Specific IFN-γ Production Research Articles

Interferon-gamma producing CD4+ T cells quantified by flow cytometry as early markers for Mycobacterium avium ssp. paratuberculosis infection in cattle

Current diagnostic methods for Johne’s disease in cattle allow reliable detection of infections with Mycobacterium avium ssp. paratuberculosis (MAP) not before animals are 2 years of age. Applying a flow cytometry-based approach (FCA) to quantify a MAP-specific interferon-gamma (IFN-γ) response in T cell subsets, the present study sought to monitor the kinetics of the cell-mediated immune response in experimentally infected calves. Six MAP-negative calves and six calves, orally inoculated with MAP at 10 days of age, were sampled every 4 weeks for 52 weeks post-inoculation (wpi). Peripheral blood mononuclear cells (PBMC) were stimulated with either purified protein derivatives (PPD) or whole cell sonicates derived from MAP (WCSj), M. avium ssp. avium or M. phlei for 6 days followed by labeling of intracellular IFN-γ in CD4+ and CD8+ T cells. No antigen-specific IFN-γ production was detectable in CD8+ cells throughout and the responses of CD4+ cells of MAP-infected and control calves were similar up to 12 wpi. However, the mean fluorescence intensity (MFI) for the detection of IFN-γ in CD4+ cells after WCSj antigen stimulation allowed for a differentiation of animal groups from 16 wpi onwards. This approach had a superior sensitivity (87.8%) and specificity (86.8%) to detect infected animals from 16 wpi onwards, i.e., in an early infection stage, as compared to the IFN-γ release assay (IGRA). Quantification of specific IFN-γ production at the level of individual CD4+ cells may serve, therefore, as a valuable tool to identify MAP-infected juvenile cattle.

IMMUNE RESPONSE TO SARS-COV-2 IN PATIENTS WITH CHRONIC HIV INFECTION

Introduction. Data for the long-term effects of SARS-CoV-2/HIV co-infection on immune restoration, as well as the level of post-exposure and post-vaccination immunity at the current stage of SARS-CoV-2 pandemic in HIV+ individuals is still scarce. We assessed SARS-CoV-2-specific immune responses, and the effects of SARS-CoV-2 infection on the immune recovery in HIV+cART+ patients with different exposure history. Materials and methods. HIV+cART+ patients 9 (2-18) months after mild/moderate COVID-19 and completed immunization with anti-SARS-CoV-2 vaccine (n=13, group A), convalescent, not immunized (n=11, group B), or with no history of exposure to SARS-CoV-2 (n=11, group C) were included in the study. CD4AC and CD4/CD8 ratio were determined before and after the documented/probable contact with SARS-CoV-2 by 4-color flow cytometry (TRUCount, MultiTest, FACSCanto II). Virus-specific immunity was characterized by the SARS-CoV-2 specific IFNγ production (SARS-CoV-2 IGRA, Euroimmun) and the levels of RBD-IgG ((Euroimmun ELISA). Results. SARS-CoV-2 specific T-cell and IgG responses were highly correlated and present, respectively, in 92% and 100%; 64% and 54%, 36% and 50% from group A, B and C patients. SARS-CoV-2 specific IFNy+T cells and RDB-IgG were significantly higher in the group with hybrid exposure (A) as compared to convalescent (B) and asymptomatic (C) patients. No significant difference existed between background and actual CD4AC (mean 836 vs 799 cells/µl, p>0.05, Mann-Whitney), and the CD4/CD8 ratio significantly increased in the group with hybrid exposure (0.92 vs 1.07, p<0.01, paired T-test). Conclusion. Over 80% of tested HIV+ individuals have mounted a SARS-CoV-2 specific immune response. Immunization and hybrid exposure provide a durable and significantly stronger SARS-CoV-2-specific immune response as compared to mild/ asymptomatic infection, without affecting the long-term immune recovery.

Evidence of exhausted lymphocytes after the third anti-SARS-CoV-2 vaccine dose in cancer patients.

Evidence is scant regarding the long-term humoral and cellular responses Q7 triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines in cancer patients after repeated booster doses. The possibility of T-cell exhaustion following these booster doses in this population has not yet been fully studied and remains uncertain. In this single-center prospective observational study, we explored the specific humoral and cellular response to S1 antigen in 36 patients with solid malignancies at baseline, and after the second and third doses of the mRNA-1273 vaccine. A dual behavior was observed: 24 (66.7%) patients showed partial specific IFN-γ response after the second dose that was further enhanced after the third dose; and 11 (30.5%) already showed an optimal response after the second dose and experienced a marked fall-off of specific IFN-γ production after the third (4 patients negativization), which might suggest T cell exhaustion due to repetitive priming to the same antigen. One (2.8%) patient had persistently negative responses after all three doses. Seroconversion occurred in all patients after the second dose. We then studied circulating exhausted CD8+ T-cells in 4 patients from each of the two response patterns, those with increase and those with decrease in cellular response after the third booster. The patients with decreased cellular response after the booster had a higher expression of PD1+CD8+ and CD57+PD1+CD8+ exhausted T cells compared with those with an increased cellular response both in vivo and in vitro. The proportion of PD1+CD8+ and CD57+PD1+CD8+ exhausted T cells inversely correlated with IFN-γ production. Our preliminary data show that the two-dose SARS-CoV-2 vaccine regimen was beneficial in all cancer patients of our study. An additional booster seems to be beneficial in suboptimal vaccine seroconverters, in contrast to maximal responders that might develop exhaustion. Our data should be interpreted with caution given the small sample size and highlight the urgent need to validate our results in other independent and larger cohorts. Altogether, our data support the relevance of immunological functional studies to personalize preventive and treatment decisions in cancer patients.

A New Live Auxotrophic Vaccine Induces Cross-Protection against Klebsiella pneumoniae Infections in Mice.

The development of a whole-cell vaccine from bacteria auxotrophic for D-amino acids present in the bacterial cell wall is considered a promising strategy for providing protection against bacterial infections. Here, we constructed a prototype vaccine, consisting of a glutamate racemase-deficient mutant, for preventing Klebsiella pneumoniae infections. The deletion mutant lacks the murI gene and requires exogenous addition of D-glutamate for growth. The results showed that the K. pneumoniae ΔmurI strain is attenuated and includes a favourable combination of antigens for inducing a robust immune response and conferring an adequate level of cross-protection against systemic infections caused by K. pneumoniae strains, including some hypervirulent serotypes with elevated production of capsule polysaccharide as well as multiresistant K. pneumoniae strains. The auxotroph also induced specific production of IL-17A and IFN-γ. The rapid elimination of the strain from the blood of mice without causing disease suggests a high level of safety for administration as a vaccine.

Cysteamine with In Vitro Antiviral Activity and Immunomodulatory Effects Has the Potential to Be a Repurposing Drug Candidate for COVID-19 Therapy.

The ongoing pandemic of coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), needs better treatment options both at antiviral and anti-inflammatory levels. It has been demonstrated that the aminothiol cysteamine, an already human applied drug, and its disulfide product of oxidation, cystamine, have anti-infective properties targeting viruses, bacteria, and parasites. To determine whether these compounds exert antiviral effects against SARS-CoV-2, we used different in vitro viral infected cell-based assays. Moreover, since cysteamine has also immune-modulatory activity, we investigated its ability to modulate SARS-CoV-2-specific immune response in vitro in blood samples from COVID-19 patients. We found that cysteamine and cystamine decreased SARS-CoV-2-induced cytopathic effects (CPE) in Vero E6 cells. Interestingly, the antiviral action was independent of the treatment time respect to SARS-CoV-2 infection. Moreover, cysteamine and cystamine significantly decreased viral production in Vero E6 and Calu-3 cells. Finally, cysteamine and cystamine have an anti-inflammatory effect, as they significantly decrease the SARS-CoV-2 specific IFN-γ production in vitro in blood samples from COVID-19 patients. Overall, our findings suggest that cysteamine and cystamine exert direct antiviral actions against SARS-CoV-2 and have in vitro immunomodulatory effects, thus providing a rational to test these compounds as a novel therapy for COVID-19.

Effects of Lactobacillus salivarius supplementation on the growth performance, liver function, meat quality, immune responses and Salmonella Pullorum infection resistance of broilers challenged with Aflatoxin B1

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins. It has been reported that dietary exposure to AFB1 is related to the low growth performance, immunosuppression, and high susceptibility to infectious diseases of chickens. The aim of the present study was to evaluate the protective effects of Lactobacillus salivarius on broiler chickens challenged with AFB1. First, AFB1 degradation ability of Lactobacillus salivarius was measured by a high-performance liquid chromatography (HPLC) method. Then, the Arbor Acres broiler chickens were randomly assigned to experimental groups. The effects of Lactobacillus salivarius supplementation on the growth performance, liver function, and meat quality were measured, and immune response was also determined after vaccination with attenuated infectious bursal disease virus (IBDV) vaccine of broilers challenged with AFB1. Besides, resistance to Salmonella Pullorum infection along with AFB1 exposure was determined in broilers. The results showed that Lactobacillus salivarius could effectively degrade AFB1. Lactobacillus salivarius supplementation improved growth performance, liver function, and meat quality of broilers challenged with AFB1. In addition, Lactobacillus salivarius supplementation resulted in enhanced specific antibody and IFN-γ production, and lymphocyte proliferation in broilers challenged with AFB1 after IBDV vaccine immunization. Furthermore, Lactobacillus salivarius supplementation enhanced Salmonella Pullorum infection resistance in broilers challenged with AFB1. Our results revealed a tremendous potential of Lactobacillus salivarius as feed additive to degrading AFB1 and increasing broilers production performance in poultry production.

Abstract 1531: Development of PBMC derived tumor effector cells with potent pan-cancer cytolytic activity for autologous cellular immunotherapy

Abstract Introduction: A unique autologous cellular therapeutic (SUPLEXA) has been developed from human PBMC. It is comprised of NK cells, γδ T cells and CD8+ T effector cells, capable of broadly lysing a variety of tumor cell lines in vitro. SUPLEXA cells are manufactured using an efficient 2 week xeno-free manufacturing procedure employing two proprietary engineered leukocyte stimulator cell lines (ENLIST) that express an array of immunomodulatory proteins. The SUPLEXA cell manufacturing process is highly reproducible and demonstrates low inter-subject variability in cellular composition. SUPLEXA cells are distinguished from many other cellular approaches in that they are derived from autologous PBMC that have only been stimulated with ENLIST cells through naturally occurring receptors without any genetic modification. Here, we report on the in vitro characterization of SUPLEXA cytolytic activity on a variety of target tumor cell lines. Results: We prepared SUPLEXA cells from the PBMC of a variety of solid tumor patients (5 melanoma and 5 prostate). Yields and cell characterization by mass cytometry (CyTOF) yielded similar profiles within the variability expected for all the major cell subtypes. SUPLEXA cells were comparatively assessed for cytolytic activity on 4 tumor cell lines; K562 (leukemia), PC3 (prostate), M14 (melanoma) and COLO205 (colorectal cancer). In all instances, cytotoxicity was observed in a quantitative flow cytometry and microscopic assay and was titratable with cell number. Dose-dependent cytolysis was observed for all tumor lines, even at low E:T ratios of 1:10 suggesting that SUPLEXA cells acquire efficient and broad tumor cell lysis activity. Supernatants from cytolysis assays were analyzed for cytokine levels by a 35-plex assay revealing a remarkably restricted cytokine profile with constitutively high levels of RANTES production by the SUPLEXA cells and titratable levels of induced IL-6, IFN-γ and IL-8 production. Conclusions: SUPLEXA cells represent an easy to manufacture autologous anti-tumor cell therapy that overcomes many of the limitations associated with previous autologous therapies. They possess a broad anti-tumor activity at exceedingly low E:T ratios with specific production of IFN-γ, IL-6 and IL-8 during tumor cell killing. Ongoing efforts are underway to understand the relative contributions of NK cells, γδ T cells and CD8+ T cells to broad tumor cytolysis and the antigen-specificities seen with SUPLEXA cells from multiple donors. Planning for a first in human clinical trial is currently underway. Citation Format: Frank Borriello, Joshua W. Keegan, James A. Lederer. Development of PBMC derived tumor effector cells with potent pan-cancer cytolytic activity for autologous cellular immunotherapy [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 1531.

Immune responses induced by inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in neonatal pigs using different adjuvants

Vaccination of neonatal pigs could be supportive to prevent porcine reproductive and respiratory syndrome virus (PRRSV), which is an important porcine pathogen causing worldwide welfare and health problems in pigs of different age classes. However, neonatal immunity substantially differs to adults, thus different vaccines may be required in neonateal pigs. We examined if the immunogenicity and efficacy of inactivated PRRSV (iPRRSV) vaccines in neonatal pigs could be improved with adjuvants containing oil-in water (O/W) emulsions with or without Toll-like receptor (TLR) agonists and by altering the delivery route from intramuscular (i.m.) to the skin. Three-day-old PRRSV-naïve piglets (n = 54, divided in 6 groups) received a prime vaccination and a booster vaccination four weeks later. The vaccine formulations consisted of different O/W emulsions (Montanide™ ISA28RVG (ISA28)), a squalene in water emulsion (SWE) for i.m. or a Stable Emulsion (SE) with squalene for skin vaccination) and/or a mixture of TLR1/2, 7/8 and 9 agonists (TLRa) combined with iPRRSV strain 07V063. These vaccines were delivered either i.m. (ISA28, SWE, TLRa or SWE + TLRa) or into the skin (skiSE + TLRa) with dissolving microneedle (DMN)-patches. All animals received a challenge with homologous PRRSV three weeks after booster vaccination. Specific antibodies, IFN-γ production and viremia were measured at several time-points after vaccination and/or challenge, while lung pathology was studied at necropsy. After booster vaccination, only ISA28 induced a specific antibody response while a specific T-cell IFN-γ response was generated in the SWE group, that was lower for ISA28, and absent in the other groups. This suggests that prime vaccination in neonates induced a specific immune response after booster vaccination, dependent on the emulsion formulation, but not dependent on the presence of the TLRa or delivery route. Despite the measured immune responses none of the vaccines showed any efficacy. Further research focused on the early immune response in draining lymph nodes is needed to elucidate the potential of TLR agonists in vaccines for neonatal pigs.

Activation-Induced Marker Expression Identifies Mycobacterium tuberculosis-Specific CD4 T Cells in a Cytokine-Independent Manner in HIV-Infected Individuals with Latent Tuberculosis.

HIV infection is a significant risk factor for reactivation of latent Mycobacterium tuberculosis infection (LTBI) and progression to active tuberculosis disease, yet the mechanisms whereby HIV impairs T cell immunity to M. tuberculosis have not been fully defined. Evaluation of M. tuberculosis–specific CD4 T cells is commonly based on IFN-γ production, yet increasing evidence indicates the immune response to M. tuberculosis is heterogeneous and encompasses IFN-γ–independent responses. We hypothesized that upregulation of surface activation-induced markers (AIM) would facilitate detection of human M. tuberculosis–specific CD4 T cells in a cytokine-independent manner in HIV-infected and HIV-uninfected individuals with LTBI. PBMCs from HIV-infected and HIV-uninfected adults in Kenya were stimulated with CFP-10 and ESAT-6 peptides and evaluated by flow cytometry for upregulation of the activation markers CD25, OX40, CD69, and CD40L. Although M. tuberculosis–specific IFN-γ and IL-2 production was dampened in HIV-infected individuals, M. tuberculosis–specific CD25+OX40+ and CD69+CD40L+ CD4 T cells were detectable in the AIM assay in both HIV-uninfected and HIV-infected individuals with LTBI. Importantly, the frequency of M. tuberculosis–specific AIM+ CD4 T cells was not directly impacted by HIV viral load or CD4 count, thus demonstrating the feasibility of AIM assays for analysis of M. tuberculosis–specific CD4 T cells across a spectrum of HIV infection states. These data indicate that AIM assays enable identification of M. tuberculosis–specific CD4 T cells in a cytokine-independent manner in HIV-uninfected and HIV-infected individuals with LTBI in a high-tuberculosis burden setting, thus facilitating studies to define novel T cell correlates of protection to M. tuberculosis and elucidate mechanisms of HIV-associated dysregulation of antimycobacterial immunity.

Assessment of Probiotic Properties of Lactobacillus salivarius Isolated From Chickens as Feed Additives.

The continued use of sub-therapeutic antibiotics as feed additives in the poultry industry improved health and growth performance. However, the resulting antibiotic resistance increasingly becomes a major threat to public health. Probiotics are promising alternatives for the antibiotics used in poultry industry. The aim of this study was to evaluate the probiotic properties of Lactobacillus salivarius as feed additive in chickens. White leghorn chickens were randomly assigned to experimental groups. Effects of Lactobacillus salivarius supplementation on growth performance, resistance to Escherichia coli O78 challenge and heat-stress, and immune response after vaccinated with attenuated infectious bursal disease virus (IBDV) vaccine were determined. The results showed that Lactobacillus salivarius supplementation improved growth performance, such as weight and longer shank length, increased relative weights of the immune organs and decreased concentrations of odor-causing compounds. In addition, Lactobacillus salivarius supplementation alleviated organ injury caused by Escherichia coli O78 challenge and heat stress. Furthermore, Lactobacillus salivarius results in enhanced immune response after IBDV vaccine immunization, enhanced specific antibody and IFN-γ production, and lymphocyte proliferation. Our results revealed a tremendous potential of Lactobacillus salivarius as antibiotics' substitute in poultry production.

The Role of MicroRNA-155 in Chlamydia muridarum Infected lungs

Our laboratory has investigated the role of an evolutionarily conserved RNA species called microRNAs (miRs) in regulation of anti-chlamydial protective immunity. MiRs including miR-155 expressed in specific immune effector cells are critical for antigen specific protective immunity and IFN-γ production. Using miR-155 deficient mice, and a murine pulmonary model for chlamydial infection, we report here 1) the effect of host miR-155 on bacterial burden, and 2) identify probable immune genes regulated by miR-155.

IL-2 Restores T-Cell Dysfunction Induced by Persistent Mycobacterium tuberculosis Antigen Stimulation

Tuberculosis (TB) is a chronic disease mainly caused by Mycobacterium tuberculosis. The function of T cells usually decreased and even exhausted in severe TB such as multiple drug resistant TB (MDR-TB), which might lead to the failure of treatment in return. The mechanism of T cell dysfunction in TB is still not clear. In this study we set up a mouse model of T cell dysfunction by persistent M. tuberculosis antigen stimulation and investigated the therapeutic role of interleukin 2 (IL-2) in it. C57BL/6 mice were primed with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and boosted repeatedly with a combination of M. tuberculosis fusion proteins Mtb10.4-HspX (MH) plus ESAT6-Ag85B-MPT64 <190−198>-Mtb8.4-Rv2626c (LT70) or MH plus ESAT6 and CFP10 with adjuvant of N, N′-dimethyl-N, N′-dioctadecylammonium bromide (DDA) plus polyinosinic-polycytidylic acid (Poly I:C). Following persistent antigen stimulation, the mice were treated with IL-2 and the therapeutic effects were analyzed. The results showed that compared with the mice that received transient antigen stimulation (boost twice), persistent antigen stimulation (boost more than 10 times) resulted in decrease of antigen specific IFN-γ and IL-2 production, reduction of memory CD8+ T cells, over-expression of immune checkpoint programmed cell death protein 1 (PD-1), and impaired the protective immunity against bacterial challenge. Treating the T cell functionally exhausted mice with IL-2 restored antigen-specific T cell responses and protective efficacy. In conclusion, persistent stimulation with M. tuberculosis antigens induced T cell dysfunction, which could be restored by complement of IL-2.

Transgenic rice seeds expressing altered peptide ligands against the M3 muscarinic acetylcholine receptor suppress experimental sialadenitis-like Sjögren’s syndrome

Objective: We previously reported that Rag1−/− mice inoculated with splenocytes from M3 muscarinic acetylcholine receptor (M3R) knockout mice immunized with an M3R peptide mixture developed sialadenitis-like Sjögren’s syndrome (M3R-induced sialadenitis [MIS]). We also found that intravenous administration of altered peptide ligand (APL) of N-terminal 1 (N1), which is one of the T-cell epitopes of M3R, suppressed MIS. In this study, we aimed to evaluate the suppressive ability and its mechanisms of rice seeds expressing N1-APL7 against MIS.Methods: Rice seeds expressing N1 and N1-APL7 were orally administered to MIS mice for 2 weeks. The changes in saliva flow and sialadenitis (salivary gland inflammation) were analyzed. The M3R-specific T-cell response in the spleen and the expression of regulatory molecules in the cervical lymph nodes and mesenteric lymph nodes were also analyzed.Results: Oral administration of N1-APL7-expressing rice seeds significantly recovered reduction in saliva flow and suppressed sialadenitis when compared with treatment with nontransgenic rice seeds and N1 rice seeds. IFNγ production from M3R-reactive T cells tended to decline in the N1-APL7 rice-treated group as compared with those in the other groups. In the N1-APL7 rice-treated group, the mRNA expression levels of Foxp3 in the cervical-lymph-node CD4+ T cells were higher than those in the other groups.Conclusion: Oral administration of N1-APL7-expressing rice suppressed MIS via suppression of M3R-specific IFNγ and IL-17 production and via enhancement of regulatory molecule expression.Key messagesWe generated N1-peptide- or N1-APL7-expressing rice seeds. Oral administration of N1-APL7-expressing rice seeds significantly recovered the reduction of saliva flow and suppressed sialadenitis via the suppression of M3R specific IFNγ and IL-17 production and via enhancement of regulatory T (Treg) cells.

Abstract B181: Adenovirus, Semliki Forest virus and vaccinia virus-induced immunogenic cell death augments oncolytic virus immunotherapy

Abstract Oncolytic viruses represent new immunotherapy agents with therapeutic effects consisting of direct oncolysis of cancer cells and anticancer immune responses induced upon oncolysis. However, the exact pathways of cell death and the subsequent immune response initiations after oncolysis are not fully elucidated. We have examined the cell death pathways and immune-stimulatory pathways involved in adenovirus (Ad), Semliki Forest virus (SFV) and vaccinia virus (VV) infection. The three viruses activate distinct but different pathways but all of them induced immunogenic cell death (ICD), which is depicted by elevated cell surface exposure of calreticulin (CRT) and extracellular secretion of ATP. Oncolysis-mediated ICD grants dying cells to be phagocytosed and it promotes DC maturation and activation with a Th1-polarized cytokine secretion profile. As a proof of concept, we validated that when DCs are incubated with Ad- or SFV-, but not VV-infected tumor cells expressing a surrogate antigen (CMVpp65), they can efficiently process and cross-present peptides of this antigen to CD8+ T-cells, as shown by specific IFN-γ production. Mice immunized with SFV- or VV-infected murine glioblastoma cells (GL261) elicit a strong and specific T-cell response against GL261. This adaptive immune response can control tumor growth and prolong mice survival after GL261 rechallenge. Citation Format: Mohanraj Ramachandran, Magnus Essand. Adenovirus, Semliki Forest virus and vaccinia virus-induced immunogenic cell death augments oncolytic virus immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B181.

Generation of Zika Virus-Specific T-Cells for Adoptive Immunotherapy

Background Zika virus (ZIKV) infection during pregnancy can cause severe birth defects, with no effective treatment. Adoptive transfer of virus-specific T-cells has proven to be safe and effective for the prevention or treatment of many viral infections, and could represent a novel treatment approach for ZIKV infection. However, this approach for ZIKV infection has been hampered by limited data on immunogenic T-cell antigens within ZIKV. Hence, we have generated ZIKV-specific T-cells and characterized the cellular immune responses against ZIKV antigens. Methods T-cell products were generated from blood of ZIKV-exposed donors (n=3), ZIKV-naive adult donors (n=6), and umbilical cord blood (n=7) by stimulation with overlapping peptide libraries (15mers overlapping by 11 amino acids) spanning four ZIKV polyproteins (C, M, E, and NS1) using a GMP-compliant protocol. Results We successfully generated T-cells targeting ZIKV antigens with a mean 56.0-fold T cell expansion at 17 days for ZIKV-exposed donors and 21 days for ZIKV-naive donors and cord blood donors. The resultant products specifically recognized C, M, E, and NS1 proteins by IFN-γ ELISpot, with mean 142.5 spot forming cells (SFCs)/1 × 105 cells, 81.7 SFCs/1 × 105 cells, 132.1 SFCs/1 × 105 cells, and 180.7 SFCs/1 × 105 cells, respectively, compared to 5.4 SFCs/1 × 105 cells for irrelevant antigen (Fig 1). ZIKV-specific T-cells were polyclonal (16.2±2.8% CD3+CD4+ and 32.4±2.4% CD3+CD8+ T-cells) and expressed markers for both central and effector memory phenotype (3.5±0.6% CD45RO+CD62L+ and 48.1±1.9% CD45RO+CD62L–), with minimal expression of PD-1, Tim-3, Lag-3, and CTLA-4. Notably, both CD4+ and CD8+ subsets were polyfunctional comprising ZIKV-directed T-cell populations, which secreted multiple cytokines (16.7±0.6% CD4+IFN-γ+TNF-α+ and 7.4±0.4% CD8+IFN-γ+TNF-α+) as measured by intracellular cytokine staining. Moreover, multiplex cytokine assay demonstrated specific production of IFN-γ (928.7±239.8 pg/mL), TNF-α (1495.5±453.3 pg/mL), IL-2 (38.2±8.4 pg/mL), and GM-CSF (67.0±22.2 pg/mL) in response to ZIKV antigens. Importantly, ZIKV-specific T-cells selectively killed autologous monocytes infected with ZIKV confirming functionality of this unique T cell therapeutic. Epitope mapping using peptide arrays in IFN-γ ELISpot identified several novel HLA class I or class II-restricted epitopes within NS1, which is essential for viral replication and immune evasion. Conclusions Our findings demonstrate that it is feasible to generate potent ZIKV-specific T-cells from a variety of cell sources including virus naive donors for future clinical use in an “off the shelf’ setting.

Novel BAFF-R CAR T-Cell Therapy for CD19 Antigen-Loss Relapsed B Cell Tumors

Background: Chimeric antigen receptor (CAR) T cells against CD19 have shown great potential in treatment of B cell malignancies. However, tumor relapse from antigen loss can limit efficacy. B-cell activating factor-receptor (BAFF-R), a tumor necrosis factor receptor superfamily protein (TNFRSF13C), is another potential B-cell specific target of B cell malignancies. BAFF-R is an especially interesting alternative to CD19 as BAFF-R signaling is a driver of B-cell survival, which may limit the capacity of clonal B-cell tumors to escape therapy by down-regulation of antigen expression. However, while the BAFF/BAFF-R axis has been successfully targeted for autoimmune diseases, the promise for cancer therapy has not yet been fulfilled.Methods and Results: A humanized, single-chain variable fragment (scFv) derivative of an anti-human BAFF-R antibody (Qin et al. Clin Can Res 2018;24:1114-1123) was engineered onto a second generation CAR construct containing 4-1BB costimulatory and CD3ζ intracellular signaling domains. BAFF-R-CAR T cells demonstrated cytotoxicity against human lymphoma and acute lymphocytic leukemia (ALL) lines. Adoptively transferred BAFF-R-CAR T cells eradicated 10-day pre-established tumor xenografts after a single treatment and were superior to CD19-CAR T cells (not shown) and retained efficacy against xenografts deficient in CD19 expression, including one primary patient-derived xenograft (PDX). Specifically, we modeled disease relapse due to the loss of CD19 by generating CRISPR CD19 gene knock-out of the ALL (Nalm-6) cell line and a gRNA-silenced CD19 gene knock-down of an ALL PDX. We confirmed the absence of CD19 and presence BAFF-R expression, which was unaffected, on the resulting cell lines by surface staining (Fig. 1A). Using transduced CD8 TN cells, we found that CD19-CAR T cells demonstrated cytotoxicity only against wild-type tumor cells, while BAFF-R-CAR T cells maintained significant cytotoxicity against both wild-type and CD19-negative tumors in vitro (Fig. 1B). The therapeutic efficacy of BAFF-R-CAR T cells was tested against human ALL Nalm-6-CD19 deficient xenografts established in NSG mice following IV tumor challenge on day 0 with luciferase-expressing cells. A single dose of 2.5 x 106 CD4 TN + 106 CD8 TN BAFF-R- or CD19-CAR T cells/mouse infused IV on day 11 post tumor implantation completely eliminated established Nalm-6-CD19KO ALL tumors and conferred long-term survival. In contrast, treatment with PBS or identical mixtures of CD19-CAR T cells or non-transduced T cells from the same donor were associated with progressive tumor growth and 100% mortality by Day 60 (Fig. 1C).Finally, four relapsed, antigen loss primary ALLs obtained after CD19-directed therapy retained BAFF-R expression and activated BAFF-R-, but not CD19-CAR T cells. Specifically, cell surface staining demonstrated CD19 and BAFF-R expression in tumors obtained prior to CD19-targeted therapy. However, post-treatment samples exhibited clear down-regulation of CD19, while retaining positive BAFF-R expression (Fig. 1D, results from a single representative patient shown). The ability of the primary tumor samples to activate either CD19- or BAFF-R-CAR T cells was determined by expression of the degranulation marker CD107a on the CAR T cells. Cryopreserved ALL samples were co-cultured with BAFF-R or CD19 CAR-T cells derived from the same healthy donor in the presence of anti-CD107a antibody for 6 h. Non-transduced T cells (non-CAR) from the same donor were used as a negative control. Activation of CD19-CAR T cells by all four CD19-negative post-blinatumomab therapy tumors was significantly reduced, compared with BAFF-R-CAR T cells and with corresponding available CD19-positive pre-therapy tumors, while BAFF-R-CAR T cells were equally activated by pre- and post-CD19-targeted therapy tumors (Fig.1E-F). We observed similar trends for both CD19- and BAFF-R-CAR T cell activation by pre- and post-CD19-targeted therapy tumors, as measured by specific intracellular CAR T-cell TNF-α and IFN-γ production (not shown).Conclusion: Taken together, our data suggest that BAFF-R is amenable to CAR T-cell therapy and that targeting it may add to existing alternative strategies to overcome relapse from CD19 antigen loss, such as CD22 CAR T cells. Future strategies combining dual targeting of CD19 and BAFF-R may also be effective. [Display omitted] DisclosuresWang:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Forman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding.

Intranasal immunization with recombinant chlamydial protease-like activity factor attenuates atherosclerotic pathology following Chlamydia pneumoniae infection in mice.

We have shown previously that intranasal vaccination with recombinant chlamydial protease-like activity factor (rCPAF: antigen) and interleukin-12 (IL-12) as an adjuvant induces robust protection against pathological consequences of female genital tract infection with Chlamydia muridarum, a closely related species and a rodent model for the human pathogen Chlamydia trachomatis. Another related species Chlamydia pneumoniae, a human respiratory pathogen, has been associated with exacerbation of atherosclerotic pathology. CPAF is highly conserved among Chlamydia spp. leading us to hypothesize that immunization with rCPAF with IL-12 will protect against high-fat diet (HFD) and C.pneumoniae-induced acceleration of atherosclerosis. rCPAF±IL-12 immunization induced robust splenic antigen (Ag)-specific IFN-γ and TNF-α production and significantly elevated serum total anti-CPAF Ab, IgG2c, and IgG1 antibody levels compared to mock or IL-12 alone groups. The addition of IL-12 to rCPAF significantly elevated splenic Ag-specific IFN-γ production and IgG2c/IgG1 anti-CPAF antibody ratio. Following intranasal C.pneumoniae challenge and HFD feeding, rCPAF±IL-12-immunized mice displayed significantly enhanced splenic IFN-γ, not TNF-α, response on days 6 and 9 after challenge, and significantly reduced lung chlamydial burden on day 9 post-challenge compared to mock- or IL-12-immunized mice. Importantly, rCPAF±IL-12-immunized mice displayed significantly reduced atherosclerotic pathology in the aortas after C.pneumoniae challenge. Serum cholesterol levels were comparable between the groups suggesting that the observed differences in pathology were due to protective immunity against the infection. Together, these results confirm and extend our previous observations that CPAF is a promising candidate antigen for a multisubunit vaccine regimen to protect against Chlamydia-induced pathologies, including atherosclerosis.

Abstract 1002: Dysfunction of HPV16-specific CD8+ T cells derived from oropharyngeal tumors is related to the expression of Tim-3 but not PD-1

Abstract Human papillomavirus (HPV) type 16 infection is one of the most important etiological agents of oropharyngeal squamous cell carcinoma. Patients with HPV-positive squamous cell carcinomas of the head and neck were reported to have a better clinical outcome than patients with HPV-negative tumors. Because HPV16 E6 and E7 oncoproteins are highly immunogenic and constitutively expressed within the tumor cells, HPV-specific T cell immunity may contribute to the better prognosis of HPV-positive tumors. We analyzed the frequency, phenotype and function of HPV16 E6/E7-specific tumor-infiltrating T cells (TILs) in oropharyngeal tumors and tested the effect of anti-PD1 mAb (nivolumab), soluble Tim-3 (sTim-3) and homeostatic in vitro expansion on these characteristics. We show that 73.1% of HPV-associated oropharyngeal tumors are positive for HPV16-specific T cells capable of producing IFNγ upon stimulation with E6/E7 peptide pools. These IFNγ-producing HPV-specific TILs were mainly PD-1+Tim-3-CD8+ T cells, identifying Tim-3 rather than PD-1 as a marker of T cell dysfunction. Consequently, specific IFNγ production was further enhanced by combined nivolumab plus sTim-3 treatment, but not with nivolumab alone. Our data provide the rationale for exploring additional setups of anti-PD-1 mAb treatment, including combinations with other check-point blockers, such as Tim-3 and/or with HPV16-directed therapeutic vaccines. Citation Format: Simona Partlova, Kamila Hladikova, Vladimir Koucky, Anna Fialova, Radek Spisek, Jan Boucek, Michal Zabrodsky, Michael Halaska, Ruth Tachezy, Marek Grega, Jean-François Fonteneau. Dysfunction of HPV16-specific CD8+ T cells derived from oropharyngeal tumors is related to the expression of Tim-3 but not PD-1 [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 1002.

Dysfunction of HPV16-specific CD8+ T cells derived from oropharyngeal tumors is related to the expression of Tim-3 but not PD-1

BackgroundHuman papillomavirus (HPV) type 16 infection is one of the most important etiological agents of oropharyngeal squamous cell carcinoma. Patients with HPV-associated carcinomas of the head and neck were reported to have a better clinical outcome than patients with HPV-negative tumors. Because HPV16 E6 and E7 oncoproteins are highly immunogenic and constitutively expressed, HPV-specific T cell immunity may play the key role in improving the prognosis of these patients. MethodsTumor-derived T cells were expanded in high levels of IL-2 and stimulated with HPV16 E6/E7 peptides in the presence or absence of anti-PD-1 monoclonal antibody nivolumab and soluble Tim-3. ResultsHPV16-specific tumor-infiltrating T cells were present in 73.1% of HPV-associated oropharyngeal tumors. HPV16 specific CD8+ TILs were able to produce IFNγ upon specific stimulation and predominantly expressed PD-1 but not Tim-3. Specific IFNγ production was further enhanced after a blockade of both PD-1 and Tim-3 pathways but not after a PD-1 blockade alone. Additionally, the specific stimulation of anti-HPV16 CD8+ T cells suppressed Tim-3 upregulation after the PD-1 blockade. ConclusionOur data provide the rationale for combination cancer immunotherapy approaches, including the dual blockade of PD-1 and Tim-3 and, potentially, the use of HPV16-directed therapeutic vaccines.

Parasite Specific Antibody Levels, Interferon-γ and TLR2 and TLR4 Transcripts in Blood from Dogs with Different Clinical Stages of Leishmaniosis.

Canine leishmaniosis has a wide range of disease severity from mild (stage I), to severe (stages II–III), or very severe disease (stage IV). The objective of the study was to evaluate and compare serum antibody levels, Leishmania infantum specific IFN-γ production and TLR2 and TLR4 transcripts in non-stimulated blood from dogs with different clinical stages at the time of diagnosis as well as blood parasitemia. Enzyme-Linked ImmunoSorbent Assay (ELISAs) were performed to determine serum antibody levels and IFN-γ production and quantitative polymerase chain reaction (qPCRs) in order to determine blood parasite load and TLR2 and TLR4 transcripts. Older dogs were significantly affected by more severe disease with higher antibody levels and blood parasitemia than dogs with mild disease. IFN-γ production was significantly higher in dogs with stage I disease when compared to dogs with more severe disease. Relative quantification of TLR2 in dogs with mild disease was similar to that of control dogs. On the other hand, TLR2 transcripts were significantly higher in dogs with severe disease as compared with that from control healthy dogs. No differences were found in TLR4 relative quantification between groups. This study demonstrates that dogs with different clinical stages of leishmaniosis present different levels of biological markers indicative of different immune responses.