDMS) elastomeric films were prepared using a vascular method. The morphology, mechanical properties, crystallinity, and chemical structure of the composite films were evaluated. The in vitro biodegradation kinetics of the films indicates their adequate physiological stability. Most of the results favored PDMS/HA/Gr as a best composite scaffold having more than 703% elongation. A simulation study of the microfluidic vascular channel of the PDMS/HA/Gr scaffold suggests that the pressure drop at the outlet became greater (from 1.19 to 0.067 Pa) unlike velocity output (from 0.071 to 0.089 m/s), suggesting a turbulence-free laminar flow. Our bioactive scaffold material, PDMS/HA/Gr, showed highest cytotoxicity toward the lung cancer and breast cancer cells through Runx3 protein-mediated cytotoxic T lymphocyte (CTL) generation. Our data and predicted mechanism also suggested that the PDMS/HA/Gr-supported peripheral blood mononuclear cells (PBMCs) not only increased the generation of CTL but also upregulated the expression of RUNX3. Since the PDMS/HA/Gr scaffold-supported Runx3 induced CTL generation caused maximum cell cytotoxicity of breast cancer (MCF-7) and lung cancer (A549) cells, PDMS/HA/Gr can be treated as an excellent potential candidate for CTL-mediated cancer therapy.

Runx3 Regulates CD8+ T Cell Local Expansion and CD43 Glycosylation in Mice by H1N1 Influenza A Virus Infection.

We have recently reported that transcription factor Runx3 is required for pulmonary generation of CD8+ cytotoxic T lymphocytes (CTLs) that play a crucial role in the clearance of influenza A virus (IAV). To understand the underlying mechanisms, we determined the effects of Runx3 knockout (KO) on CD8+ T cell local expansion and phenotypes using an inducible general Runx3 KO mouse model. We found that in contrast to the lungs, Runx3 general KO promoted enlargement of lung-draining mediastinal lymph node (mLN) and enhanced CD8+ and CD4+ T cell expansion during H1N1 IAV infection. We further found that Runx3 deficiency greatly inhibited core 2 O-glycosylation of selectin ligand CD43 on activated CD8+ T cells but minimally affected the cell surface expression of CD43, activation markers (CD44 and CD69) and cell adhesion molecules (CD11a and CD54). Runx3 KO had a minor effect on lung effector CD8+ T cell death by IAV infection. Our findings indicate that Runx3 differently regulates CD8+ T cell expansion in mLNs and lungs by H1N1 IAV infection. Runx3 is required for CD43 core 2 O-glycosylation on activated CD8+ T cells, and the involved Runx3 signal pathway may mediate CD8+ T cell phenotype for pulmonary generation of CTLs.

Production of donor-derived cytotoxic T lymphocytes with potent anti-leukemia activity for adoptive immunotherapy in high-risk pediatric patients given haploidentical hematopoietic stem cell transplantation

Background aimsSomatic cell therapy based on the infusion of donor-derived cytotoxic T lymphocytes (CTL) able to recognize patients’ leukemia blasts (LB) is a promising approach to control leukemia relapse after allogeneic HSCT. The success of this approach strongly depends on the ex vivo generation of high-quality donor-derived anti-leukemia CTL in compliance with Good Manufacturing Practices (GMP). We previously described a procedure for generating large numbers of donor-derived anti-leukemia CTL through stimulation of CD8-enriched lymphocytes with dendritic cells (DCs) pulsed with apoptotic LB in the presence of interleukin (IL)-12, IL-7 and IL-15. Here we report that the use of IFN-DC and the addition of IFNα2b during the priming phase significantly improve the generation of an efficient anti-leukemia T cells response in vitro. MethodsUsing this approach, 20 high-risk pediatric patients given haploidentical HSCT for high-risk acute leukemia were enrolled and 51 batches of advanced therapy medical products (ATMP), anti-leukemia CTL, were produced. ResultsQuality controls demonstrated that all batches were sterile, free of mycoplasma and conformed to acceptable endotoxin levels. Genotype analysis confirmed the molecular identity of the ATMP based on the starting biological material used for their production. The majority of ATMP were CD3+/CD8+ cells, with a memory/terminal activated phenotype, including T-central memory populations. ATMP were viable after thawing, and most ATMP batches displayed efficient capacity to lyse patients’ LB and to secrete interferon-γ and tumor necrosis factor-α. ConclusionsThese results demonstrated that our protocol is highly reproducible and allows the generation of large numbers of immunologically safe and functional anti-leukemia CTL with a high level of standardization.

Cytoplasmic Transduction Peptide-Fused Recombinant Tumor-Associated Antigens Elicit Potent Myeloma-Specific Cytotoxic T Lymphocytes By Loading Onto Dendritic Cells: Implications of Feasible Tumor Antigens for Clinical Application Against Multiple Myeloma

Dendritic cell (DC)-based vaccines are one of promising tools in patients with multiple myeloma (MM). However, the source of tumor antigens is still challenging in this field. Here, we evaluated the feasibility of tumor-associated antigens (TAAs) to be loaded onto DCs for improving the efficacy of immunotherapy against MM. Quantitative PCR was performed to evaluate the expression of TAAs from malignant plasma cells isolated from bone marrow mononuclear cells of MM patients. Based on quantitative PCR analysis, 4 cancer testis antigens (SSX2, SSX4, MAGE-A3 and MAGE-C2), hTERT, BCMA, and CD38 were selected to make CTP-fused recombinant TAAs. Then, we made cytoplasm transduction peptide (CTP)-fused recombinant proteins (CTP-SSX2, CTP-SSX4, CTP-MAGEA3, CTP-MAGEC2, CTP-hTERT, CTP-BCMA, and CTP-CD38) for the efficient delivery of TAAs to DCs. After determination of optimal protein dose for loading onto DCs, several functional studies were performed by the DCs. The CTP-fused recombinant TAAs did not deteriorate the function of DCs in terms of phenotype expressions and cytokine productions, including a Th1-polarizing capacity of naïve T cells. DCs loaded with each CTP-fused recombinant TAA had successfully generated a myeloma-specific cytotoxic T lymphocyte (CTL) to kill myeloma cell lines and primary myeloma cells. This study showed that CTP-fused recombinant myeloma-associated proteins were a useful tumor antigen source to be loaded onto DCs for generation of myeloma-specific CTLs in vitro . Therefore, CTP-fused recombinant TAAs will become a feasible tumor antigen for the clinical application of DC-based cancer immunotherapy in the field of MM. DisclosuresNo relevant conflicts of interest to declare.

A vaccine inducing solely cytotoxic T lymphocytes fully prevents Zika virus infection and fetal damage.

SUMMARYAs vaccine-induced non-neutralizing antibodies may cause antibody-dependent enhancement of Zika virus (ZIKV) infection, we test a vaccine that induces only specific cytotoxic T lymphocytes (CTLs) without specific antibodies. We construct a DNA vaccine expressing a ubiquitinated and rearranged ZIKV non-structural protein 3 (NS3). The protein is immediately degraded and processed in the proteasome for presentation via major histocompatibility complex (MHC) class I for CTL generation. We immunize Ifnar1−/− adult mice with the ubiquitin/NS3 vaccine, impregnate them, and challenge them with ZIKV. Our data show that the vaccine greatly reduces viral titers in reproductive organs and other tissues of adult mice. All mice immunized with the vaccine survived after ZIKV challenge. The vaccine remarkably reduces placenta damage and levels of pro-inflammatory cytokines, and it fully protects fetuses from damage. CD8+ CTLs are essential in protection, as demonstrated via depletion experiments. Our study provides a strategy to develop safe and effective vaccines against viral infections.

Complex Interplay Between MAZR and Runx3 Regulates the Generation of Cytotoxic T Lymphocyte and Memory T Cells.

The BTB zinc finger transcription factor MAZR (also known as PATZ1) controls, partially in synergy with the transcription factor Runx3, the development of CD8 lineage T cells. Here we explored the role of MAZR as well as combined activities of MAZR/Runx3 during cytotoxic T lymphocyte (CTL) and memory CD8+ T cell differentiation. In contrast to the essential role of Runx3 for CTL effector function, the deletion of MAZR had a mild effect on the generation of CTLs in vitro. However, a transcriptome analysis demonstrated that the combined deletion of MAZR and Runx3 resulted in much more widespread downregulation of CTL signature genes compared to single Runx3 deletion, indicating that MAZR partially compensates for loss of Runx3 in CTLs. Moreover, in line with the findings made in vitro, the analysis of CTL responses to LCMV infection revealed that MAZR and Runx3 cooperatively regulate the expression of CD8α, Granzyme B and perforin in vivo. Interestingly, while memory T cell differentiation is severely impaired in Runx3-deficient mice, the deletion of MAZR leads to an enlargement of the long-lived memory subset and also partially restored the differentiation defect caused by loss of Runx3. This indicates distinct functions of MAZR and Runx3 in the generation of memory T cell subsets, which is in contrast to their cooperative roles in CTLs. Together, our study demonstrates complex interplay between MAZR and Runx3 during CTL and memory T cell differentiation, and provides further insight into the molecular mechanisms underlying the establishment of CTL and memory T cell pools.

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.

B cell depletion in murine lupus using cytotoxic T lymphocytes in vivo: Feasibility and benefit

Given the promising results in human lupus with B cell depletion, we tested whether in vivo cytotoxic T lymphocyte (CTL) could eliminate autoreactive B cells in the setting of murine lupus. Using the parent-into-F1 (P → F1) model to generate CTL that eliminate B cells, we found that transfer ofNZB parental splenocytes into lupus-prone female NZB/W F1 mice resulted in profound B cell reduction whereas NZW → F1 mice exhibited defective B cell elimination. Using pre-disease or early disease B/W mice as hosts, NZB → F1 mice exhibited B cell depletion and improved proteinuria but no improvement in survival whereas NZW → F1 mice had significantly reduced proteinuria and prolonged survival. Thus, despite the defective IL-2 environment in B/W F1 mice, generation of CTL and B cell depletion is feasible in NZB → F1 mice. The surprising increase in survival for NZW → F1 mice despite defective B cell elimination suggests that NZW splenocytes may contain a beneficial down regulatory cell.

CD4 Cytotoxic T cells play a critical role in anti-tumor protection

Abstract The CD4 T cell lineage is established in the thymus by the transcription factor, ThPOK. In the periphery, ThPOK maintains the CD4 T helper commitment and allows the differentiation to different helper fates in response to stimulation with cognate antigens. We demonstrated that in the intestine, chronic stimulation of CD4 T cells results in the loss of ThPOK expression leading to the reprogramming of the T helper cells to cytotoxic T lymphocytes (CTL), expressing notably CD8 T cells attributes. Continuous activation in a non-immunogenic environment is also a hallmark of tumor progression. We investigated if the tumor environment supports the generation of CD4 CTL and moreover if they play essential roles in anti-tumor protection. Using the well-defined melanoma B16 model, we observed that CD4 T cells responding to the tumor uniquely lose ThPOK expression. To address the importance of the CD4 CTL in the tumor immune surveillance, we engineered two unique mouse models, in which either all the CD4 T cells are forced to reprogram to CTL or else, prevented from reprogramming to CTL. Using these models, we observed that the forced CD4 CTL eradicate melanoma cells, whereas the CD4 cells which cannot lose ThPOK and reprogram to CTL are ineffective against the tumor growth. These findings underscore the importance of CD4 CTL reprogramming in protective immunity. Finally, we developed a culture system to induce the reprograming of CD4 Th to CTL in vitro. We injected in vitro-generated CD4 CTL alone or with a co-transfer of CD8 T cells into Rag deficient hosts bearing tumor. As compared to the transfer of the CD4 CTL or CD8 CTL alone, the cooperation of the cytotoxic effector cells displayed a strong synergized effect on long term protection against tumor growth.

Impaired T cell proliferation by ex vivo BET-inhibition impedes adoptive immunotherapy in a murine melanoma model

ABSTRACTActivation of naïve CD8+ T cells stimulates proliferation and differentiation into cytotoxic T-lymphocytes (CTLs). Adoptive T Cell Therapy (ACT) involves multiple rounds of ex vivo activation to generate enough CTLs for reinfusion into patients, but this drives differentiation into terminal effector T cells. Less differentiated CTL populations, such as stem cell memory T cells, are more ideal candidates for ACT because of increased self-renewal and persistent properties. Ex vivo targeting of T cell differentiation with epigenetic modifiers is a potential strategy to improve cytotoxic T-lymphocyte (CTL) generation for ACT. We established a pipeline to assess the effects of epigenetic modifiers on CD8+ T cell proliferation, differentiation, and efficacy in a preclinical melanoma model. Single treatment with epigenetic modifiers inhibited T cell proliferation in vitro, producing CD44hiCD62Lhi effector-like T cells rather than a stem cell memory T cell phenotype. Most epigenetic modifying agents had no significant effect on ACT efficacy with the notable exception of the bromodomain and extraterminal (BET)-inhibitor JQ1 which was associated with a decrease in efficacy compared to unmodified T cells. These findings reveal the complexity of epigenetic targeting of T cell differentiation, highlighting the need to precisely define the epigenetic targeting strategies to improve CTL generation for ACT.

Bcma Heteroclitic Peptide Encapsulated Nanoparticle Enhances Antigen Stimulatory Capacity and Tumor-Specific CD8+ cytotoxic T Lymphocytes Against Multiple Myeloma

Background:B-cell Maturation Antigen (BCMA), a member of the tumor necrosis factor (TNF) receptor superfamily and the receptor for binding of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), is a promising therapeutic target for MM. Due to its restricted expression pattern on MM cells and plasma cells along with its role in promoting MM cells growth, survival, and drug resistance, BCMA is being targeted by several immunotherapeutic strategies including antibodies, immunotoxins, bispecific T-cell engagers, and CAR-T cells. Recently, we have identified nanomedicine-based therapeutics targeting BCMA as a promising area of translational research to effectively evoke and augment anti-tumor responses in MM patients. Several nanomedicines are available and more advanced nanoparticle constructs are under development for antigen encapsulation. To this end, we have designed a heteroclitic BCMA peptide encapsulated nanoparticle-based cancer vaccine to overcome the limitations of free peptide vaccines including poor peptide stability, susceptibility to enzyme degradation, and low antigen uptake and delivery. Furthermore, the nanotechnology-based cancer vaccine was developed to induce more robust BCMA-specific CD8+ cytotoxic T lymphocytes (CTL) activities in MM patients, with more sustained antigen release and increased bioavailability and presentation of the immunogenic peptide. Here, we examine the potential of a novel nanomedicine-based therapeutic delivery system specific to BCMA antigen to treat the patients with MM.Objective:The purpose of this study was to design the optimal nanoparticle encapsulated BCMA antigen constructs to efficiently evoke and develop the BCMA-specific CD8+ CTL with functional anti-myeloma activities.Findings:Nanoparticles [liposome or poly(D,L-lactide-co-glycolide) (PLGA)] with different antigen-release kinetics demonstrated their capacity to effectively deliver heteroclitic BCMA peptideto antigen-presenting cells and evoke BCMA antigen-specific CTL with anti-MM activities. The heteroclitic BCMA peptide encapsulated nanoparticles demonstrated a higher uptake by human dendritic cells than free peptide, with the highest uptake mediated with liposome-based nanoparticles. In contrast, BCMA-specific CTL induced with PLGA-based nanoparticle demonstrated the highest functional activities and specific immune responses against MM cells. The PLGA/BCMA peptide nanoparticle induced BCMA-specific CTL displayed the highest increases in CD107a degranulation, the antigen-specific CD8+ T cells proliferation and Th-1 type cytokines (IFN-g, IL-2, TNF-a) production to MM patients' tumor cells and MM cell lines compared to BCMA-CTL generated with free BCMA peptide or liposome/BCMA peptide nanoparticle. These observations were aligned with the highest level of CD28 costimulatory molecules upregulation, Tetramer+ CTL generation and peptide-specific responses within the BCMA-CTL generated by PLGA/BCMA nanoparticles. Furthermore, the PLGA/BCMA nanoparticles triggered a more robust induction of antigen-specific memory CD8+ T cells, which demonstrated significantly higher anti-tumor activities, evidenced by CD107a degranulation and IFN-g production, compared to non-memory CD8+ T cells within the BCMA-CTL. Especially, the increased central memory CTL development and their anti-tumor activities evoked by PLGA/BCMA peptide were associated with the optimal peptide release kinetics and enhanced immunogenicity of the antigen via this nanotechnology. Thus, these results demonstrate that the heteroclitic BCMA peptide encapsulated nanoparticle strategy supports the peptide delivery into dendritic cells and then subsequently to T cells, resulting in effective induction of BCMA-specific central memory CTL with poly-functional activities against MM.Significance:These results demonstrate the utility of nanotechnology using encapsulated heteroclitic BCMA peptide to enhance the immunogenicity of BCMA peptide-specific therapeutics against MM. Importantly, our observations provide the framework for therapeutic application of PLGA-based heteroclitic BCMA peptide delivery to enhance the BCMA-specific memory T cell immune responses, overcome the limitations of current peptide-based cancer vaccine, and improve the patient outcome in MM. DisclosuresMunshi:OncoPep: Other: Board of director. Anderson:Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Millennium Takeda: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Gilead: Membership on an entity's Board of Directors or advisory committees.

Photochemical Treatment of Drosophila APCs Can Eliminate Associated Viruses and Maintain the APC Function for Generating Antigen-Specific CTLs Ex Vivo.

Drosophila cells transfected with MHC class I and a number of costimulation molecules including B7.1, ICAM, LFA-3, and CD70 are potent antigen-presenting cells (APCs) for the generation of antigen-specific cytotoxic T cells (CTLs) in vitro. Using Drosophila APCs, CTLs specific for melanoma antigens have been generated in vitro and adoptively transferred to melanoma patients. However, the recent discovery that Drosophila cells can carry insect viruses raises the potential risk of Drosophila APCs transmitting xenogenic viruses to patient CTLs. In this study, we have investigated photoreactive methods to inactivate insect viruses in APC. A clinical grade psoralen compound, 8-MOP (UVADEX) in combination with UVA treatment (5 joules/cm2) can be used to inactivate Drosophila cell viruses. UVADEX treatment is sufficient to inactivate insect viruses but does not affect the expression of MHC class I molecules and costimulation molecules on Drosophila APCs. In fact, UVADEX treatment prevents Drosophila APC growth while maintaining APC function. Furthermore, UVADEX-treated Drosophila APCs maintain or have enhanced APC function as determined by enhanced T cell activation, proliferation, and CTL generation. Thus, the use of UVADEX-treated Drosophila APCs may provide a valuable tool for immunotherapy to generate tumor antigen-specific CTLs.

Novel epitopes identified from Mycobacterium tuberculosis antigen Rv2629induces cytotoxic T lymphocyte response

There is an urgent need for a more effective vaccine against tuberculosis (TB). Cytotoxic T lymphocytes (CTLs) play a critical role in combating Mycobacterium tuberculosis (M.tb). The identification of novel CTL epitopes is essential for the design of peptide-based vaccines. In this study, we predicted CTL epitope peptides of M.tb antigen Rv2629 restricted by HLA-A2, using bioinformatics methods. The affinity and stability of binding of these peptides with HLA-A2 molecules were detected by flow cytometry. Their ability to induce CTLs generation was determined in peripheral blood mononuclear cells (PBMCs) from healthy uninfected subjects, Latent tuberculosis infection (LTBI) subjects, and TB patients ex vivo. The cytotoxic activity induced by the epitope peptides was tested by lactate dehydrogenase (LDH) release assay. Finally, we found four novel CTL epitope peptides, Rv2629-p190-2L, Rv2629-p190-1Y2L, Rv2629-p274, and Rv2629-p315, which had high-affinity and stability of binding with T2 cells. Their ability of inducing CTLs was highest in PBMCs from TB patients (P < 0.05). In addition, these peptides could induce the CTLs to generate specific cytotoxic activity. They showed higher immunogenicity in TB patients and had the potential to become candidate vaccines for TB therapy.

Protective Cancer Vaccine Using Genetically Modified Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) yield both the myeloid and lymphoid lineages of blood cells and can be reprogrammed into tumor antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) to prevent tumor growth. However, the optimal approach for differentiating tumor Ag-specific CTLs from HSCs, such as HSC-CTLs, remains elusive. In the current study, we showed that a combination of genetic modification of HSCs and in vivo T cell development facilitates the generation of Ag-specific CTLs that suppressed tumor growth. Murine HSCs, which were genetically modified with chicken ovalbumin (OVA)-specific T cell receptor, were adoptively transferred into recipient mice. In the following week, mice were administered with intraperitoneal injections of an agonist α-Notch 2 antibody and cytokines (rFlt3L and rIL-7) three times. After another two weeks, mice received a subcutaneous inoculation of B16-OVA melanoma cells that express OVA as a surrogate tumor Ag, before the anti-tumor activity of HSC-derived T cells was assessed. OVA-specific CTLs developed in vivo and greatly responded to OVA Ag stimulation ex vivo. In addition, mice receiving genetically modified HSCs and in vivo priming established anti-tumor immunity, resulting in the suppression of tumor growth. These results reported in this present study provide an alternative strategy to develop protective cancer vaccines by using genetically modified HSCs.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development.

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Role of MDA5 and interferon-I in dendritic cells for T cell expansion by anti-tumor peptide vaccines in mice.

Cytotoxic T lymphocytes (CTLs) are effective components of the immune system capable of destroying tumor cells. Generation of CTLs using peptide vaccines is a practical approach to treat cancer. We have previously described a peptide vaccination strategy that generates vast numbers of endogenous tumor-reactive CTLs after two sequential immunizations (prime-boost) using poly-ICLC adjuvant, which stimulates endosomal toll-like receptor 3 (TLR3) and cytoplasmic melanoma differentiation antigen 5 (MDA5). Dendritic cells (DCs) play an important role not only in antigen presentation but are critical in generating costimulatory cytokines that promote CTL expansion. Poly-ICLC was shown to be more effective than poly-IC in generating type-I interferon (IFN-I) in various DC subsets, through its enhanced ability to escape theendosomal compartment and stimulate MDA5. In our system, IFN-I did not directly function as a T cell costimulatory cytokine, but enhanced CTL expansion through the induction of IL15. With palmitoylated peptide vaccines, CD8α+ DCs were essential for peptide crosspresentation. For vaccine boosts, non-professional antigen-presenting cells were able to present minimal epitope peptides, but DCs were still required for CTL expansions through the production of IFN-I mediated by poly-ICLC. Overall, these results clarify the roles of DCs, TLR3, MDA5, IFN-I and IL15 in the generation of vast and effective antitumor CTL responses using peptide and poly-IC vaccines.

Development and characterization of naive single-type tumor antigen-specific CD8+ T lymphocytes from murine pluripotent stem cells

ABSTRACTOptimal approaches to differentiate tumor antigen-specific cytotoxic T lymphocytes (CTLs) from pluripotent stem cells (PSCs) remain elusive. In the current study, we showed that combination of in vitro priming through Notch ligands and in vivo development facilitated the generation of tumor Ag-specific CTLs that effectively inhibited tumor growth. We co-cultured the murine induced PSCs (iPSCs) genetically modified with tyrosinase-related protein 2 (TRP2)-specific T cell receptors with OP9 cell line expressing both Notch ligands Delta-like 1 and 4 (OP9-DL1/DL4) for a week before adoptively transferred into recipient C67BL/6 mice. Three weeks later, B16 melanoma cells were inoculated subcutaneously, and the antitumor activity of the iPSC-derived T cells was assessed. We observed the development of the TRP2-specific iPSC-CD8+ T cells that responded to Ag stimulation and infiltrated into melanoma tissues, significantly inhibited the tumor growth, and improved the survival of the tumor-bearing mice. Thus, this approach may provide a novel effective strategy to treatment of malignant tumors.

Abstract OT3-01-03: A phase I trial of the safety and immunogenicity of a DNA plasmid based vaccine (WOKVAC) encoding epitopes derived from three breast cancer antigens (IGFBP2, HER2, and IGF1R) in patients with breast cancer

Abstract Background: Three proteins insulin like growth factor binding protein 2 (IGFBP2), human epidermal growth factor receptor 2 (HER2), and insulin like growth factor receptor-1 (IGF1R) are overexpressed in in pre-invasive and high risk breast lesions and are associated with progression to invasive breast cancer. These proteins are immunogenic and elicit both humoral and cellular immunity in breast cancer patients. It is hypothesized that immunization with a plasmid vaccine (WOKVAC) targeting antigens from these proteins will be safe and immunogenic. WOKVAC has been designed to include extended sequences of the immunizing antigens that are predominantly associated with eliciting Type I immune responses. Type I immunity results in immune cells called T-cells secreting high levels of inflammatory cytokines (called Th1) that stimulate tumor destruction as well as the generation of cytotoxic T-cells that can directly kill the tumors. Trial design: Phase I dose escalation study of 3 doses of WOKVAC admixed with 100mcg of GM-CSF. Patients will be assigned sequentially to one of three arms (10 patients/arm): Arm 1=150mcg, Arm 2=300mcg, Arm 3=600mcg. Each dose arm will have a staggered enrollment to assess toxicity. If the Arm 1 dose is determined to be safe, Arm 2 patients can be enrolled. If the Arm 2 dose is safe and immunologically more efficacious than Arm 1 then Arm 3 patients can be enrolled. Study treatment includes 3 monthly vaccines, two evaluations at 1 and 6 months post vaccine and a 5 year follow-up to collect reports from the patient's primary oncologist. Toxicity is assessed at baseline through the end of the study. Serial blood draws for immunologic monitoring is done. Eligibility criteria: Patients with non-metastatic, node positive, HER2 negative breast cancer that is in remission and defined as no evidence of disease. Patients must have a good performance status, be at least 28 days from last cytotoxic chemotherapy and/or radiotherapy and 28 days from any use of systemic steroids. Specific aims: (1) Determine safety of 3 escalating doses of WOKVAC, (2) Determine the most immunogenic dose, (3) Determine whether a WOKVAC Th1 polyepitope plasmid based vaccine elicits a persistent memory T-cell and (4) Determine whether WOKVAC vaccination modulates T regulatory cells and myeloid derived suppressor cells. Statistical methods: (1) Safety will be assessed per NCI CTCAE v. 4.0, (2) Immunogenicity will be defined by the magnitude of the Th1 IFN-gamma antigen specific immune response. Successful immunization is a protein specific IFN-g precursor frequency greater than 1:20,000 PBMC for each antigen or 2 fold increase if baseline immune response (3) The IGFBP2, HER2, and IGF1R specific IFN-g/IL-10 ratios by ELISPOT will be evaluated to determine that a predominantly Th1 immune response is stimulated, and (4) Humoral immune response will be measured by ELISA and serum antibody avidity for IGFBP2, HER2, and IGF1R to determine an avidity index (AI) before and after vaccination. Targeted Accrual: 30 patients Contact information: University of Washington: 866-392-8588/TrialTVG@uw.edu University of Wisconsin: 608-265-2493/prevention@uwcarbone.wisc.edu. Citation Format: Childs JS, Higgins DM, DeShong K, Heckman-Stoddard BM, Wojtowicz ME, Stanton SE, Bailey HH, Wisinski KB, Disis ML. A phase I trial of the safety and immunogenicity of a DNA plasmid based vaccine (WOKVAC) encoding epitopes derived from three breast cancer antigens (IGFBP2, HER2, and IGF1R) in patients with breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT3-01-03.

Delivery of foreign cytotoxic T lymphocyte epitopes to tumor tissues for effective antitumor immunotherapy against pre-established solid tumors in mice.

Cytotoxic T lymphocyte (CTL) can have remarkable abilities to kill tumor cells. However, the establishment of successful CTL-based anticancer therapy has met with many challenges. Within tumor cells, there exist subpopulations with low or no expression of the targeted antigen (termed as antigen-loss variants). In addition, tumor cells can downregulate the levels of major histocompatibility complex class I (MHC-I) molecules on cell surface due to immune pressure. As a result, some tumor cells can escape the immune pressure bestowed by CTLs, resulting in treatment failure. To address these difficulties, a new approach is developed to deliver foreign high-affinity CTL epitopes to tumor tissues utilizing pH-responsive "smart" microparticles (MPs). These MPs could encapsulate CTL peptide epitope, release the peptide under acidic condition encountered in tumor tissues and enhance CTL activation. Mice bearing pre-established tumor as "antigen-loss variant" solid tumor models were administered intratumorally with MPs containing the CTL peptide, which showed 100% survival following the treatment. In contrast, all control mice died from tumor. Significant protection from tumor-induced death was also observed with systemic administration of CTL peptide-MPs. The therapeutic efficacy can be attributed to enhanced delivery of the epitope to tumor tissues, presentation of the epitope by tumor cells as well as tumor stromal cells and/or generation of epitope-specific CTLs by the peptide-containing MPs. These findings offer a promising new direction for treating established solid tumor using CTL therapy.

Regeneration of CD8αβ T Cells from T-cell-Derived iPSC Imparts Potent Tumor Antigen-Specific Cytotoxicity.

Although adoptive transfer of cytotoxic T lymphocytes (CTL) offer a promising cancer therapeutic direction, the generation of antigen-specific CTL from patients has faced difficulty in efficient expansion in ex vivo culture. To resolve this issue, several groups have proposed that induced pluripotent stem cell technology be applied for the expansion of antigen-specific CTL, which retain expression of the same T-cell receptor as original CTL. However, in these previous studies, the regenerated CTL are mostly of the CD8αα+ innate type and have less antigen-specific cytotoxic activity than primary CTL. Here we report that, by stimulating purified iPSC-derived CD4/CD8 double-positive cells with anti-CD3 antibody, T cells expressing CD8αβ were generated and exhibited improved antigen-specific cytotoxicity compared with CD8αα+ CTL. Failure of CD8αβ T-cell production using the previous method was found to be due to killing of double-positive cells by the double-negative cells in the mixed cultures. We found that WT1 tumor antigen-specific CTL regenerated by this method prolonged the survival of mice bearing WT1-expressing leukemic cells. Implementation of our methods may offer a useful clinical tool. Cancer Res; 76(23); 6839-50. ©2016 AACR.