Antigen-specific Cytotoxicity Research Articles

Intranasal Vaccination against HIV-1 with Adenoviral Vector-Based Nanocomplex Using Synthetic TLR-4 Agonist Peptide as Adjuvant.

Recombinant type 5 adenovirus (rAd5) vaccines hold the promise to prevent HIV-1 infections. Intranasal vaccination not only stimulates systemic immunity but also elicits mucosal immunity that provides first defense for mucosally transmitted diseases like HIV-1. Adjuvants such as TLR agonists are usually codelivered with antigens to enhance the immunogenicity of vaccines. Here, we present a rAd5 vaccine delivery system using DEG-PEI as the carrier. Adenovirus encoding HIV gag was used as antigen, and was complexed with DEG-PEI polymer via electrostatic interaction. A novel synthetic TLR-4 agonist, RS09, was either chemically linked with DEG-PEI (DP-RS09) or physically mixed with it(DP/RS09) to enhance the immunogenticity of rAd5 vaccine. After intranasal immunization, the systemic antigen-specific immune responses and cytotoxicity T lymphocytes responses induced by DP-RS09-rAd5 and DP/RS09-rAd5 were analyzed. The mucosal secretory IgA level was detected in both nasal and vaginal washes to determine the mucosal immunity. Furthermore, cytokine productions on RAW264.7 cells were tested after preincubation with TLR-4 pathway inhibitors. The results indicated that DEG-PEI could facilitate the intranasal delivery of rAd5 vaccine. Both chemically linked (DP-RS09) and physically mixed RS09 (DP/RS09) could further enhance the mucosal immunity of rAd5 vaccine via TLR-4 pathway. This RS09 adjuvanted DEG-PEI polymer represents a potential intranasal vaccine delivery system and may have a wider application for other viral vectors.

In Vitro Generation of Antigen-Specific T Cells from Induced Pluripotent Stem Cells of Antigen-Specific T Cell Origin.

Induced pluripotent stem (iPS) cells derived from T lymphocyte (T-iPS cells) preserve the T cell receptor (TCR) α and β gene rearrangements identical to the original T cell clone. Re-differentiated CD8 single positive αβ T cells from the T-iPS cells exhibited antigen-specific cytotoxicity, improved proliferative response, and elongation of telomere indicating rejuvenation of antigen specific T cell immunity in vitro. To regenerate antigen specific cytotoxic T lymphocytes (CTL), first, we have optimized a method for reprogramming-resistant CD8 T cell clones into T-iPS cells by using sendaiviral vectors. Second, we have optimized stepwise differentiation methods for inducing hematopoietic progenitor cells, T cell progenitors, and functionally matured CD8 single positive CTL. These protocols provide useful in vitro tools and models both for research of antigen-specific T cell immunotherapy and for research of normal and pathological thymopoiesis.

Antitumor cytotoxicity induced by bone-marrow-derived antigen-presenting cells is facilitated by the tumor suppressor protein p53 via regulation of IL-12

ABSTRACTActivated antigen-presenting cells (APC) deliver the three signals cytotoxic T cells require to differentiate into effector cells that destroy the tumor. These comprise antigen, co-stimulatory signals and cytokines. Once these cells have carried out their function, they apoptose. We hypothesized that the tumor suppressor protein, p53, played an important role in generating the antitumor response facilitated by APC. CD11c+ APC derived from p53 wild-type (wt) mouse (wt p53) GM-CSF bone marrow cultures (BMAPC) and activated had reduced survival compared to BMAPC from p53 null consistent with p53-mediated apoptosis following activation. There was a lower percentage of antigenic peptide/MHC I complexes on antigen-pulsed p53 null cells suggesting p53 played a role in antigen processing but there was no difference in antigen-specific T cell proliferative responses to these cells in vivo. In contrast, antigen-specific cytotoxicity in vivo was markedly reduced in response to p53 null BMAPC. When these cells were pulsed with a model tumor antigen and delivered as a prophylactic vaccination, they provided no protection against melanoma cell growth whereas wt BMAPC were very effective. This suggested that p53 might regulate the requisite third signal and, indeed, we found that p53 null BMAPC produced less IL-12 than wt p53 BMAPC and that p53 bound to the promoter region of IL-12. This work suggests that p53 in activated BMAPC is associated with the generation of IL-12 required for the differentiation of cytotoxic immune responses and an effective antitumor response. This is a completely new role for this protein that has implications for BMAPC-mediated immunotherapy.

Cancer Germline Antigen Specific CTL As Immunotherapy for Neuroblastoma

Cancer germline antigens (CGA) are expressed on several solid tumors including neuroblastoma. Their expression can be epigenetically upregulated upon treatment with decitabine (DAC), a demethylating chemotherapy drug. We have previously shown that DAC treatment improves the recognition of neuroblastoma and sarcoma cells to CGA-specific cytotoxic T lymphocytes (CTL) by upregulating the expression of MAGE-A1, MAGE-A3 and NY-ESO-1.[1,2] We have conducted a Phase I/II clinical trial in which low dose DAC is followed by administration of autologous dendritic cell (DC) vaccine pulsed with MAGE-A1, MAGE-A3 and NY-ESO-1 peptide antigens. [3] An alternative therapy that does not depend upon the ability of a patient to respond to a vaccine is the use of tumor antigen-specific T cells. Nevertheless it can be difficult to expand CTL with tumor antigen specific killing functions, and not just cytokine secretion. Li and group reported that a combination of CD25 depletion followed by culture of monocyte depleted lymphocytes with tumor antigens and IL-21 could result in T cell responses to cancer antigens. [4,5]IL-21 exerts potent antitumor effects by expanding cytotoxic CD8+ T cells, and suppressing FOXp3 expression and expansion of regulatory T cells (Tregs). The first objective of this study was to determine if CD25 depletion followed by IL-21 in culture could facilitate the generation of CGA-CTL. We have successfully cultured MAGE-A1, MAGE-A3 and NY-ESO-1 CTL following 3-5 weekly stimulations of CD25 depleted peripheral blood lymphocytes (PBL) with autologous dendritic cells (DC) pulsed with overlapping peptides derived from each of these antigens from 4/4 healthy donors. After the second antigen stimulation, T cells were supplemented with IL-2, IL-7 along with IL-21. Antigen-specific cytotoxicity was detected against autologous B cell blasts (BB) pulsed with MAGE-A1, MAGE-A3 or NY-ESO-1 peptides as well as tumor cell lines that have been treated with DAC and/or IFN-γ. Without performing CD25 depletion and culture in IL-21, we were unable to generate CGA-CTL from these donors, indicating that CD25 depletion and IL-21 in culture favors the generation of CGA-CTL. The second objective was to study if the addition of IL-21 alone, without CD25 depletion, may be sufficient for the expansion of these CTL. In a translational setting, omitting CD25 depletion would greatly simplify the expansion of CGA-CTL from patients. CTL cultured from PBL (without CD25 depletion) displayed equal or greater antigen-specific cytotoxicity when compared to CTL cultured from the CD25 depleted cell fraction in all donors, indicating that CD25 depletion is not necessary and the effects of IL-21 in culture seem sufficient to facilitate the expansion of CGA specific CTL. These CGA-CTL preferentially lysed HLA partially matched neuroblastoma cell lines treated with DAC and/or IFN-γ, the later to upregulate MHC class I expression on neuroblastoma cells. To understand potential mechanisms whereby DAC and IFN-γ could enhance tumor cell susceptibility to killing by CGA-specific CTL, we stained three neuroblastoma lines, BE2C, SKH-SH and IMR-32 for Fas, Fas ligand, TRAIL-R1, TRAIL-R2, mannose-6-phosphate receptor (M6PR) and intracellular-adhesion molecule (ICAM-1) that are associated with cytotoxicity. The increased percentages of Fas+, M6PR+ and ICAM-1+ on tumor lines post-treated with DAC and IFN-γ for five days indicates enhanced cytotoxicity could be Fas, M6PR or ICAM-1 mediated. Further studies are underway to determine the mechanisms whereby DAC and IFN-γ treated tumor cells are killed by CGA CTL cultured in this manner. DisclosuresNo relevant conflicts of interest to declare.

Wilms´Tumor 1 Protein Is Highly Expressed on Malignant Plasma Cells and Provides a Novel Target for Immunotherapeutic Approaches

Introduction: The Wilms' tumor 1 (WT1) protein is a tumor associated antigen that is potentially targetable by immunotherapeutic approaches. We have demonstrated the overexpression of WT1 in myeloma cells by IHC and in HLA-A*0201+ pts by staining with a high-affinity fully human IgG1 mAb (ESK1) specific to the RMFPNAPYL/HLA-A*0201 complex on malignant plasma cells. We report initial results from pts with plasma cell leukemia (PCL) or relapsed/refractory multiple myeloma (rMM) who have been treated with CD34-selected allo transplants followed by the administration of donor-derived WT1-specific T-cell infusions to induce an immunotherapeutic effect.Methods: In situ expression of WT1 was assessed by IHC analyses using a sequential double staining technique of MoAbs specific for CD138 and WT1.For staining with the RMFPNAPYL/HLA-A*0201 complex, BM samples were blocked with human FcR Blocking Reagent and then directly stained with MoAbs specific for CD38, CD56, CD45 and ESK1 or its isotype control human IgG1 and were analyzed by flow cytometry. WT1-specific T cells were generated from the original stem cell donors by sensitization of CD3+ enriched T-cell fractions with autologous APCs loaded with the pool of overlapping pentadecapeptides of WT1 (Invitrogen, Boston, MA). Cells were propagated in vitro with weekly restimulation and supplementation with IL-2 beginning at day 10-16. After 35-49 days, T-cells were harvested, counted and tested for antigen specific cytotoxicity, HLA-restriction, lack of alloreactivity and sterility. Pts received CD34-selected PBSC allografts after myeloablative cytoreduction with busulfan, melphalan and fludarabine. Pts were treated with 3 infusions of donor-derived WT1-specific T-cell infusions (5x10e6 cells/kg) starting 6 weeks post allo HSCT and at 4 weekly thereafter.Results: Marrow from all pts with immunohistochemical documented plasma cell involvement stained positive for WT1 IHC while WT1 staining remained negative in pts in CR. Only pts expressing HLA-A*0201 that stained positively for WT1 by IHC also demonstrated expression of WT1 by the RMFPNAPYL/HLA-A*0201 complex, whereas pts lacking HLA-A*0201 but with active disease stained positive for WT1 IHC but not ESK1 staining. Of 7 pts, 3 PCL and 4 rMM, treated with WT1-specific T cells, 4 pts had persistent disease post CD34-selected allotransplant. Of these 4 pts 2 pts developed a striking rise of WT1-specific T-cell frequencies and developed a complete remission post WT1 CTL infusions lasting for >2years.Conclusion: WT1 is overexpressed on malignant plasma cells and serves as a target for potential immunotherapeutic approaches in pts with multiple myeloma. Pts with persistent PCL following CD34-selected allografts treated with adoptive transfer of donor-derived WT1-specific cytotoxic T cells can achieve long lasting remission underscoring the therapeutic potential of T-cells specific for immunogenic WT1 peptides expressed on malignant plasma cells. DisclosuresO'Reilly:Atara Biotherapeutics: Research Funding.

Pre-Clinical Development of Gene Modification of Hematopoietic Stem Cells with Chimeric Antigen Receptors for Cancer Immunotherapy

Background: Patients with refractory or recurrent B-lineage hematological malignancies have less than 50% of chance of cure, despite intensive therapy. Innovative approaches are needed to decrease their morbidity and mortality. Chimeric Antigen Receptors (CAR) successfully engineer antigen specificity in immune cells, with clinical trials currently being conducted using ex vivo expanded gene-modified mature T cells. Results from preclinical studies and clinical trials show that effector cells usually have transient in vivo persistence that could significantly limit clinical efficacy and allow tumor recurrence. Building upon the standard of care and seeking an approach that could foster persistence of the CAR-modified cells, we have published studies using anti-CD19 CAR-modified human hematopoietic stem cells (HSC) engrafted in NSG immunodeficient mice. We hypothesize that modification of HSC with CD19-specific CAR will generate persistent multi-lineage anti-tumor activity for immunotherapy of CD19+ hematological malignancies. To increase the safety of the modification of HSC, a suicide gene can be inserted into the vector to eradicate the modified cells in the setting of toxicity. Thorough evaluation of this approach in relevant study models is required for advancement to clinical trials.Significance: This approach is untested in clinical translation to this date, and implies harnessing a patient's own HSC to create a whole self-renewing immune system directed to destroy cancer, a concept that can be applied to different cancers just by adjusting the target specificity. The prospect of modifying autologous cells to enhance graft-versus-cancer activity bears the possibility of decreased morbidity and mortality, being desirable for specifically vulnerable populations, as children and elderly patients, and offering alternative therapy for those without cell sources available for allogeneic HSC transplantation, benefiting patients from ethnic minorities. This approach can be easily adopted in the clinical setting for patients planned to receive autologous HSC transplant as their standard therapy, enhancing graft-versus-cancer activity with anti-CD19 specificity.Methods: High-titer third-generation lentiviral constructs were produced carrying second-generation CD19-specific CAR co-stimulated by CD28. These vectors also co-delivered HSV-sr39TK to provide a suicide gene to allow ablation of gene-modified cells if necessary. Human HSC isolated from umbilical cord blood and G-CSF-mobilized apheresed peripheral blood stem cells (PBSC) were transduced with such lentiviral vectors and injected into NSG pups after irradiation for in vivo evaluation of engraftment, function and suicide gene activation.Results: We have consistently achieved engraftment of human cells in about 95% of study mice, with engraftment of CAR+ cells in about 80% of the animals. Human HSC were successfully transduced with lentiviral vectors carrying anti-CD19 CAR with no impairment of differentiation or proliferation in vitro and in vivo. Immune cells differentiated in vivo from CAR+ HSC had antigen-specific cytotoxicity directed by CAR. CAR+ human cells were detected in BM, spleen, blood and thymus of injected mice. CAR+ T cells were stably detected in the blood of engrafted mice up to 40 weeks post-injection, demonstrating lymphopoiesis of CAR+ T cells successfully escaping thymic deletion and persisting throughout murine lifetime. As a surrogate of the antigen specificity and efficacy, CD19+ cells were significantly decreased in all mice engrafted with anti-CD19 CAR demonstrating that CAR+ immune cells were not inactivated or developed tolerance. Mice humanized with at least 4% of CAR+ cells in blood had significant protection against challenge with CD19+ tumor cell line, with inhibition or elimination of tumor development and consequent survival advantage. Activation of HSV-sr39TK suicide gene by ganciclovir treatment successfully led to ablation of gene-modified cells in vitro and in vivo.Conclusions: Our results demonstrate feasibility of CAR modification of human HSC for cancer immunotherapy. It could be easily employed in the context of HSC transplantation to augment the anti-cancer activity, with CAR-expressing myeloid and NK cells to ensure tumor-specific immunity until de novo production of T cells from CAR-modified HSC. DisclosuresLarson:BMS: Consultancy.

Combined Trabectedin and anti-PD1 antibody produces a synergistic antitumor effect in a murine model of ovarian cancer.

BackgroundMonoclonal antibodies (mAb) that block programmed death (PD)-1 signaling pathway hold great potential as a novel cancer immunotherapy. Recent evidence suggests that combining with conventional, targeted or other immunotherapies, these mAb can induce synergistic antitumor responses. In this study, we investigated whether Trabectedin (ET-743), a novel anticancer agent currently used for treating relapsed ovarian cancer, can synergize with anti (α)-PD-1 mAb to increase antitumor activity in the murine ID8 ovarian cancer model.MethodsMice with established peritoneal ID8 tumor were treated with either single or combined Trabectedin and α-PD-1 mAb, their overall survival was recorded; tumor-associated immune cells and immune gene expression in tumors from treated mice were analyzed by flow cytometry and quantitative RT-PCR, respectively, and antigen-specific immunity of effector CD8+ T cells was evaluated by ELISA and cytotoxicity assay. In addition, the effect of Trabectedin on tumoral PD-L1 expression was analyzed by both flow cytometry and immunofluorescence staining.ResultsThough single treatment showed a modest antitumor effect in mice bearing 10-day-established ID8 tumor, combined Trabectedin and α-PD-1 mAb treatment induced a strong antitumor immune response, leading to a significant tumor regression with half of mice tumor-free 90 days after tumor inoculation. Mechanistic investigation revealed that combination treatment induces a systemic tumor-specific immunity with an indispensable role of both CD4+ and CD8+ T cells, and effector CD8+ T cells exhibited the antigen-specific cytokine secretion and cytotoxicity upon tumor antigen stimulation; additionally, combination treatment increased the IFN-γ-producing effector T cells and decreased the immunosuppressive cells in peritoneal cavity; accordingly, it enhanced the expression of Th1-associated immune-stimulating genes while reducing the transcription of regulatory/suppressive immune genes, reshaping tumor microenvironment from a immunosuppressive to a stimulatory state. Finally, in vivo Trabectedin treatment has been shown to induce IFN-γ-dependent PD-L1 expression within tumor, possibly constituting a mechanistic basis for its synergistic antitumor effect with α-PD-1 mAb therapy.ConclusionThis study provides the evidence that α-PD-1 mAb can produce a synergistic antitumor efficacy when combined with Trabectedin, a clinically available anticancer agent, supporting a direct translation of this combination strategy in clinic for the treatment of ovarian cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0613-y) contains supplementary material, which is available to authorized users.

Combined immunotherapy with whole tumor lysate–pulsed interleukin-15–activated dendritic cells and cucurbitacin I promotes strong CD8+ T-cell responses and cures highly aggressive lymphoma

Background aimsDendritic cell (DC)-based therapies could be important strategies for lymphoma treatment. MethodsAKR/J mice with Dalton's lymphoma were treated with recombinant interleukin-15 (rIL-15)-activated autologous DCs and pulsed with whole tumor cell lysates in the presence or absence of suboptimal doses of the STAT3 inhibitor cucurbitacin I. One group of treated mice received an additional dose of rIL-15 to boost the DC-based adoptive cell therapy (ACT). Kaplan-Meier survival analysis and multiple immunologic and enzymatic parameters were assessed to demonstrate the efficacy of the vaccination protocol. ResultsTherapy with tumor lysate–pulsed, rIL-15–activated DCs plus cucurbitacin I significantly prolongs the survival of tumor-bearing mice but fails to provide a complete cure. Additional treatment of vaccinated mice with rIL-15 dramatically improves the therapeutic efficacy and provides a lifelong cure with no relapse. DCs derived from the surviving vaccinated mice regained their anti-tumor potential against the lymphoma cells with respect to growth inhibition and cytotoxicity. Both cluster of differentiation (CD)4+ and CD8+ T cells were mobilized in metastatic organs of successfully vaccinated mice in large numbers and demonstrated antigen-specific proliferation and tumor cell cytotoxicity. ACT also augments DC function by upregulating tumor necrosis factor-related apoptosis-inducing ligand and tumor necrosis factor-α expression. In addition, combinatorial immunotherapy restores the levels of antioxidant enzymes and liver function enzyme activities that are severely repressed in untreated tumor-bearing mice. ConclusionsEffective vaccination for a complete cure against aggressive lymphoma requires DC-based ACT in combination with chemotherapy and cytokine therapy.

Wilms’ tumor 1 protein is highly expressed on malignant plasma cells and provides a novel target for immunotherapeutic approaches

Abstract Introduction: The Wilms' tumor 1 (WT1) protein is a tumor associated antigen that is potentially targetable by immunotherapeutic approaches. We have demonstrated the overexpression of WT1 in myeloma cells by IHC and in HLA-A*0201+ pts by staining with a high-affinity fully human IgG1 mAb (ESK1) specific to the RMFPNAPYL/HLA-A*0201 complex on malignant plasma cells. We report initial results from pts with plasma cell leukemia (PCL) or relapsed/refractory multiple myeloma (rMM) who have been treated with CD34-selected allo transplants followed by the administration of donor-derived WT1-specific T-cell infusions to induce an immunotherapeutic effect. Methods: In situ expression of WT1 was assessed by IHC analyses using a sequential double staining technique of MoAbs specific for CD138 and WT1.For staining with the RMFPNAPYL/HLA-A*0201 complex, BM samples were blocked with human FcR Blocking Reagent and then directly stained with MoAbs specific for CD38, CD56, CD45 and ESK1 or its isotype control human IgG1 and were analyzed by flow cytometry. WT1-specific T cells were generated from the original stem cell donors by sensitization of CD3+ enriched T-cell fractions with autologous APCs loaded with the pool of overlapping pentadecapeptides of WT1 (Invitrogen, Boston, MA). Cells were propagated in vitro with weekly restimulation and supplementation with IL-2 beginning at day 10-16. After 35-49 days, T-cells were harvested, counted and tested for antigen specific cytotoxicity, HLA-restriction, lack of alloreactivity and sterility. Pts received CD34-selected PBSC allografts after myeloablative cytoreduction with busulfan, melphalan and fludarabine. Pts were treated with 3 infusions of donor-derived WT1-specific T-cell infusions (5x10e6 cells/kg) starting 6 weeks post allo HSCT and at 4 weekly thereafter. Results: Marrow from all pts with immunohistochemical documented plasma cell involvement stained positive for WT1 IHC while WT1 staining remained negative in pts in CR. Only pts expressing HLA-A*0201 that stained positively for WT1 by IHC also demonstrated expression of WT1 by the RMFPNAPYL/HLA-A*0201 complex, whereas pts lacking HLA-A*0201 but with active disease stained positive for WT1 IHC but not ESK1 staining. Of 7 pts, 3 PCL and 4 rMM, treated with WT1-specific T cells, 4 pts had persistent disease post CD34-selected allotransplant. Of these 4 pts 2 pts developed a striking rise of WT1-specific T-cell frequencies and developed a complete remission post WT1 CTL infusions lasting for >2years. Conclusion: WT1 is overexpressed on malignant plasma cells and serves as a target for potential immunotherapeutic approaches in pts with multiple myeloma. Pts with persistent PCL following CD34-selected allografts treated with adoptive transfer of donor-derived WT1-specific cytotoxic T cells can achieve long lasting remission underscoring the therapeutic potential of T-cells specific for immunogenic WT1 peptides expressed on malignant plasma cells. Disclosures O'Reilly: Atara Biotherapeutics: Research Funding.

A Small-Molecule c-Rel Inhibitor Reduces Alloactivation of T Cells without Compromising Antitumor Activity

Preventing unfavorable GVHD without inducing broad suppression of the immune system presents a major challenge of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We developed a novel strategy to ameliorate GVHD while preserving graft-versus-tumor (GVT) activity by small molecule-based inhibition of the NF-κB family member c-Rel. Underlying mechanisms included reduced alloactivation, defective gut homing, and impaired negative feedback on interleukin (IL)-2 production, resulting in optimal IL-2 levels, which, in the absence of competition by effector T cells, translated into expansion of regulatory T cells. c-Rel activity was dispensable for antigen-specific T-cell receptor (TCR) activation, allowing c-Rel-deficient T cells to display normal GVT activity. In addition, inhibition of c-Rel activity reduced alloactivation without compromising antigen-specific cytotoxicity of human T cells. Finally, we were able to demonstrate the feasibility and efficacy of systemic c-Rel inhibitor administration. Our findings validate c-Rel as a promising target for immunomodulatory therapy and demonstrate the feasibility and efficacy of pharmaceutical inhibition of c-Rel activity.

Immunosuppression by N-Methyl-d-Aspartate Receptor Antagonists Is Mediated through Inhibition of Kv1.3 and KCa3.1 Channels in T Cells

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ion channels that play an important role in neuronal development, plasticity, and excitotoxicity. NMDAR antagonists are neuroprotective in animal models of neuronal diseases, and the NMDAR open-channel blocker memantine is used to treat Alzheimer's disease. In view of the clinical application of these pharmaceuticals and the reported expression of NMDARs in immune cells, we analyzed the drug's effects on T-cell function. NMDAR antagonists inhibited antigen-specific T-cell proliferation and cytotoxicity of T cells and the migration of the cells toward chemokines. These activities correlated with a reduction in T-cell receptor (TCR)-induced Ca(2+) mobilization and nuclear localization of NFATc1, and they attenuated the activation of Erk1/2 and Akt. In the presence of antagonists, Th1 effector cells produced less interleukin-2 (IL-2) and gamma interferon (IFN-γ), whereas Th2 cells produced more IL-10 and IL-13. However, in NMDAR knockout mice, the presumptive expression of functional NMDARs in wild-type T cells was inconclusive. Instead, inhibition of NMDAR antagonists on the conductivity of Kv1.3 and KCa3.1 potassium channels was found. Hence, NMDAR antagonists are potent immunosuppressants with therapeutic potential in the treatment of immune diseases, but their effects on T cells have to be considered in that Kv1.3 and KCa3.1 channels are their major effectors.

PLGA-nanoparticle mediated delivery of anti-OX40 monoclonal antibody enhances anti-tumor cytotoxic T cell responses

OX40 (CD134) is a tumor necrosis factor (TNF) receptor expressed mainly on activated T cells and transmits a potent costimulatory signal once engaged. Agonistic anti-OX40 monoclonal antibody (mAb) enhances tumor immune response leading to therapeutic effects in mouse tumor models. However, when tested in phase I clinical trials it did not show objective clinical activity in cancer patients. In this study, we examined the feasibility of nanoparticle (NP)-mediated delivery of anti-OX40 mAb to efficiently induce cytotoxic T lymphocyte (CTL) responses. The biodegradable poly(dl-lactide-co-glycolide) nanoparticle (PLGA-NP) carrying anti-OX40 mAb, anti-OX40-PLGA-NP, was prepared by double emulsion method and showed an average diameter of 86nm with a loading efficiency of 25%. We found that anti-OX40-PLGA-NP induced CTL proliferation and tumor antigen-specific cytotoxicity as well as cytokine production more strongly than free anti-OX40 mAb. These results suggest that PLGA-based nanoparticle formulation may provide efficient delivery system of anti-OX40 mAb for cancer immunotherapy.

Anti-CD19 Chimeric Antigen Receptor Controlled By The Suicide Gene HSVsr39TK In Hematopoietic Stem Cells For Immunotherapy Of B-Lineage Malignancies

BackgroundAlthough significant improvements have been made, patients with relapsed or refractory B-cell malignancies continue to have unfavorable clinical outcomes. We hypothesize that transduction of hematopoietic stem cells (HSCs) with an anti-CD19 Chimeric Antigen Receptor (CAR) will produce a multi-lineage, persistent immunotherapy that can be controlled by the HSVsr39TK suicide gene. MethodsFirst generation anti-CD19 CAR lentiviral constructs containing the HSVsr39TK suicide gene were developed to compare vectors containing the human elongation factor alpha short (EFS) or myeloproliferative sarcoma virus U3 (MNDU3) promoters for transduction efficiency, antigen-specific cytotoxicity and ganciclovir (GCV)-induced cell death in primary human T-cells. The CD28 costimulatory domain was added to the selected construct, and high titer lentiviral vectors were generated to evaluate transduction of human umbilical cord blood (UCB) HSCs for in vitro and in vivo assays. In vitro assays were performed after culture under myeloid differentiation conditions, followed by assessment of phenotype, transduction efficiency, cytotoxic function and GCV-induced cell death. In vivo assays were conducted through transplantation of gene-modified human HSCs into irradiated NSG pups, compared to humanized NSG injected with non-modified human HSCs. Once engraftment was identified, mice from each cohort were further separated into GCV treated and untreated groups. Following GCV administration, mice were harvested to evaluate the presence of human and CAR-modified cells in the bone marrow, spleen and peripheral blood. ResultsIn human primary T cells, the MNDU3 promoter resulted in higher percentage of CAR expressing cells and mean fluorescence intensity compared to the EFS promoter. Cytotoxicity by the transduced T cells against the huCD19+Raji cell line showed similar target cell specific lysis among the constructs. Treatment with GCV effectively decreased the in vitro survival of the cells containing the HSVsr39TK gene compared to the non-transduced and control vector. The construct with MNDU3 promoter was then used with a CD28-containing second-generation anti-CD19 CAR (CCL-MND-αCD19/z/28-sr39). Once transduction efficiency and CAR function were validated in primary human T cells, this vector was used to transduce human UCB CD34+ cells. Following transduction, these cells were evaluated in vitro and in vivo. The cells used for the in vitro studies were cultured under myeloid differentiation conditions. The average number of CAR expressing cells was 45% at the clinically relevant vector copy number of 0.5-1 copies/cell. The myeloid cells transduced with the CCL-MND-αCD19/z/28-sr39 vector demonstrated CD19-specific killing and were eliminated by GCV. In vivo studies demonstrated successful engraftment of transduced HSC with CAR-expressing cells in the different hematopoietic lineages (T, NK, myeloid) detected among human cells in the bone marrow (1.2-15.4%, mean 7.6%), spleen (0.3-15.4%, mean 5.6%), and peripheral blood (0.5-30%, mean 9.2%). Mice engrafted with anti-CD19 CAR-modified HSCs exhibited decreased huCD19+ populations, compared to the mice engrafted with non-modified HSCs. Treatment with GCV resulted in significant decrease in CAR-expressing cells only in the mice transplanted with CD34+ cells transduced with the HSVsr39TK-containing vector. DiscussionHere we demonstrate that HSCs can be effectively transduced with an anti-CD19 CAR linked to the HSVsr39TK suicide gene. The CAR was detected in human cells in the bone marrow, spleen and peripheral blood and resulted in decreased B-lineage populations as an index of antigen-specific cytotoxicity; the HSVsr39TK gene conferred sensitivity to ganciclovir which eliminated transduced cells. These results provide pre-clinical support for the use of a CD19 targeted CAR in HSCs for the treatment of B-cell malignancies. Disclosures:Larson:Millenium: Speakers Bureau.

Targeted Cytolysins Synergistically Potentiate Cytoplasmic Delivery of Gelonin Immunotoxin

Targeted endocytic uptake is a first step toward tissue-specific cytoplasmic macromolecular delivery; however, inefficient escape from the endolysosomal compartment makes this generally impractical at present. We report here a targeted cytolysin approach that dramatically potentiates endosomal release of an independently targeted potent gelonin immunotoxin. Fibronectin domains engineered for affinity to EGF receptor or carcinoembryonic antigen were fused to the plant toxin gelonin or bacterial pore-forming cytolysins. These fusion proteins display synergistic activity in both antigen-specific cytotoxicity in vitro, enhancing potency by several orders of magnitude, and in tumor growth inhibition in vivo. In addition, the number of internalized gelonin molecules required to induce apoptosis is reduced from approximately 5 × 10(6) to less than 10(3). Targeted potentiation shows promise for enhancing cytoplasmic delivery of other macromolecular payloads such as DNA, siRNA, and miRNA.

Quantifying Biomass Changes of Single CD8+ T Cells during Antigen Specific Cytotoxicity

Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20–60% over 1–4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2–3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.

Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion

Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i) CD8+ T cells with specificity against the MART-1aa26–35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii) clonal CMV pp65aa495–503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495–503 specific T cell receptor were analyzed. Our data demonstrate that human CD8+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency.

Broadening of the T-cell repertoire to HIV-1 Gag p24 by vaccination of HLA-A2/DR transgenic mice with overlapping peptides in the CAF05 adjuvant.

Induction of broad T-cell immune responses is regarded as critical for vaccines against the human immunodeficiency virus type 1 (HIV-1) which exhibit high diversity and, therefore, focus has been on inducing cytotoxic CD8 T-cell responses against the more conserved parts of the virus, such as the Gag protein. Herein, we have used the p24 protein which contains a range of conserved T-cell epitopes. We demonstrate that a vaccine of HIV-1 subtype B consensus group-specific antigen (Gag) p24 protein with the CD8-inducing liposomal cationic adjuvant formulation (CAF) 05, induces both CD4 and CD8 T-cell responses in CB6F1 mice. The adjuvanted vaccine also induced functional antigen-specific cytotoxicity in vivo. Furthermore, we found that when fragmenting the Gag p24 protein into overlapping Gag p24 peptides, a broader T-cell epitope specificity was induced in the humanized human leukocyte antigen (HLA)-A2/DR-transgenic mouse model. Thus, combining overlapping Gag p24 peptides with CAF05 appears to be a promising and simple strategy for inducing broader T-cell responses to multiple conserved epitopes which will be relevant for both prophylactic and therapeutic HIV-1 vaccines.

Contrasted TCRβ Diversity of CD8+ and CD8− T Cells in Rainbow Trout

Teleost fish express highly diverse naive TCRβ (TRB) repertoires and mount strong public and private clonal responses upon infection with pathogens. Fish T cells express typical markers such as CD8, CD4-1 and CD4-2, CD3, CD28 and CTLA4. Fish CD8+ T cells have been shown to be responsible for antigen-specific cell-mediated cytotoxicity in in vitro systems using histo-compatible effector and target cells. We compare here the complexity of TRB repertoires between FACS sorted CD8+ and CD8− T cells from spleen and pronephros of rainbow trout. In contrast to human, while the TRB repertoire is highly diverse and polyclonal in CD8+ T cells of naïve fish, it appeared very different in CD8− lymphocytes with irregular CDR3 length distributions suggesting a dominance of activated clones already in naïve fish or the presence of non conventional T cells. After infection with a systemic virus, CD8+ T cells mount a typical response with significant skewing of CDR3 length profiles. The infection also induces significant modifications of the TRB repertoire expressed by the CD8− fraction, but for a different set of V/J combinations. In this fraction, the antiviral response results in an increase of the peak diversity of spectratypes. This unusual observation reflects the presence of a number of T cell expansions that rise the relative importance of minor peaks of the highly skewed distributions observed in unchallenged animals. These results suggest that the diversity of TRB expressed by CD8+ and CD8− αβ T cells may be subjected to different regulatory patterns in fish and in mammals.

Liver-Primed Memory T Cells Generated under Noninflammatory Conditions Provide Anti-infectious Immunity

Development of CD8(+) T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1(+) memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire.

Characterization of Novel Multiantigenic Vaccine Candidates with Pan-HLA Coverage against Mycobacterium tuberculosis

The low protection by the bacillus Calmette-Guérin (BCG) vaccine and existence of drug-resistant strains require better anti-Mycobacterium tuberculosis vaccines with a broad, long-lasting, antigen-specific response. Using bioinformatics tools, we identified five 19- to 40-mer signal peptide (SP) domain vaccine candidates (VCs) derived from M. tuberculosis antigens. All VCs were predicted to have promiscuous binding to major histocompatibility complex (MHC) class I and II alleles in large geographic territories worldwide. Peripheral mononuclear cells (PBMC) from healthy naïve donors and tuberculosis patients exhibited strong proliferation that correlated positively with Th1 cytokine secretion only in healthy naïve donors. Proliferation to SP VCs was superior to that to antigen-matched control peptides with similar length and various MHC class I and II binding properties. T-cell lines induced to SP VCs from healthy naïve donors had increased CD44(high)/CD62L(+) activation/effector memory markers and gamma interferon (IFN-γ), but not interleukin-4 (IL-4), production in both CD4(+) and CD8(+) T-cell subpopulations. T-cell lines from healthy naïve donors and tuberculosis patients also manifested strong, dose-dependent, antigen-specific cytotoxicity against autologous VC-loaded or M. tuberculosis-infected macrophages. Lysis of M. tuberculosis-infected targets was accompanied by high IFN-γ secretion. Various combinations of these five VCs manifested synergic proliferation of PBMC from selected healthy naïve donors. Immunogenicity of the best three combinations, termed Mix1, Mix2, and Mix3 and consisting of 2 to 5 of the VCs, was then evaluated in mice. Each mixture manifested strong cytotoxicity against M. tuberculosis-infected macrophages, while Mix3 also manifested a VC-specific humoral immune response. Based on these results, we plan to evaluate the protection properties of these combinations as an improved tuberculosis subunit vaccine.