High Expression Of Co-stimulatory Molecules Research Articles

Identification and validation of the association of Janus kinase 2 mutations with the response to immune checkpoint inhibitor therapy

BackgroundJanus kinase 2 (JAK2) mutation plays an important role in T cell immunity. However, the effect of JAK2 mutation on immunotherapy is largely uncharacterized.MethodsIn this study, we analyzed the effect of JAK2 mutation on the efficacy and outcomes of immune checkpoint inhibitor (ICI) therapy in the discovery cohort (n = 662) and the verification cohort (n = 1423). Furthermore, we explored the association of JAK2 mutation with the tumor immune microenvironment in a multiomics cohort.ResultsIn the discovery cohort (n = 662), JAK2 mutant-type patients had a better objective response rate (58.8% vs. 26.7%, P = 0.010), durable clinical benefit (64.7% vs. 38.9%, P = 0.043), progression-free survival (hazard ratio [HR] = 0.431, P = 0.015), and overall survival (HR = 0.378, P = 0.025), relative to JAK2 wild-type patients. Moreover, we further verified the prognostic significance of JAK2 mutation in an independent ICI treatment cohort with a larger sample size (n = 1423). In addition, we discovered that the JAK2 mutation was remarkably related to increased immunogenicity, such as a higher TMB, higher expression of costimulatory molecules and stimulation of antigen processing mechanisms. In addition, JAK2 mutation was positively correlated with activated anticancer immunity, such as infiltration of various immune cells and higher expression of chemokines.ConclusionOur study demonstrates that JAK2 mutation is a novel marker that can be used to effectively predict prognosis and response to ICI therapy.

Defining a TCF1-expressing progenitor allogeneic CD8+ T cell subset in acute graft-versus-host disease

Graft-versus-host disease (GvHD) is a severe complication of hematopoietic stem cell transplantation driven by activated allogeneic T cells. Here, we identify a distinct subset of T cell factor-1 (TCF1)+ CD8+ T cells in mouse allogeneic and xenogeneic transplant models of acute GvHD. These TCF1+ cells exhibit distinct characteristics compared to TCF1- cells, including lower expression of inhibitory receptors and higher expression of costimulatory molecules. Notably, the TCF1+ subset displays exclusive proliferative potential and could differentiate into TCF1- effector cells upon antigenic stimulation. Pathway analyses support the role of TCF1+ and TCF1- subsets as resource cells and effector cells, respectively. Furthermore, the TCF1+ CD8+ T cell subset is primarily present in the spleen and exhibits a resident phenotype. These findings provide insight into the differentiation of allogeneic and xenogeneic CD8+ T cells and have implications for the development of immunotherapeutic strategies targeting acute GvHD.

Gamma-Irradiated Non-Capsule Group B Streptococcus Promotes T-Cell Dependent Immunity and Provides a Cross-Protective Reaction.

Group B Streptococcus (GBS) is a Gram-positive bacterium commonly found in the genitourinary tract and is also a leading cause of neonatal sepsis and pneumonia. Despite the current antibiotic prophylaxis (IAP), the disease burdens of late-onset disease in newborns and non-pregnant adult infections are increasing. Recently, inactivation of the pathogens via gamma radiation has been proven to eliminate their replication ability but cause less damage to the antigenicity of the key epitopes. In this study, the non-capsule GBS strain was inactivated via radiation (Rad-GBS) or formalin (Che-GBS), and we further determined its immunogenicity and protective efficacy as vaccines. Notably, Rad-GBS was more immunogenic and gave rise to higher expression of costimulatory molecules in BMDCs in comparison with Che-GBS. Flow cytometric analysis revealed that Rad-GBS induced a stronger CD4+ IFN-γ+ and CD4+IL-17A+ population in mice. The protective efficacy was measured through challenge with the highly virulent strain CNCTC 10/84, and the adoptive transfer results further showed that the protective role is reversed by functionally neutralizing antibodies and T cells. Finally, cross-protection against challenges with prevalent serotypes of GBS was induced by Rad-GBS. The higher opsonophagocytic killing activity of sera against multiple serotypes was determined in sera from mice immunized with Rad-GBS. Overall, our results showed that the inactivated whole-cell encapsulated GBS could be an alternative strategy for universal vaccine development against invasive GBS infections.

Curcumin mediated dendritic cell maturation by modulating cancer associated fibroblasts-derived exosomal miRNA-146a.

Though cancer associated fibroblasts (CAFs), being a main component of tumor microenvironment (TME), are known to modulate immune response through secretion of various growth hormones, exosomes carrying miRNAs and cytokines; their effect on dendritic cells (DCs) are yet to be elucidated. Thus, aim of this study was to assess the effect of miRNAs and cytokines released by lung-CAFs and to evaluate immunomodulatory potential of curcumin on DC maturation through modulating their TME. To check the effect of CAFs derived exosomes on DC maturation, we cultured imDCs in the presence of CAFs derived conditioned media (CAFs-CM) and characterized by the presence of maturation markers CD80, CD83, CD86 and CTLA4 using qRT-PCR. Additionally, expression of miR-221, miR-222, miR-155, miR-142-3p and miR-146a was assessed to evaluate the role of epigenetic regulators on DC maturation. Likewise, cytokine profiling of CAFs-CM as well as CAFs-CM treated with curcumin was also conducted using ELISA. Results revealed the generation of regulatory DCs which were characterized by decreased expression of maturation markers in the presence of CAFs-CM. In addition, such DCs showed higher expression of epigenetic regulator miR-146a which was positively correlated with increased expression of anti-inflammatory cytokines like IL-6, IL-10, TGF-β and decreased expression of TNF-α (pro-inflammatory). Moreover, curcumin had the potential to convert regulatory DCs generated by CAFs into mDCs, which were characterized by high expression of co-stimulatory molecules, low expression of CTLA4, lower levels of immune suppressive cytokines production and lower levels of miR-146a. Collectively, these findings provide insight into understanding the immunomodulatory role of curcumin in targeting CAFs and modulating TME, thus enhancing antitumor immune response in DC based therapy.

Altered Phenotype of Circulating Dendritic Cells and Regulatory T Cells from Patients with Acute Myocarditis

Dendritic cells (DCs) and regulatory T cells (Tregs) play an essential role in myocarditis. However, a particular DC phenotype in this disease has not been assessed. Herein, we aim to evaluate myeloid (mDCs) and plasmacytoid DC (pDC) phenotype, as well as Treg levels from myocarditis patients and healthy controls. Using multiparametric flow cytometry, we evaluated the levels of myeloid DCs (mDCs), plasmacytoid DCs (pDCs), and Tregs in peripheral blood from myocarditis patients (n = 16) and healthy volunteers (n = 16) and performed correlation analysis with clinical parameters through Sperman test. DCs from myocarditis patients showed a higher expression of costimulatory molecules while a diminished expression of the inhibitory receptors, ILT2 and ILT4. Even more, Treg cells from myocarditis patients displayed higher levels of FOXP3 compared to controls. Clinically, the increased levels of mDCs and their higher expression of costimulatory molecules correlate with a worse myocardial function, higher levels of acute phase reactants, and higher cardiac enzymes. This study shows an activating phenotype of circulating DCs from myocarditis patients. This proinflammatory status may contribute to the pathogenesis and immune deregulation in acute myocarditis.

β-glucan from Aureobasidium pullulans augments the anti-tumor immune responses through activated tumor-associated dendritic cells

Dendritic cells (DCs) are recognized as the most potent antigen-presenting cells, capable of priming both naïve and memory T cells. Thus, tumor-resident DCs (tumor-associated DCs: TADCs) play a crucial role in the immune response against tumors. However, TADCs are also well known as a “double-edged sword” because an immunosuppressive environment, such as a tumor microenvironment, maintains the immature and tolerogenic properties of TADCs, resulting in the deterioration of the tumor. Therefore, it is essential to maintain and enhance the anti-tumoral activity of TADCs to aid tumor elimination. This study demonstrated the potential for tumor growth inhibition of Aureobasidium pullulan-derived β-glucan (AP-BG). Administration of AP-BG dramatically limited the development of different types of tumor cell lines transplanted into mice. Examination of the tumor-infiltrating leukocytes revealed that AP-BG caused high expression of co-stimulatory molecules on TADCs and enhanced the production of cytolytic granules as well as pro-inflammatory cytokines by the tumor-resident T cells. Furthermore, the syngeneic mixed lymphoid reaction assay and popliteal lymph node assay showed the significant ability of AP-BG to improve DCs’ antigen-specific priming of T cells in vitro and in vivo. Taken together, β-glucan might be an immune-potentiating adjuvant for cancer treatment. This highly widely-used reagent will initiate a new way to activate DC-targeted cancer immune therapy.

Clinically applicable CD34+-derived blood dendritic cell subsets exhibit key subset-specific features and potently boost anti-tumor T and NK cell responses

Allogeneic stem cell transplantation (alloSCT), following induction chemotherapy, can be curative for hemato-oncology patients due to powerful graft-versus-tumor immunity. However, disease recurrence remains the major cause of treatment failure, emphasizing the need for potent adjuvant immunotherapy. In this regard, dendritic cell (DC) vaccination is highly attractive, as DCs are the key orchestrators of innate and adaptive immunity. Natural DC subsets are postulated to be more powerful compared with monocyte-derived DCs, due to their unique functional properties and cross-talk capacity. Yet, obtaining sufficient numbers of natural DCs, particularly type 1 conventional DCs (cDC1s), is challenging due to low frequencies in human blood. We developed a clinically applicable culture protocol using donor-derived G-CSF mobilized CD34+ hematopoietic progenitor cells (HPCs) for simultaneous generation of high numbers of cDC1s, cDC2s and plasmacytoid DCs (pDCs). Transcriptomic analyses demonstrated that these ex vivo-generated DCs highly resemble their in vivo blood counterparts. In more detail, we demonstrated that the CD141+CLEG9A+ cDC1 subset exhibited key features of in vivo cDC1s, reflected by high expression of co-stimulatory molecules and release of IL-12p70 and TNF-α. Furthermore, cDC1s efficiently primed alloreactive T cells, potently cross-presented long-peptides and boosted expansion of minor histocompatibility antigen-experienced T cells. Moreover, they strongly enhanced NK cell activation, degranulation and anti-leukemic reactivity. Together, we developed a robust culture protocol to generate highly functional blood DC subsets for in vivo application as tailored adjuvant immunotherapy to boost innate and adaptive anti-tumor immunity in alloSCT patients.

Cryo-thermal therapy induces macrophage polarization for durable anti-tumor immunity

Many cancer therapies are being developed for the induction of durable anti-tumor immunity, especially for malignant tumors. The activation of antigen-presenting cells (APCs), including macrophages and dendritic cells (DCs), can bridge innate and adaptive immune responses against tumors. However, APCs have an immunosuppressive phenotype and reversing it for effective tumor-specific antigen presenting is critical in developing new cancer treatment strategies. We previously developed a novel cryo-thermal therapy to treat malignant melanoma in a mouse model; long-term survival and durable anti-tumor immunity were achieved, but the mechanism involved was unclear. This study revealed cryo-thermal therapy-induced macrophage polarization to the M1 phenotype and modulated the phenotypic and functional maturation of DCs with high expression of co-stimulatory molecules, increased pro-inflammatory cytokine production, and downregulated immuno-inhibitory molecule expression. Further, we observed CD4+ T-cell differentiation into Th1 and cytotoxic T-cell sub-lineages and generation of cytotoxic CD8+ T cells, in which M1 macrophage polarization had a direct, important role. The results indicated that cryo-thermal-induced macrophage polarization to the M1 phenotype was essential to mediate durable anti-tumor immunity, leading to long-term survival. Thus, cryo-thermal therapy is a promising strategy to reshape host immunosuppression, trigger persistent memory immunity for tumor eradication, and inhibit metastasis in the long term.

CD1d highly expressed on DCs reduces lung tumor burden by enhancing antitumor immunity.

Dendritic cells (DCs), as professional antigen-presenting cells are essential for the initial activation of adaptive antitumor immunity. CD1d is considered to present phospholipid and glycosphingolipid antigens to NKT cells. However, it is currently unknown whether CD1d expression on DCs is capable of enhancing antitumor immunity, particularly T-cell related immunity. We observed that CD1d was predominantly expressed on DCs in 3LL tumor-bearing mice, whilst a deficiency of CD1d promoted tumor growth. Notably, CD1d expression on DCs was not only required for presenting antigen to NKT cells, but also markedly promoted CD4+T and CD8+T cell activation, particularly cytotoxic T cells. All the T cells (NKT, CD4+T and CD8+T cells) upregulated CD69, CD107a and IFN-γ after the adoptive transfer of CD1d-positive DCs (CD1d+DCs) and tumor growth was suppressed. With regard to the mechanism, we revealed that CD1d+DCs were concomitant with a higher expression of costimulatory molecules (CD40, CD80 and CD86) and MHCI/II, which are essential for DCs to present antigens to T cells. Consistently, CD1d+DCs displayed stronger activation-associated-ERK1/2 and NF-κB signals; whereas JAK2-STAT3/6 signaling was required for maintaining a high level of CD1d on DCs. In lung cancer patients, the antitumor activities of all the T cells were enhanced with the increase of CD1d+DCs. Analysis of TCGA data revealed that high levels of CD1d indicated better outcomes for patients. Collectively, CD1d enhanced DC-based antitumor immunity, not only by targeting NKT, but also by activating CD4+T and CD8+T cells. CD1d+DCs may be superior to the bulk population of DCs in cancer immunotherapy.

Abstract 4695: Murine tumor growth suppression through CD8+CTLs via activated DEC-205+dendritic cells by sequential administration of α-galactosylceramide in vivo

Abstract Cancer immunotherapy is mediated by the effective priming and activation of tumor-specific class I major histocompatibility complex molecule-restricted CD8+ cytotoxic T lymphocytes. DEC-205+ dendritic cells can cross-present the epitopes of captured tumor antigens associated with class I MHC molecules alongside co-stimulatory molecules to prime and activate tumor-specific CD8+ CTLs. Immunosuppressive tolerogenic DCs with reduced co-stimulatory molecules may be a cause of insufficient CTL induction. Hepa1-6-1 cells were established from the mouse hepatoma cell line Hepa1-6; these cells grow continuously after subcutaneous implantation into syngeneic C57BL/6 mice and do not prime CD8+ CTLs. Here, we show that the growing of tumors was suppressed by activated CD8+ CTLs with tumor-specific cytotoxicity through the administration of the glycolipid α-galactosylceramide, which is a compound known to stimulate invariant natural killer T cells and selectively activate DEC-205+ DCs. Moreover, we demonstrated that sequential repetitive intraperitoneal inoculation of α-GalCer every 48 hour appeared to convert tolerogenic DEC-205+ DCs into immunogenic DCs with a higher expression of co-stimulatory molecules and a greater cross-presentation capacity, which primed CTL precursors and induced tumor-specific CD8+ CTLs within the tumor environment without activating iNKT cells. These findings provide a new basis for cancer immunotherapy to convert tolerogenic DEC-205+ DCs within tumors into immunogenic DCs via the sequential administration of an immuno-potent lipid/glycolipid, and activated immunogenic DCs expressing sufficient suitable co-stimulatory molecules will prime and activate tumor-specific CD8+ CTLs within the tumor to control tumor growing. Citation Format: Hideki Kogo, Hidemi Takahashi, Eiji Uchida. Murine tumor growth suppression through CD8+CTLs via activated DEC-205+dendritic cells by sequential administration of α-galactosylceramide in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4695.

Impact of lenalidomide maintenance on the immune environment of multiple myeloma patients with low tumor burden after autologous stem cell transplantation.

Lenalidomide is a potent anti-myeloma drug with immunomodulatory properties. It is increasingly used in a low-dose maintenance setting to prolong remission duration after standard treatment. Data on the in vivo effects of lenalidomide are scarce and sometimes different from the well-described in vitro effects. We therefore evaluated the numerical, phenotypical and functional impact of lenalidomide maintenance on several immune cell types in a cohort of seventeen homogeneously treated myeloma patients achieving a low residual myeloma burden after a bortezomib based-induction followed by autologous stem cell transplantation. Lenalidomide maintenance: 1) increased the fraction of naïve CD8+ T cells and several memory T-cell subsets, 2) reduced the numbers of terminal effector CD8+ T cells, 3) resulted in a higher expression of co-stimulatory molecules on resting T cells and of the inhibitory checkpoint molecules LAG-3 on CD4+ T cells and TIM-3 on CD4+ and CD8+ T cells, 4) reduced the number of TIGIT+ CD8+ T cells, 5) increased the number of regulatory T cells with a phenotype associated with strong suppressive capacity. Purified CD8+ T cells showed increased and more polyfunctional recall viral responses. However, PBMC responses were not enhanced during lenalidomide maintenance and CD4+ T-cell responses specific for the myeloma-associated antigen MAGE-C1 even tended to become lower. We conclude that lenalidomide maintenance after autologous stem cell transplantation has complex pleotropic effects on the immune environment. Immune interventions such as anti-myeloma vaccination should include measures to tackle an expanded inhibitory Treg compartment.

Abstract 3060: Role of curcumin in targeting cancer-associated fibroblasts and modulation of tumor microenvironment in dendritic cell-based immunotherapy

Abstract INTRODUCTION: Cancer associated fibroblasts (CAFs), main component of tumor microenvironment (TME), modulate the recruitment and functions of tumor-associated immune cells by secreting various growth hormones, miRNAs and cytokines; thus having an important role in generation of immunosuppressive TME which is yet to be elucidated. Curcumin is known to have various properties including capability to modulate numerous target proteins including transcription factors, receptors, kinases, cytokines, enzymes and growth factors. Thus, aim of the study was to evaluate the effect of miRNAs and cytokines released by lung cancer patients’ derived CAFs and to assess immunomodulatory potential of curcumin on DC maturation by targeting these CAFs through modulating their TME. METHODOLOGY: CAFs were cultured from lung cancer patient derived tumor tissue biopsy and characterised using CAF-specific markers. Further, immature DCs (imDCs) were differentiated in presence of rGM-CSF and rIL-4 for 5-6 days. These imDCs were cultured in the presence of conditioned media derived from CAFs (CAFs-CM) as well as NFs (Normal Fibroblasts, NFs-CM) at day 6 for 48 hours to evaluate the effect of CAFs on DC maturation. Mature DCs (mDCs) were characterized by the presence of maturation markers CD80, CD83, CD86 and CTLA4 using qRT-PCR. Moreover, expression of miR-221, miR-222, miR-155, miR-142-3p and miR-146a was assessed to evaluate the role of epigenetic regulators on DC maturation. Cytokine profiling of CAFs-CM as well as CAFs-CM treated with curcumin was conducted. RESULTS: α-SMA+Vimentin+ cells were considered as CAFs. A significant upregulation of CD80, CD83 and CD86 was observed when cultured in the presence of NFs-CM while a remarkable downregulation of these markers was found when cultured in CAFs-CM. CTLA-4 was down regulated in NFs-CM as compared to CAFs-CM, suggesting the role of CAFs in generation of regulatory DCs. Amongst all miRNAs, miR-146a was shown to be up regulated dramatically in CAF-DCs (DCs cultured in CAFs-CM) as well as in CAFs-CM, suggesting the immunosuppressive role of miR-146a. Further, an increased expression of miR-146a was positively correlated with increased expression of anti-inflammatory cytokines like IL-6, IL-10, TGF-β and decreased expression of TNF-α (pro-inflammatory) in CM derived from CAF-DCs. Moreover, curcumin had the potential to convert regulatory DCs facilitated by CAFs into mDCs, which were characterized by high expression of co-stimulatory molecules, low expression of CTLA4, lower levels of immune suppressive cytokines production, lower levels of miR-146a. CONCLUSION: These findings provide insight into understanding the immunomodulatory role of curcumin in targeting CAFs and modulating the tumor microenvironment, thus enhancing antitumor immune response in DC based therapy. Citation Format: Sheefa Mirza, Nayan Jain, Rakesh Rawal. Role of curcumin in targeting cancer-associated fibroblasts and modulation of tumor microenvironment in dendritic cell-based immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3060. doi:10.1158/1538-7445.AM2017-3060

CD103+ lung dendritic cells (LDCs) induce stronger Th1/Th17 immunity to a bacterial lung infection than CD11bhi LDCs.

Recent studies suggest differential roles for CD103+ and CD11bhi lung dendritic cells (LDCs) in host defense against viral and bacterial infections. In this study, we examined the contribution of these LDC subsets in protective immunity to chlamydial lung infection using a Chlamydia muridarum mouse infection model. We found that CD103+ LDCs showed higher expression of costimulatory molecules (CD40, CD80 and CD86) and increased production of cytokines (IL-12p70, IL-10, IL-23 and IL-6) compared with CD11bhi LDCs, but the expression of programmed death-ligand 1 (PD-L1) was similar between the two subsets. More importantly, we found, in adoptive transfer experiments, that the mice receiving CD103+ LDCs from Chlamydia-infected mice exhibited better protection than the recipients of CD11bhi LDCs, which was associated with more robust Th1/Th17 cytokine responses. In addition, in vitro experiments showed that CD103+ LDCs induced stronger IFN-γ and IL-17 responses, when cocutured with chlamydial antigen-primed CD4+ T cells, than CD11bhi LDCs. Furthermore, the blockade of PD1 in the culture of CD4+ T cells with either CD103+ or CD11bhi LDCs enhanced production of IFN-γ and IL-17. In conclusion, our data provide direct evidence that CD103+ LDCs are more potent in promoting Th1/Th17 immunity to chlamydial lung infection than CD11bhi LDCs.

Cell enrichment-free massive ex-vivo expansion of peripheral CD20+ B cells via CD40-CD40L signals in non-human primates

Non-human primates (NHPs) are valuable as preclinical resources that bridge the gap between basic science and clinical application. B cells from NHPs have been utilized for the development of B-cell targeted drugs and cell-based therapeutic modalities; however, few studies on the ex-vivo expansion of monkey B cells have been reported. In this study, we developed a highly efficient ex-vivo expansion protocol for monkey B cells resulting in 99% purity without the requirement for prior cell-enrichment procedures. To this end, monkey peripheral blood mononuclear cells (PBMCs) were stimulated for 12 days with cells constitutively expressing monkey CD40L in expansion medium optimized for specific and massive expansion of B cells. The B cells expansion rates obtained were 2–5 times higher than those previously reported in humans, with rates ranging from 7.9 to 16.6 fold increase. Moreover, expanded B cells sustained high expression of co-stimulatory molecules including CD83 and CD86 until day 12 of culture, and the simple application of a brief centrifugation resulted in a CD20+ B cell purity rate of greater than 99%. Furthermore, small amounts of CD3+CD20+BT-like cells were generated and CD16 was expressed at moderate levels on expanded B cells. Thus, the establishment of this protocol provides a method to produce quantities of homogeneous, mature B cells in numbers sufficient for the in vitro study of B cell immunity as well as for the development of B cell-diagnostic tools and cell-based therapeutic modalities.

Natural Toll-like Receptor Agonists for in Situ Vaccination Lymphoma Immunotherapy

Introduction: There is an unmet need for novel and effective treatments for lymphoma, the fifth most common malignancy in the US. In situ vaccination-an immunotherapeutic maneuver involving local irradiation, intratumoral (i.t.) injections of Flt3L and Toll-like receptor (TLR) agonist-has been shown in recent clinical trials (NCT00185965, NCT00880581, NCT00226993) to induce partial and complete remissions in patients with low-grade lymphoma. The strength of anti-tumor response correlates with the potency of immunogenic dendritic cells (DCs) to efficiently uptake and present tumor antigens to T cells. While the latest clinical trial NCT01976585 employs Poly-ICLC-a synthetic TLR3 agonist-to activate DCs, we hypothesize that "natural" TLR agonists (nTLRa) contained within prophylactic vaccines (e.g. live/attenuated bacteria or viruses) could simultaneously target multiple TLRs and be repurposed as clinical-grade DC activators for the in situ vaccination maneuver.Methods: Twenty-four prophylactic vaccines from our hospital's pharmacy were screened using in vitro assays for TLR ligand activity. DC subsets CD11c+ MHC-II+, CD11c+ CD11b+, and CD11c+ CD103+ were gated for flow-cytometric readouts of: (i.) activation markers (e.g. CD40, CD80, CD86, MHC class I and II), (ii.) ability to co-stimulate T cells in the context of TCR activation, as assessed by T cell activation markers (e.g. CD69, intracellular IFNγ), and (iii.) ability of nTLRa-activated DCs to induce T cell proliferation. TLR knock-out cell lines were used for dissecting the mechanistically-distinct properties of each prophylactic vaccine. Best single and combination nTLRa candidates were evaluated with the in situ vaccination maneuver in an in vivo A20 murine lymphoma model.Results: We identify a combination of vaccines Typhim, BCG-TICE, and MMR that demonstrates the ability to induce high expression of costimulatory molecules on DCs (Figure A) in vitro, as well as possessing the ability to co-stimulate in vitro T cell activation and proliferation. DC costimulation of T cell activation and proliferation is highly dependent on the "live" status of several vaccines, including BCG, MMR, Zostavax, which suggests the involvement of other pattern-recognition receptors in addition to TLRs. Cohorts of mice implanted with A20 lymphoma tumors that received i.t. injections of Typhim, BCG, MMR demonstrated slower A20 growth status post in situ vaccination maneuver, as compared to the control cohort receiving only Flt3L and irradiation (XRT).Conclusion: Prophylactic vaccines contain natural ligands to TLRs and are immediately translatable as sources of clinical-grade stimulators of dendritic cells. Combinations of the best nTLRa candidates display synergistic activation of DCs and demonstrate in vivo anti-A20 murine lymphoma immunity. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Phase I/II Clinical Trial of CpG-Activated Whole Cell Vaccine in Mantle Cell Lymphoma (MCL): Results in Safety and Efficacy from Planned Interim Analysis

IntroductionMCL has a poor prognosis. In eligible patients, intensifying frontline, CHOP-like regimens (e.g., cytarabine) as well as high-dose chemotherapy and autologous stem cell transplant (HDT/ASCT) consolidation in first remission have improved progression free survival (PFS) but less so, overall (OS). Preclinical animal models show benefits of adding tumor-specific T-cells to ASCT. CpG (PF-3512676) is a toll-like receptor 9 (TLR9) agonist and an effective vaccine adjuvant that induces costimulatory molecule expression on both antigen-presenting and MCL cells. This phase I/II clinical trial (NCT00490529) adds autologous T-cell transfer, harvested from patients after vaccination with CpG-activated autologous MCL cells - a maneuver termed immunotransplant. This is a planned interim analysis for safety and efficacy triggered by the first 20 patients reaching 1 year post ASCT.MethodsPrior to therapy, subjects' tumor cells are collected by biopsy or apheresis and patient-specific vaccine is created by incubating fresh tumor cells with CpG (3 mcg/mL PF-3512676, 72-hr culture), then irradiated and cryopreserved. Patients receive induction chemoimmunotherapy of physician's choice. Patients achieving at least partial response (PR) then receive 3 subcutaneous autologous tumor vaccinations (1 x 108 cells/dose) mixed with CpG (18 mg) every 4-7 days. Primed T-cells (≥ 1 x 1010 CD3 cells) were collected by apheresis 2-4 weeks following vaccine 3 and a rituximab (375 mg/m2) B-cell purge. After standard HDT/ASCT (conditioning = BCNU, cyclophosphamide, etoposide), primed T-cells and a 4th vaccination are given on day+1. A 5th CpG-MCL vaccination followed 3 months post ASCT. The primary endpoint of this study is freedom from minimal residual disease (MRD) at 1 year post ASCT, measured by presence of patient-specific, malignant B-cell VDJ sequence in peripheral blood (ClonoSeq™, analyzed at a sensitivity of ≥ 1 clone/10,000 leukocytes) -an endpoint previously shown to be highly prognostic. Secondary endpoints include PFS, OS, and immune response to vaccine. 59, transplant-eligible, MCL patients are targeted for accrual in this 2-stage design.ResultsIn this interim analysis of 24 patients accrued, 20 have surpassed 1 year post ASCT. All patients had Stage IV disease. Median values included (range): follow-up 43.5 months (11.5-60.1), age 60 years (47-70), and MIPI score 5.9 (5.1-7.8). Vaccine was made from biopsy alone (n=12), apheresis alone (n=9), or both (n=1). Frontline therapy included R-CHOP (n=7), R-hyperCVAD (n=14), alternating R-CHOP/R-DHAP (n=2), and R-EPOCH (n=1). 19 patients achieved complete response while another 3 had PR. All responding patients were vaccinated, able to yield sufficient T-cells for adoptive transfer, and proceeded through standard HDT/ASCT. At 1-year post ASCT, freedom from MRD was 90.5% (n=21). 2 patients did not reach 1-year post ASCT. One died from an ASCT complication (metapneumovirus) while the other relapsed 6 months following ASCT (included in MRD analysis). The most common toxicity due to CpG-MCL vaccine was erythematous rash at injection site (90.9%, n=20, each grade 1). No serious adverse events were seen related to vaccines or adoptive T-cell transfer. All patients had successful hematopoietic recovery, but two were delayed and received backup stem cell infusions with eventual recovery. Though median PFS and OS have not been reached, 3-year PFS and OS at interim analysis are 54.5% and 63.6%, respectively (intention to treat). In this data set, higher expression of co-stimulatory molecules (such as CD40, CD80, and CD86) on a subject's MCL in response to CpG was associated with freedom from MRD (p =0.02). Post-ASCT, higher peripheral T-cell granzyme and perforin response to ex vivo re-challenge with autologous MCL cells was also associated with freedom from MRD (p =0.01).ConclusionsThe addition of CpG-activated, whole MCL vaccination and autologous, adoptive T-cell transfer to standard therapy appears both feasible and safe. At interim analysis, the 1-year freedom from MRD surpasses rates previously reported in other studies and warrants further investigation. To date, 46 patients have either completed and/or are continuing to undergo study treatment and the study remains open to accrual for patients newly diagnosed with MCL. DisclosuresMiklos:Pharmacyclics: Research Funding. Rezvani:Pharmacyclics: Research Funding. Faham:Adaptive Biotechnologies: Employment. Levy:Immune Design: Research Funding; Dynavax: Research Funding.

Abstract 456: Defined co-mutation subgroups of KRAS-mutated NSCLC display distinct immune profiles

Abstract Traditional pathological divisions of lung cancer are giving rise to molecular classifications that identify unique drivers of tumor malignancy. Of these molecular classifications, KRAS represents about 30% of all lung adenocarcinomas. Our group has recently investigated if co-mutations within KRAS-mutated lung tumors can aid in identifying a unique therapeutic strategy for patient treatment. Given that tumors exert a strong influence over the immune system and the recent successes in immune targeting via checkpoint blockade therapy in various cancers, including lung, we hypothesized that co-mutational subgroups of KRAS-mutant NSCLC elicit distinct immune profiles and support a rationale for evaluating these subgroups in conjunction with appropriate immunotherapies for further clinical testing. To investigate this phenomenon, we performed a supervised analysis of 86 immune-related genes from The Cancer Genome Atlas data set in lung adenocarcinomas with KRAS mutations and TP53 or STK11(LKB1) co-mutations. The results from our analysis revealed that KRAS-mutant NSCLC with LKB1 (KL) and TP53 (KP) co-mutations have distinct immune profiles. KP tumors have an increase in (1) PD-L1 and PD-L2; (2) co-stimulation via CD40, CD80, CD86; (3) antigen presentation HLA-I, HLA-II, and CD1; (4) FOXP3 and CD4, indicating T regulatory populations; (5) CD33, CD11b, and CD11c, which are potentially suppressive myeloid cell populations. We conclude that KRAS co-mutations can create significantly different immune microenvironments; KRAS + TP53 (KP) mutant tumors may specifically benefit from checkpoint blockade, depletion of immunosuppressive myeloid and T regulatory populations, but could support de novo anti-tumor T cell expansion due to high expression of co-stimulatory molecules and HLA-I and HLA-II expression. Conversely, KRAS + LKB1 (KL) mutant tumors have an immunosuppressed or inert phenotype and may potentially benefit from adoptive cell transfer of anti-tumor T cells in conjunction with or in lieu of checkpoint blockade. Citation Format: Warren L. Denning, Lixia Diao, Ferdinandos Skoulidis, Yanyan Lou, Lauren Byers, Jing Wang, John Weinstein, Don Gibbons, John Heymach. Defined co-mutation subgroups of KRAS-mutated NSCLC display distinct immune profiles. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 456. doi:10.1158/1538-7445.AM2015-456

Intranodal vaccination with mRNA-optimized dendritic cells in metastatic melanoma patients

Autologous dendritic cell (DC) therapy is an experimental cellular immunotherapy that is safe and immunogenic in patients with advanced melanoma. In an attempt to further improve the therapeutic responses, we treated 15 patients with melanoma, with autologous monocyte-derived immature DC electroporated with mRNA encoding CD40 ligand (CD40L), CD70 and a constitutively active TLR4 (caTLR4) together with mRNA encoding a tumor-associated antigen (TAA; respectively gp100 or tyrosinase). In addition, DC were pulsed with keyhole limpet hemocyanin (KLH) that served as a control antigen. Production of this DC vaccine with high cellular viability, high expression of co-stimulatory molecules and MHC class I and II and production of IL-12p70, was feasible in all patients. A vaccination cycle consisting of three vaccinations with up to 15×106 DC per vaccination at a biweekly interval, was repeated after 6 and 12 months in the absence of disease progression. mRNA-optimized DC were injected intranodally, because of low CCR7 expression on the DC, and induced de novo immune responses against control antigen. T cell responses against tyrosinase were detected in the skin-test infiltrating lymphocytes (SKIL) of two patients. One mixed tumor response and two durable tumor stabilizations were observed among 8 patients with evaluable disease at baseline. In conclusion, autologous mRNA-optimized DC can be safely administered intranodally to patients with metastatic melanoma but showed limited immunological responses against tyrosinase and gp100.

Epstein–Barr virus induces the differentiation of semi-mature dendritic cells from cord blood monocytes

BackgroundEpstein–Barr virus (EBV) is a tumorigenic virus which has effectively infected nearly all human beings with over 95% adult being seropositive. The persistence of latent EBV infection is not fully understood. Recent studies point towards a hypothesis of immune suppression and immune evasion involving regulatory T cells (Tregs) and dendritic cells (DCs). We sought to explore the mechanism of EBV suppression and immune evasion. MethodsWe compared the effects of EBV on cord blood (CB) and adult DCs differentiation and maturation including phenotype by flow cytometry, cytokine by ELISA and RT-PCR. And we evaluated the function of DC by co-culture DC and Treg by detection the expression of Foxp3, the phenotype and the cytokine profile of Tregs by flow cytometry. ResultsCB DCs derived from EBV-infected CB monocytes or from EBV-infected CB immature DCs (iDCs) displayed distinct phenotypes of “semi-mature” DCs with high expression of co-stimulatory molecules, such as CD40, CD80 and CD86 but low cytokine production, related to immune tolerance and homeostasis. While the EBV-infected adult iDCs resemble that of “pathogen-driven regulatory mature DCs” with high expression of co-stimulatory molecules, down-regulation of IL-12 secretion and up-regulation of IL-10 secretion, related to protection of host and immune evasion of pathogens. EBV infected cord blood monocytes-derived DCs drived Tregs development by driving the expression of Foxp3, increasing the expression of CTLA-4, decreasing the expression of GITR and promoted the generation of intracellular IL-2 and IL-10 by Tregs. ConclusionEpstein–Barr virus induces the differentiation of semi-mature dendritic cells from cord blood monocytes. The differences between CB and adult DCs suggested that the developmental maturity of the cells may affect their immune responses to EBV infection.

Gamma delta T cells are activated by polysaccharide K (PSK) and contribute to the anti-tumor effect of PSK

Polysaccharide K (PSK) is a widely used mushroom extract that has shown anti-tumor and immunomodulatory effects in both preclinical and clinical studies. Therefore, it is important to understand the mechanism of actions of PSK. We recently reported that PSK can activate toll-like receptor 2 and enhances the function of NK cells. The current study was undertaken to study the effect of PSK on gamma delta (γδ) T cells, another important arm of the innate immunity. In vitro experiments using mouse splenocytes showed that γδ T cells produce IFN-γ after treatment with PSK and have up-regulated expression of CD25, CD69, and CD107a. To investigate whether the effect of PSK on γδ T cells is direct or indirect, purified γδ T cells were cultured either alone or together with bone marrow-derived DC in a co-culture or trans-well system and then stimulated with PSK. Results showed that direct cell-to-cell contact between γδ T cells and DC is required for optimal activation of γδ T cells. There was also reciprocal activation of DC by PSK-activated γδ T cells, as demonstrated by higher expression of costimulatory molecules and enhanced production of IL-12 by DC in the presence of γδ T cells. PSK can also co-stimulate γδ T cells with anti-TCR and anti-CD3 stimulation, in the absence of DC. Finally, in vivo treatment with PSK activates γδ T cells among the tumor infiltrating lymphocytes, and depleting γδ T cells during PSK treatment attenuated the anti-tumor effect of PSK. All together, these results demonstrated that γδ T cells are activated by PSK and contribute to the anti-tumor effect of PSK.