IFN-gamma Production Research Articles

Characterisation and analysis of IFN-gamma producing cells in rainbow trout Oncorhynchus mykiss

IFN-γ is one of the key cytokines involved in Th1 immune responses. It is produced mainly by T cells and NK cells, which drive both innate and adaptive responses to promote protection against infections. IFN-γ orthologues have been discovered to be functionally conserved in fish, suggesting that type I immunity is present in early vertebrates. However, few studies have looked at IFN-γ protein expression in fish and its role in cell mediated immunity due to a lack of relevant tools. In this study, four monoclonal antibodies (mAbs) V27, N2, VAB3 and V91 raised against short salmonid IFN-γ peptides were developed and characterised to monitor IFN-γ expression. The results show that the IFN-γ mAbs specifically react to their peptide immunogens, recognise E. coli produced recombinant IFN-γ protein and rainbow trout IFN-γ produced in transfected HEK 293 cells. The mAb VAB3 was used further, to detect IFN-γ at the cellular level after in vitro and in vivo stimulation. In flow cytometry, a basal level of 3–5% IFN-γ secreting cells were detected in peripheral blood leucocytes (PBL), which increased significantly when stimulated in vitro with PAMPs (Aeromonas salmonicida bacterin), a mitogen (PHA) and recombinant cytokine (IL-2). Similarly, after injection of live bacteria (Aeromonas salmonicida) or poly I:C the number of IFN-γ+ cells increased in the lymphoid population of PBL, as well as in the myeloid population after infection, with the myeloid cells increasing substantially after both treatments. Immunohistochemistry was used to visualise the IFN-γ+ cells in spleen and head kidney following vaccination, which increased in intensity of staining and number relative to tissue from saline-injected control fish. These results show that several types of cells can produce IFN-γ in trout, and that they increase following infection or vaccination, and likely contribute to immune protection. Hence monitoring IFN-γ producing cells/protein secretion may be an important means to assess the effectiveness of Th1 responses and cell mediated immunity in fish.

Development of a novel anti-B7-H4 antibody enhances anti-tumor immune response of human T cells

BackgroundB7-H4 is a member of the B7 superfamily that is expressed on the surface of tumors and exhibits limited expression on normal tissue. B7-H4 negatively regulates tumor immunity by interacting with the B7-H4 receptor, which is expressed by activated CD8 + T cells. Hence, we sought to generate an immunomodulatory antibody that targets B7-H4 and blocks the immunosuppressive activity of B7-H4. MethodsAnti-B7-H4 antibodies were generated using the hybridoma technique and screened by a binding assay based on B7-H4-expressing tumor cells. The B7-H4 antagonistic antibodies were further screened based on their checkpoint blockade activity using a SEB-stimulated peripheral blood mononuclear cell (PBMC) assay, which comprised B7-H4-expressing antigen presenting cells (APCs) and activated T cells. To assess the immunomodulatory activity of anti-B7-H4 antibodies, activated human CD8+ T cells were cultured in B7-H4 protein-coated plates, and the production of IL-2 and the proliferation rate of CD8+ T cells were measured. In addition, we evaluated the ADCC effect of anti-B7-H4 antibodies against tumor cell lines. The in vivo antitumor efficacy of the anti-B7-H4 antibody was also evaluated in human T cell-engrafted NOG mice. ResultsA panel of anti-B7-H4 antibodies was generated. The top 23 antibodies were screened to identify antibodies that disabled B7-H4-mediated inhibition. Antibody 17 exhibited the greatest induction of the production of IL-2 and IFN-gamma in SEB-stimulated PBMCs. Antibody 17 was constructed as a chimeric antibody (CH17) with a human IgG1 constant domain. CH17 showed high affinity for human B7-H4 and fully cross-reacted with cynomolgus B7-H4. Additionally, CH17 mediated potent antibody-dependent cell cytotoxicity (ADCC) against different B7-H4-positive tumor cell lines. More importantly, CH17 relieved B7-H4-mediated T cell suppression by enhancing IL2 production and promoting T cell proliferation. In an MDA-MB-468-bearing mouse model in which human pan-T cells were engrafted, CH17 delayed tumor growth by engaging T cells and exerted a synergistic effect in combination with an anti-human PD-1 antibody. ConclusionsWe successfully generated an immunomodulatory antibody targeting B7-H4 that possesses both T cell immune checkpoint inhibitory activity and ADCC activity in B7-H4-positive tumors. B7-H4-targeting antibodies might represent a promising immunotherapy for B7-H4-expressing tumors.

Abstract 1770: Ectopic expression of ULBP2 promotes tumor progression in syngeneic mice

Abstract ULBP2 is one of the ligands for NKG2D (NKG2DLs). ULBP2 expression is induced in transformed cells and is recognized by immune effector cells via the activating NKG2D immunoreceptor. Previously, we reported high concentrations of serum-soluble ULBP2 (sULBP2) were correlated with poor prognosis in NSCLC patients. As a possible mechanism, we demonstrated that sULBP2 directly suppressed the cytolytic activity of peripheral blood mononuclear cells (PBMCs). However, little is known about the in vivo function of human NKG2DLs on tumor cells. The present study investigates the function of human NKG2DLs in the syngeneic C57BL/6 mice. We generated ULBP2 stably expressing B16 melanoma cell lines (ULBP2-B16). As previously reported, 4-hour 51Cr releasing assay showed NK cell cytotoxicity against the tumor cells was enhanced by ectopic expression of ULBP2 on the tumor cells. IFN-gamma production in NK cells was decreased by non-contact co-culture with ULBP2-B16. To investigate the in vivo function of ULBP2, C57BL/6 mice were inoculated s.c. with mock-B16 cells or ULBP2-B16 cells. ULBP2-B16 tumors grew rapidly compared to mock-B16 tumors. NKG2D blockade by anti-NKG2D antibody injected i.p. suppressed tumor growth of ULBP2-B16 tumors. Spleen cells and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. NKG2D was expressed on splenic NK cells, while it was rarely expressed on splenic CD8+ T cells. NKG2D was downregulated on NK cells in TILs (NK-TILs) from ULBP2-B16. NKG2D was highly expressed on CD8+ T cells in TILs (CD8+-TILs) from mock-B16 tumors, while its expression was suppressed on these from ULBP2-B16 tumors. Degranulation analysis revealed that ULBP2-B16 reduced activity of NK-TILs and CD8+-TILs compared to mock-B16. In conclusion, ectopic expression of ULBP2 on tumor cells promotes tumor progression via suppression of the activity of NK cells and CD8+ T cells. These findings suggest that neutralization of NKG2DLs may be a promising approach for the treatment of NKG2DLs-expressing tumors. Citation Format: Kosuke Yamaguchi, Naomi Miyake, Yasuhiko Teruya, Kohei Yamane, Naoki Kinoshita, Tomohiro Sakamoto, Masahiro Kodani, Hiroki Chikumi, Akira Yamasaki. Ectopic expression of ULBP2 promotes tumor progression in syngeneic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1770.

Abstract LB181: Autologous glioblastoma tumor cells treated with an antisense oligonucleotide against insulin-like growth factor type 1 receptor protect mice against GL261 tumor challenge

Abstract Background: Despite the current standard of care (SOC) established by Stupp et al (N Engl J Med. 2005:10;352:987-96), patients with glioblastoma continue to have a poor prognosis. IGV-001 is a novel immunotherapy that combines irradiated, patient-derived glioblastoma tumor cells and an antisense oligonucleotide against insulin-like growth factor type 1 receptor (IMV-001) in biodiffusion chambers (0.1-micron pore size). We recently evaluated IGV-001 in patients with newly diagnosed glioblastoma (Andrews et al. Clin Cancer Res. 2021; in press). In a subgroup of IGV-001-treated, Stupp-eligible patients with methylated O6-methylguanine-DNA methyl-transferase promoter, median progression free survival was 38.4 months (compared with 6.5 months in a historical SOC; P=0.0008). We sought to unravel the potential mechanism associated with the high degree of effectiveness of IGV-001 in patients using the GL261-luciferase (-Luc) glioblastoma orthotopic murine model. Methodology: Biodiffusion chambers (0.1- or 5-micron pore size for comparison) containing phosphate-buffered saline (PBS) alone or IGV-001 prepared with 1×106 GL261-Luc cells were implanted in the flanks of C57BL/6 albino female mice. The chambers were explanted after 48 hours (as done in the clinical protocol), followed by intracranial tumor challenge with GL261-Luc cells 26 days after chamber removal. Mice were monitored for survival and tumor growth. FluoroSpot assays were conducted to determine IFN-gamma production as a surrogate measure of antitumor Th1 cytotoxic lymphocyte (CTL) activity. Results: No mice died of disease in either IGV-001-treated group and continued to gain weight until the termination of the study, 28 days post-intracranial tumor challenge. In comparison, there was only one survivor in the PBS group at termination with a median survival time (MST) of 20 days (P<0.001). At the endpoint, 54% of all IGV-001-treated mice (0.1- and 5-micron chambers combined) presented with only a minimal GL-261-Luc bioluminescent signal (<1×108 photons/sec). FluoroSpot assays revealed that on day 29, post-chamber implantation, the number of IFN-gamma-producing cells in the peripheral blood was significantly greater (P<0.01) in IGV-001-treated versus PBS-treated mice, regardless of chamber size. Conclusions: These data support the antitumor activity of IGV-001 in newly diagnosed glioblastoma as demonstrated in the phase 1 study. The results suggest that IGV-001 effectiveness is at least in part associated with a systemic immunological effect, resulting in the generation of Th1 antitumor CTLs. Follow-up studies are aimed to determine the MST of mice receiving this immunotherapy and the factors triggering complete versus partial responses using phenotypic evaluation of T-cell activation/exhaustion markers and Th1/Th2 cytokine production. Citation Format: Jenny Zilberberg, Samantha Garcia, Amelia Zellander, David W. Andrews, Mark A. Exley. Autologous glioblastoma tumor cells treated with an antisense oligonucleotide against insulin-like growth factor type 1 receptor protect mice against GL261 tumor challenge [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB181.

Phase I/II Trial of Lenalidomide and Ofatumumab for the Treatment of Relapsed or Refractory Non-Hodgkin's Lymphoma (NHL)

Introduction:Outcomes for patients with NHL who are refractory to first line therapy as well as relapsed disease are poor. Lenalidomide, an immunomodulatory drug, stimulates T-cell proliferation, IL2, IL10 and IFN-gamma production. Ofatumumab, a monoclonal anti-CD20 antibody has shown to have activity as monotherapy in B-cell NHL. This phase I/II study evaluates the safety and clinical activity of the combination treatment of lenalidomide and ofatumumab for relapsed/refractory NHL.Methods:Patients with histologically confirmed diagnosis of CD20+ NHL, recurrent/ refractory to at least one prior therapy were eligible for the study. All the patients received 8 weekly infusions of ofatumumab 1000mg. Dose escalation of lenalidomide was done in three planned dose cohorts. Cohort 1 received 15mg per day, cohort 2 and 3 received 20mg and 25mg per day respectively on days 1-21 every 28 days. The study design involved a standard 3+3 approach. Dose reduction Cohort (-1) at 10mg/day was built into the protocol in the event of a dose limiting toxicity in cohort 1. Phase II evaluated lenalidomide at maximum tolerated dose (MTD) and ofatumumab in 36 patients. Flow analysis for total T-cells, sIL2r, IL12, VEGF, TNFa levels were checked at baseline, 6 months, 12 months on therapy and at the time of discontinuation of treatment.Results:The MTD for lenalidomide was 10mg. Six patients in phase 1 treated at the MTD and 34 patients enrolled on Phase II were eligible for evaluation. Data was analyzed for all patients treated at the MTD (n=40). Twelve (30%) patients had diffuse large B-cell lymphoma (DLBCL), 12 (30%) follicular lymphoma (FL), 6 (15%) mantle cell lymphoma (MCL), 6 (15%) chronic lymphocytic leukemia/small lymphocytic leukemia (CLL/SLL), 4(10%) other lymphomas. The median age was 64.5 years (35.5-80.7). Thirty one (78%) were male, 40 (100%) caucasians, 45% had stage IV disease. Number of lenalidomide cycles ranged from 1-63. In the phase I cohort, grade 3-4 adverse events included neutropenia (10%), thrombocytopenia (10%) and secondary malignancies (20%). Grade 3-4 adverse events on Phase II were neutropenia (42%), thrombocytopenia (8%), infusion reactions (8%) and secondary malignancies (17%). Overall response rate [stable disease (SD), partial response (PR) + complete response CR] was 67% (95% %CI:52%-80%) including 11 (92%) patients with FL, 5 (83%) MCL, 3 (25%) DLBCL and 4(66%) CLL/SLL. CR was noted in 3 patients which included DLBCL, FL and MCL. The durability of response in patients who achieved CR was 7.02 years (DLBCL), 2.89 years (FL) and 2.54 years (MCL). At 6 months, the mean change from baseline in sIL2r was 389.8 [SD=421 p=0.002], CD3+T = -0.43 [SD=0.77, p=0.04]. The median change in sIL2r and CD3+CD4+G was lower for patients with CR when compared to patients with PR or SD [sIL2r:-82.7 vs 543.5, p=0.02; CD3_CD4_G: -26.1 vs. -0.82, p=0.02]. Median progression free survival (PFS) was 6.8 months (2.3-12.1). Median overall survival (OS) was 19.8 months (7.4-42.8). Estimated 1 year PFS was 38% and 2 years was 20%. Estimated 1 year OS was 56%, 2-year survival was 49%.Conclusion:The combination of lenalidomide at 10 mg for 21/28 days and ofatumumab has manageable toxicities and produces some durable response in patients with relapsed/refractory indolent NHL including FL, MCL, and CLL/SLL. DisclosuresLunning:Celgene: Consultancy; BMS: Consultancy; Genentech: Consultancy; Onyx: Consultancy; TG Therapeutics: Consultancy; AbbVie: Consultancy; Juno: Consultancy; Epizyme: Consultancy; Gilead: Consultancy; Spectrum: Consultancy; Pharmacyclics: Consultancy. Armitage:Tesario bio Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Samus Therapeutics: Consultancy; Conatus- IDMC: Consultancy. Vose:Incyte: Research Funding; Acerta: Research Funding; Allos Therapeutics: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Kite: Research Funding; Merck: Research Funding; Onyx: Research Funding; Seattle Genetics: Research Funding; US Biotest: Research Funding; Bristol-Myers Squibb: Research Funding.

Direct Comparison of Anti-CD33 and Anti-CD123 Chimeric Antigen Receptor T Cells with CD28 or 41BB Co-Stimulatory Domains for Treatment of Acute Myeloid Leukemia

Introduction: Acute Myeloid Leukemia (AML) is an aggressive malignancy with very poor prognosis and limited treatment options. Chimeric antigen receptor expressing T (CART) cell therapy has been highly successful against refractory B cell acute lymphoblastic leukemia and lymphoma. Effective use of CART cells in AML has not yet been well established. Differences in efficacy and potency of CD28 or 41BB co-stimulatory domains of the CART cells have not been thoroughly studied particularly in the context of AML. Here, we systematically compare potency and efficacy of anti-CD123 and anti-CD33 CART cells with CD28 or 41BB co-stimulatory domains against AML both in vitro and in vivo .Methods: anti-CD33 and anti-CD123 CART cells were developed using two different anti-CD33 and a single anti-CD123 single chain fragment variable sequences and cloning into a third-generation lentiviral plasmid containing a CD8 transmembrane domain, a co-stimulatory signaling domain (either CD28 or 41BB) and a CD3zeta domain. CART surface expression on T cell surface was measured by flow cytometry. Efficacy of CAR expressing T cells was tested against a panel of AML cell lines with relatively uniform expression of CD33 and variable expression of CD123 including MOLM14, MV4;11, THP1 and U937. Enzyme-linked immunosorbent assay kits for interleukin-2 (IL2) and interferon-gamma (IFN-gamma) were used to assess in vitro CART functionality, 105 tumor cells were co-incubated with 105 transduced CART cells or mock T cells for 16 hours in 96-well plate. In vitro killing of tumor by CART cells was evaluated by measuring green and red fluorescence by IncuCyte ZOOM, 105 tumor cells that were co-incubated with 105 transduced CART cells or mock T cells for 40 hours in 96-well plates. In vivo CART functionality was assessed by monitoring leukemia burden through bioluminescent imaging using mice engrafted with AML cell lines that received anti-CD33 and anti-CD123 CART cell therapy (tumor challenge of 1x106 cells was given and CART cell therapy dose of 7x106 transduced cells was given). Bone marrow and spleen of mice were assessed by flow cytometry for CART cell and leukemia presence.Results: In vitro cytokine production of IL2 and IFN-gamma when anti-CD123 and anti-CD33 CART cells are co-incubated with various AML cell lines indicate greater production of cytokine in CART cells with a CD28 co-stimulatory domain. In vitro killing assay indicates no significant difference in killing capacity of anti-CD33 and anti-CD123 with CD28 or 41BB co-stimulatory domains, however there is some evidence that the CD28 co-stimulatory domain may yield more killing at earlier time points than 41BB co-stimulatory domain. In vivo activity of anti-CD33 and anti-CD123 CART cells against MOLM14, THP1 and U937 shows that clearance of AML occurs more potently and at earlier time points with the CD28 co-stimulatory domain when compared to the 41BB co-stimulatory domain. Bone marrow and spleen analyses show the lack of presence of leukemia but presence of CART cells with CD28 co-stimulatory domain.Conclusions: CART cells targeting surface antigen CD33 and CD123 in AML demonstrate in vitro and in vivo activity against AML, showing clearance of leukemia and presence of CART cells. Additionally, in vitro and in vivo comparison of CD28 and 41BB co-stimulatory domains indicates that the CD28 may be more potent and efficacious in clearing AML. Further experiments are ongoing to more fully evaluate relative potency of CAR T cells incorporating different co-stimulatory domains. DisclosuresNo relevant conflicts of interest to declare.

In vitro evaluation of antimicrobial activity and measuring its antigenic specific immune response in mice model studies: Allium cepa

Allium cepa (onion; Liliaceae family), herbaceous annual plant and reported Sulphur based amino acids with many minerals including vitamins. Inspite of various medicinal uses of this plant as food and also showed various beneficial effects. The antimicrobial activity of aqueous extract from Allium cepa (onion bulb) was evaluated against bacterial strains using disc diffusion method. In contrast, inflammatory response or inhibition of T cell antigenic response was evaluated using typhoid vaccine and swine flu vaccine in Swiss mice. These studies were conducted according to ethical guidelines and determining T cell proliferation including estimation of cytokines from cell culture supernatant and also measuring its hemolytic activity. The results showed that aqueous extract show the highest inhibition rate against Pseudomonas aeruginosa and Salmonella enteritidis. In mice model studies, aqueous extract at higher doses showed inhibition in antigen specific T cell proliferation and also declining in the level of IFN-gamma production but enhancement in IL-4 production. In addition, aqueous extract at higher concentration does not showed any hemolytic activity. Overall, this study provides evidence for the presence of secondary metabolites in aqueous extract that probably interferes with bacterial growth (antimicrobial) and also declining in antigenic specific proliferation (i.e. anti-inflammatory) which would prevent and inhibit initiation and progression of many diseases.

Chlamydia pneumoniae-Induced IFN-Gamma Responses in Peripheral Blood Mononuclear Cells Increase Numbers of CD4+ but Not CD8+ T Effector Memory Cells.

BackgroundChlamydia pneumoniae causes respiratory infection in adults and children. Previous studies in our laboratory identified significantly higher in vitro T lymphocyte responses to C. pneumoniae in children with asthma compared to healthy controls which may indicate the presence of T effector memory (TEM) lymphocytes.AimIn the present study, healthy subjects were screened for the presence of TEM cells and their cytokines. CCR7 negative effector TEMs may indicate persistent infection with C. pneumoniae.MethodsPeripheral blood mononuclear cells (PBMC) (1×106/mL) from adult non-asthmatic subjects were infected for 1h ± C. pneumoniae TW-183 at a multiplicity of infection (MOI) = 0.1 and cultured (48 hrs). Distributions of lymphocytes (CD4+, CD8+) and TEM cells (CD4+CCR7+CD45RA+CD154+, CD8+CCR7+CD45RA+CD154+) were determined. Levels of intracellular interleukin (IL)-2, IL-4, and interferon (IFN)-gamma were measured (flow microfluorimetry); IFN-gamma was measured in supernatants (ELISA).ResultsC. pneumoniae infection led to a decrease in numbers of CD8+ TEM and CD8+CD154+ cells; CD4+TEM and CD4+CD154+ cells did not change. Numbers of TEM cells (CD4+IL-2+, CD8+ IL-2+) also decreased. However, number of TEM cells (CD4+IL4-+, CD8+ IL-4+) and (CD4+ IFN-gamma+, CD8+IFN-gamma+) did not change. When stratified according to IFN-gamma+ status, numbers of CD4+ IL-2+ and CD4+IL-4+ TEMs increased; CD8+IL-2+ and CD8+ IL-4+ TEMs decreased.ConclusionC. pneumoniae-induced PBMC IFN-gamma+ responses increased numbers of CD4+ IL-2+ and CD4+IL-4+ TEM cells, while CD8+IL-2+ and CD8+IL-4+ TEMs decreased. Production of IFN-gamma by C. pneumoniae infected PBMC should be further studied as a biomarker of persistent infection in humans.

Eos differentially regulates Th1 versus Tfh differentiation through feed-forward propagation of the IL-2/STAT5 signaling pathway

Abstract The Ikaros zinc finger (IkZF) protein Eos is a transcription factor with known roles in driving regulatory T cell (Treg) suppressive function. However, whether it regulates the differentiation of effector CD4+ T cell subsets is currently unknown. Here, we find that Eos deficiency compromises both expression of the T helper 1 (Th1) differentiation program and effector function in vitro and in vivo, including production of IFN-gamma and TNF-alpha. Despite historical associations between IkZF factors and changes in gene expression via the alteration of chromatin structure, our ATAC-seq analysis indicated that there were minimal changes in chromatin accessibility between wildtype and Eos-deficient cells cultured under Th1-polarizing conditions. Rather, we find that IL-2 signaling promotes the STAT5-dependent induction of Eos expression, and that Eos both interacts with STAT5 and supports tyrosine phosphorylation-mediated STAT5 activation. Thus, STAT5 enrichment is reduced at Th1 target genes in the absence of Eos, including Prdm1, which encodes Blimp-1, an antagonist of the T follicular helper (Tfh) cell transcriptional program. Consequently, we find that Eos-deficient T cells also exhibit increased expression of signature Tfh genes. Together, our findings indicate that modulation of IL-2/STAT5 signaling represents a novel mechanism by which Eos regulates the Th1 and Tfh gene programs. Given the important role for IL-2/STAT5 signaling in both the positive and negative regulation of other T helper cell subsets, it will be of interest to determine whether this mechanism is conserved across additional CD4+ T cell populations.

Effect of patient immunodeficiencies on the diagnostic performance of serological assays to detect Aspergillus-specific antibodies in chronic pulmonary aspergillosis

BackgroundPrevalence of chronic pulmonary aspergillosis (CPA) is ~3 million patients worldwide, and detection of Aspergillus-specific antibody is a critical diagnostic component. Some patients with CPA have subtle immune deficits possibly contributing to poor Aspergillus antibody production and false negative results. Materials/methodsWe analyzed patient data from 167 cases of clinically confirmed CPA previously evaluated by ImmunoCAP Aspergillus-specific IgG EIA, Bordier ELISA and LDBio Aspergillus IgG/IgM ICT lateral flow assay, to identify deficiencies in: mannose binding lectin (MBL), IgG, IgA, IgM, IFN gamma, IL12 or IL17 production, and/or low cell marker counts (CD4, CD19, CD56). We defined patients as ‘sero-negative’ if ImmunoCAP Aspergillus IgG was consistently and repeatedly negative (<40 mg A/L). ‘Sero-positive’ was defined as all other CPA cases. ResultsWe found the rate of false negatives by ImmunoCAP Aspergillus IgG EIA (n = 23) to be more prevalent in patients with immunodeficiency markers, especially multiple defects. MBL deficiency combined with low CD19 cells (p < 0.001), pneumococcal antibody levels (p = 0.043), IgM (p = 0.047) or three combined (p = 0.001–0.018) or all four together (p = 0.018) were significant. The performance LDBio Aspergillus IgG/IgM ICT appears to be relatively unaffected by immunodeficiency (92.7% of ImmunoCap sero-negatives were positive). The Bordier assay performed significantly better than the ImmunoCAP assay (P = 0.0016) for sero-negative CPA cases. ConclusionsIn select cases of CPA, ImmunoCAP EIA yields a false negative result, making serological diagnosis difficult. ImmunoCAP false negatives are more prevalent in patients with multiple immunological defects, who may still be positive with the LDBio Aspergillus ICT or Bordier EIA.

IMMU-14. ONCOLYTIC ADENOVIRUS DELTA-24-RGD ENGINEERED TO EXPRESS 4-1BBL AS A THERAPEUTIC APPROACH FOR DIPG

Abstract Despite the advances in our understanding of pediatric diffuse midline gliomas (DMG) they remain the leading cause of pediatric death caused by cancer. Our group has demonstrated that the administration of Delta-24-RGD in DIPG models is safe and therapeutically efficacious. An on-going clinical trial with this virus has proven to be safe for these patients. To further improve the anti-tumoral response of the virus, we armed Delta-24-RGD with 4-1BBL (Delta-24-ACT). 41BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. Here, we showed that in vitro Delta-24-ACT can infect and express 4-1BBL in murine and human DIPG cell lines. Importantly, 4-1BBL expression in DIPG cell lines was able to activate lymphocytes and increase their IFN-gamma production. In addition, Delta-24-ACT triggered immunogenic cell death in DIPG cell lines, as shown by the release of DAMPs such as ATP, HMGB1, Hsp90a and calreticulin translocation. Delta-24-ACT administration in orthotopic DIPG models was well tolerated and safe. We confirmed the expression of the ligand within the tumor. Moreover, using flow cytometry and multispectral immunohistochemistry we observed profound changes in the tumor microenvironment with an increase in the T-cell populations and a remodeling of the myeloid component before and after treatment. Functional studies showed no differences in lymphocytes isolated from mice treated splenocytes but uncovered TILS that showed a significantly increased in the production of IFN-gamma. Delta-24-ACT treatment of mice bearing orthotopic DIPG murine tumors resulted in a significant increase in the median survival and led to free of disease long-term survivors. In summary, Delta-24-ACT is a virus that builds in our clinical experience with Delta-24-RGD in DIPG patients going a step further to boost the antitumor effect of viral therapy while maintaining a safe profile.

Abstract 4513: The role of HPK1 in the regulation of T cell function and anti-tumor immune activity

Abstract Antigen recognition and T-cell receptor (TCR) activation are fundamental processes that drive anti-tumor T cell responses. HPK1 has been identified as a negative regulator of TCR activation, as well as BCR activation, and is a potential anticancer target for immuno-oncology. We show that genetic ablation of HPK1 in human T cells resulted in increased cytokine production upon cell stimulation. Jurkat cells with genetic HPK1 knockout were unable to phosphorylate SLP76, a direct downstream target of HPK1, upon cell stimulation which was associated with increased IL-2 production. Genetic deletion of HPK1 in human RAMOS B cells reduced phosphorylation of BLNK upon IgM stimulation and led to increased TNF alpha and TNF beta production. Treatment of HPK1 knockout primary human T cells with pembrolizumab enhanced IFN gamma secretion compared to the knockout cells alone. Based on these genetic data, HPK1 may be an attractive target for immuno-oncology. We describe herein the in vitro and in vivo profile of several small molecule HPK1 inhibitors and report a surprising disparity between in vitro and in vivo findings. In vitro, the HPK1 inhibitors tested phenocopied the genetic data by potently inhibiting pSLP76 and enhancing IL-2 production in Jurkat cells following stimulation. Similarly, HPK1 inhibitors enhanced IL-2 production in human PBMCs, and increased IFN gamma production in combination with atezolizumab in a primary T cell co-culture assay. In vivo, the HPK1 small molecule inhibitors inhibited tumor growth in the MC38 model, which was further enhanced in combination with anti-PD-L1 in 3 different syngeneic models (MC38, CT26 and MBT-2). Despite these results, tumor growth inhibition was not observed in the GL261 glioma syngeneic model whose growth was recently shown to be inhibited when implanted into HPK1 kinase-dead mice. Further, although several in vitro assays demonstrated increased functional cytokine production with HPK1 inhibitor treatment, the compounds did not increase in vivo cytokine production in tumors. Moreover, combining HPK1 inhibitors with a PD-L1 antibody in vivo abolished the anti-PD-L1-induced production of IFN gamma in the CT26 model. In summary, while our genetic data support the role of HPK1 as a negative regulator of T and B cells, the in vitro activity of HPK1 kinase activity inhibitors was not correlated with functional effects in in vivo syngeneic tumor models. These results underline the complexity of interpreting HPK1 biology and also highlight challenges for the development of clinically active compounds targeting this pathway. Citation Format: Yaoyu Chen, Jonathan Rios-Doria, Michelle Pusey, Kerri Lasky, Min Ye, Pramod Thekkat, Karen Gallagher, Kristine Stump, Patricia Conlen, Christine Gardiner, Hui Wang, Alexander Sokolsky, Mark Rupar, Luping Lin, Elham Behshad, Maryanne Covington, Holly Koblish, Oleg Vechorkin, Wenqing Yao, Sunkyu Kim, Yingnan Chen. The role of HPK1 in the regulation of T cell function and anti-tumor immune activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4513.

Abstract 991: Platelets inhibit hepatocellular carcinoma growth in the context of nonalcoholic fatty liver disease

Abstract Purpose: Non-alcoholic fatty liver disease (NAFLD) is a major risk factor for hepatocellular carcinoma (HCC) in the US and globally. Platelets have been recognized as an important element of immune system and recently, platelets have been shown to be activated and aggerate in the livers of mice and humans with NAFLD. However, the role of platelets in HCC disease progression in NAFLD is still unknown. Methods: In this study, mice underwent an intrahepatic injection of an HCC cell line and were fed a methionine choline deficient (MCD) diet to generate an HCC NAFLD mouse model. The mouse model was then used to interrogate the interplay between platelets, immune responses and HCC growth within a liver with NAFLD. Anti-GP1bα or clopidogrel were used to deplete or inhibit platelet function and effects on tumor growth and immune cell activation were studied. Platelets express leukotriene receptors, and the possible involvement of leukotrienes was tested with zileuton, a leukotriene inhibitor. Finally, antibody dependent T cell depletion was used to identify the effector cells controlling HCC tumor development. Results: NAFLD mice with HCC treated with clopidogrel showed a close to 3-fold increase in tumor weight compared to vehicle control (50.6g vs 147.8g P&amp;lt;0.05) as well as tumor-to-liver weight ratio (0.05 vs 0.13 P&amp;lt;0.05). CD4+ and CD8+ T-cells from the livers of NAFLD mice showed a 2- to 3-fold increase in CD69+ T-cells compared to non-NAFLD mice (24.2% vs 9.1% P&amp;lt;0.05 in CD4+ and 23.7% vs 8.9% P&amp;lt;0.05 in CD8+, respectively). Interestingly, in NAFLD mice treated with clopidogrel, there was a decrease in CD69+ compared to NAFLD mice treated with control in both CD4+ (18.3% vs 24.2% P&amp;lt;0.05, respectively) and CD8+ (15.32% vs 23.7% P&amp;lt;0.05, respectively) T-cells. Consistently, when NAFLD mice were depleted of platelets with anti-GP1bα, there was a significant decreased in T cell activation compared to IgG control. Similarly, when NAFLD mice were treated with zileuton, there was a decrease in IFN-gamma production in liver isolated CD4+ (5.4% vs 3.1% P=0.12) and CD8+ (23.7% vs 16.7% P&amp;lt;0.05) T-cells compared to vehicle control treated mice. Finally, the clopidogrel treatment increased HCC tumor-to-liver weight ratio was abolished in mice treated with anti-CD8 (0.29 vs 0.23 P&amp;gt;0.05) but not in mice treated with anti-CD4 (0.11 vs 0.16 P&amp;lt;0.05). Conclusion: The results of this study show that in NAFLD livers, platelets increase immune cell activation and inhibit HCC in a CD8+ T-cell dependent manner. The results also suggest that leukotrienes may play a key role in modulating this response, but further studies are required to better characterize this observation. Citation Format: John C. McVey, Chi Ma, Laurence Diggs, Qiong Fu, Tim F. Greten. Platelets inhibit hepatocellular carcinoma growth in the context of nonalcoholic fatty liver disease [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 991.

Abstract 2178: Expansion of tumor-specific tumor-infiltrating lymphocytes (TIL) from head and neck tumors

Abstract Background: Until recently, treatment options for patients with head and neck cancer include a combination of surgery, chemotherapy and radiation. Although the combined use of these treatment options modestly improves survival, combination therapy increases toxicity. Overall survival of these treatment regimens for patients with advanced head and neck cancer is just over 10 months. Even with the newly approved and vastly more tolerable use of anti-PD-1 immunotherapy, survival is only extended by 3-5 months. In comparison, adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has improved the median overall survival in patients with metastatic melanoma to 52 months. The goal of this study was to test the feasibility of expanding tumor-reactive TIL from head and neck tumors for the development of a clinical trial. Methods: Primary tumors were resected from 20 head and neck cancer patients. Tumors were minced into small fragments (1-3 mm3) and plated in high dose IL-2. After 4 weeks, expanded TIL were phenotyped by flow cytometry and tested for tumor reactivity by IFN-gamma production after co-culture of TIL with autologous tumor digest. IFN-gamma levels were determined by ELISA. Reactive TIL were expanded using a rapid expansion protocol (REP). Results: The mean expansion of TIL per fragment was 1.96 × 106. From 20 primary tumors, seven were contaminated and discarded. TIL were successfully expanded from 11/13 (85%) evaluable tumors. The phenotype of expanded TIL consisted of CD3− CD56+ NK cells and CD3+ T cells. Tumor-specific reactivity was analyzed for seven samples. TIL expanded from three (43%) tumors were predominantly CD8+ and were reactive against autologous tumor as determined by IFN-gamma ELISA. Expansion of TIL during REP resulted in an average 600-fold increase in cell numbers (range of 61 - 1,774 fold) and led to the depletion of NK cells and an increase of CD3+ T cells. Conclusions: In this study, we have demonstrated the feasibility of growing tumor-reactive TIL from head and neck tumors. This study raises the potential for the implementation of ACT with TIL for the treatment of head and neck cancer patients. Citation Format: Luz Nagle, Amy Mackay Weber, MacLean Hall, Matthew Beatty, J. Trad Wadsworth, Caitlin McMullen, Krupal Patel, Kathryn Vorwald, Christine Chung, Shari Pilon-Thomas. Expansion of tumor-specific tumor-infiltrating lymphocytes (TIL) from head and neck tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2178.

Abstract 3257: CTLA-4 blockade drives loss of regulatory T cell functional stability in glycolysis defective tumors

Abstract Only a limited fraction of patients derives durable clinical benefit upon immune checkpoint blockade. Deepening our understanding of mechanisms of response and resistance to these therapies is thus needed to optimize their employment in rational combinations. Cellular energy metabolism reprogramming is a critical hallmark of cancer. High glucose consumption and lactate production by tumor cells restrict nutrient availability in the tumor microenvironment (TME) for effector T cells, which also rely on glycolysis to replicate and function. In addition, immune checkpoints and co-stimulatory molecules are emerging as important regulators of T cell metabolism. Exploiting the capacity of immune checkpoint blockade to perturb T cell metabolism in combination with inhibition of tumor glycolysis may thus be a rational and more effective anti-cancer approach. We investigated the link between tumor glycolysis and immune cell function using RNA sequencing data sets from patients treated with checkpoint blockade and in tumor:T cell co-culture systems. We then studied the effects of checkpoint blockade in syngeneic glycolysis-defective murine tumor models and explored mechanisms underlying anti-tumor activity. We found that expression of glycolysis-related genes is inversely correlated with infiltration of most immune cell types in melanomas from patients before CTLA-4 blockade. However, after treatment, immune-related and glycolysis-related genes were more often co-expressed, suggesting that anti-CTLA-4 may partially restore immune cell fitness in the glycolytic TME. To directly assess the effect of tumor metabolism on T cell function, we co-cultured activated T cells with the highly glycolytic murine mammary carcinoma 4T1 and observed that tumor cells, or similar non-toxic concentrations of exogenous lactate, significantly limited T cell activation and viability. We thus investigated whether inhibition of tumor glycolysis could potentiate the activity of CTLA-4 blockade. We found that neoadjuvant treatment with anti-CTLA-4 significantly prolonged survival in mice bearing glycolysis defective 4T1 tumors, where lactate dehydrogenase A (LDH-A) - the critical enzyme controlling lactate production in aerobic glycolysis - was knocked down (4T1-KD). Intriguingly, tumor protection was associated with intratumoral regulatory T cell (Treg) functional phenotypic destabilization towards IFN-gamma and TNF-alpha producing Tregs. By mimicking the LDH-A-KD and control 4T1 TME in vitro using higher and lower concentrations of glucose, respectively, we showed that CTLA-4 blockade promotes glucose uptake by Tregs and more efficiently counteracts Treg suppression and enhances CD28 co-stimulation at higher glucose concentrations. These findings indicate that rebalancing glucose utilization in favor of immune cells in the TME may facilitate the activity of CTLA-4 blockade and reveal an additional mechanism through which anti-CTLA-4 interferes with Treg function in the presence of glucose. Citation Format: Roberta Zappasodi, Inna Serganova, Ivan Cohen, Masatomo Maeda, Masahiro Shindo, Yasin Senbabaoglu, Avigdor Leftin, Rachana Maniyar, Svena Verma, Matthew Lubin, Myat Kyaw Ko, Mayuresh M. Mane, Arnab Ghosh, Ellen Ackerstaff, Jason A. Koutcher, Ping-Chih Ho, Greg M. Delgoffe, Ronald Blasberg, Jedd D. Wolchok, Taha Merghoub. CTLA-4 blockade drives loss of regulatory T cell functional stability in glycolysis defective tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3257.

Abstract A01: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) for the treatment of bladder cancer

Abstract Bladder cancer remains a leading cause of death and one of the most expensive cancers to treat in the United States. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) is a personalized therapeutic approach that has the potential to treat bladder cancer at every stage of disease. We have previously demonstrated the ability to expand tumor reactive T cells from transurethral resection, radical cystectomy, and metastatic lymph node tumor specimens collected from bladder cancer patients. In this study, our goal is to investigate different delivery approaches of ACT in vivo to determine the best strategy to decrease tumor burden and advise clinical trial design. Experiments were performed using MB49 murine bladder cancer cells transfected to express ovalbumin (MB49OVA). MB49OVA cells are recognized by OVA-specific OT-I T cells as determined by IFN-gamma production in vitro. Our methods include both subcutaneous and orthotopic bladder tumor models. In vivo, we first tested systemic delivery of OT-I T cells (5.0 × 10e6) by tail vein injection after lymphodepletion with a regimen of cyclophosphamide followed by fludarabine in mice bearing subcutaneous MB49OVA tumors. ACT-treated mice experienced significant tumor regression (p=0.02), improved survival (median survival PBS=47 days, ACT=undefined, p=0.01), and increased tumor infiltration of CD3+, CD4+, and CD8+ T cells. OT-I T cells as well as endogenous OVA-specific CD8+ T cells were detectable in the spleens and tumors of treated mice. Next we tested the efficacy of intravesical delivery of OT-I T cells in mice bearing orthotopic MB49OVA tumors, without prior lymphodepletion. OT-I T cells (4.0 × 10e6) were delivered directly to the bladder via catherization and tumor growth was monitored weekly via ultrasound imaging. Infiltration of CellTrace Violet-labeled OT-I T cells into orthotopic tumors was detected after 3 hours of incubation by flow cytometry analysis of resected tumor. Orthotopic bladder tumor growth was significantly abrogated in ACT-treated mice compared with PBS control-treated mice (p=0.001) and improved survival was observed (median survival PBS=35 days, ACT=undefined). An increase in CD8+ T-cell infiltration was measured in tumors of mice at day 60 post treatment. In order to evaluate the efficacy of TIL transfer in these bladder tumor models, T cells infiltrating subcutaneous MB49OVA tumors were isolated and cultured in vitro in media containing IL-2. After 1 week, bladder TIL demonstrated reactivity against MB49OVA and MB49 parental cells as determined by IFN-gamma production. Future work will implement the adoptive transfer of MB49 TIL for the treatment of orthotopic bladder tumors alone or in combination with anti-PD-L1 therapy. Taken together, these data suggest that ACT using bladder tumor-specific T cells has the potential to reduce bladder tumor burden when given either systemically or intravesically directly into the bladder. Citation Format: Brittany L. Bunch, Jennifer Morse, Sarah Asby, Michael Poch, Shari Pilon-Thomas. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) for the treatment of bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A01.

Immune responses in beta-thalassaemia: heme oxygenase 1 reduces cytokine production and bactericidal activity of human leucocytes

Patients with beta-thalassaemia increase the risk of bacterial infections, particularly Burkholderia pseudomallei (Bp), the causative agent of melioidosis in Thailand. Impaired immune cell functions may be the cause of this susceptibility, but detailed mechanisms have not been defined. In this study, we observed impaired production of IFN-gamma and IL-10 by whole blood from beta-thalassaemia patients upon stimulation with a range of bacteria-derived stimuli. In contrast, IFN-gamma response via TCR and plasma IgG specific for Bp were still intact. Importantly, mRNA expression of heme oxygenase 1 (HO-1), a potential modulator of immune function, was increased in whole blood from beta-thalassaemia patients, either with or without stimulation with Bp in vitro. Induction of HO-1 by hemin or CoPP in vitro reduced production of IFN-gamma and IL-10 from healthy human PBMCs and decreased bacterial clearance activity of whole blood from healthy controls and beta-thalassaemia, while inhibition of HO-1 by SnPP enhanced both functions in healthy controls. These results were confirmed to some extent in purified human monocytes of healthy controls. Our results suggest a mechanism that excess hemin of beta-thalassaemia patients is a significant cause of immune suppression via HO-1 induction and may underlie the susceptibility of these individuals to severe bacterial infection.

Modeling a human STAT4 mutation that predisposes to disseminated Coccidioidomycosis in mice

Abstract Coccidioidomycosis is a common fungal respiratory disease caused by the fungus Coccidioides spp. fungi in Arizona and the central valley of California. Disseminated Coccidioidomycosis (DCM) occurs in 8% of identified cases, causing significant morbidity and mortality. DCM occurred in 3 generations of a family in Tucson, Arizona who all shared a heterozygous missense mutation (p.E626G) in STAT4. Transfection of STAT4 E626G in WT human cells showed dampened responses to STAT4-dependent cytokine gene induction, suggesting the mutation is a dominant negative. To understand the function of E626G mutation, we generated the homologous mutation in Stat4 in B6 mice. Mice carrying E626G were grossly normal with normal numbers of innate and adaptive immune cells in primary and secondary lymphoid organs. To examine the effect of E626G on susceptibility to Coccidioides infection, we infected mice with C. posadasii strain 1038 intranasally. After infection E626G heterozygous mice all died by 50 days post infection (dpi), median time to death 45.5 days; range 30–50 dpi. 87% of WT B6 controls were alive when the experiment ended at a predetermined 70 dpi, resulting in a median time to death of &amp;gt;70 days. Mediastinal lymph nodes in E626G heterozygous mice had reduced numbers of B cells and activated (CD62Llow) T cells compared to B6 controls on 14 dpi. Cells from infected lungs from E626G heterozygotes produced less IFN-gamma after infection on 28, 35 and 42 dpi. Interferon stimulated cytokines (IP-10, MIG, MIP-2) were also significantly reduced compared to WT controls. Ongoing work includes examining the cells requiring STAT4 signaling and IFN-gamma production as well as the mechanism of reduced accumulation of immune cells in the draining lymph nodes.

Metabolic regulation of cytokine production in leishmania resistance and susceptibility

Abstract Leishmania, a protozoan parasite, ensures its survival inside its mammalian host by modulating host metabolism and immune response. Recent advances in the field of immune-metabolism have shown that the metabolic enzymes and metabolites have immune-modulatory functions. Leishmania infection of a susceptible host skews immune response towards Th2-type, enhances production of pro-parasitic cytokines IL-10 and IL-4 but reduces IFN-gamma and IL-12 production. By contrast, Leishmania infection of a resistant host sets an immune response bias towards Th1-type, results in high production of pro-inflammatory IL-12 and IFN-gamma, which activates macrophages to kill the intracellular amastigotes. To understand the role of metabolic factors in Leishmania susceptibility or resistance, we used L. donovani susceptible or resistant mouse strains BALB/c or 129/Sv, respectively. The mice were first intravenously infected with L. donovani, followed by intra-peritoneal treatment with different inhibitors of metabolic pathways. The splenocytes from the treated resistant and susceptible mouse strains were studied for in-situ cytokine response and T cell subsets by FACS. Also the mouse serum was used for cytokine estimation by ELISA. The results show that different metabolic pathways regulate cytokine production in L. donovani-infected mouse suggesting their role in resistance and susceptibility to Leishmania infection.

IFNAR signaling augments T cell and plasmacytoid dendritic cell activation during costimulation blockade resistant rejection

Abstract Costimulation blockade (CoB) is a promising new transplant immunosuppression strategy offering improved long-term allograft survival without the nephrotoxicity of calcineurin inhibitors. However, increased risks of T cell mediated acute rejection have impeded the widespread adoption of CoB. Type I interferon (IFN) produced predominantly by plasmacytoid dendritic cells (pDCs) in response to innate immune signals, induce systemic inflammation that may prime the adaptive immune system for acute rejection. Here we investigate how signaling through the type I interferon receptor (IFNAR) contributes to CoB-resistant allograft rejection. We performed fully MHC-mismatched skin grafts from Balb/cJ donors to C57BL/6 recipients. Mice were treated with CoB alone (250 μg CTLA4-Ig + 250 μg anti-CD154 given by IP injection day 0, 2, 4, 6) or in conjunction with anti-IFNAR (250 μg given day 0, 2, 4, 6). CoB treatment improved graft survival but resulted in CoB-resistant rejection, while CoB+anti-IFNAR significantly improved graft survival. We found that CoB+anti-IFNAR led to a reduction in the activation and effector function of CD8+ T cells compared to CoB alone, demonstrated by reduced frequencies of CD44+CD8+ T cells and an inhibition of IFN-gamma and TNF-alpha production by CD8+ T cells. In addition, we observed reduced CD80 and MHC I expression on conventional DCs (cDCs) and pDCs treated with CoB+anti-IFNAR compared to treatment with CoB alone. These data suggest that signaling through IFNAR augments cDC and pDC activation and is associated with CD8+ T cell activation and effector functions in order to promote CoB-resistant allograft rejection.