Stimulation Of PBMC Research Articles

Identification of HLA-A2-Restricted Mycobacterial Lipoprotein Z Peptides Recognized by T CellsFrom Patients With ActiveTuberculosis Infection.

Identification of HLA-restricted peptides derived from mycobacterial antigens that are endowed with high affinity and strong antigenicity is not only of interest in tuberculosis (TB) diagnostics and treatment efficacy evaluation, but might also provide potential candidates for the development of therapeutic vaccines against drug-resistant TB. Our previous work demonstrated that lipoprotein Z (LppZ) displayed high immunogenicity and antigenicity in active TB patients. In the present study, ten HLA-A2-restricted LppZ peptides (LppZp1-10) were predicted by bioinformatics, among which LppZp7 and LppZp10 were verified to possess high affinity to HLA-A2 molecules using T2 cell-based affinity binding assay. Moreover, results from ELISpot assay showed that both LppZp7 and LppZp10 peptides were able to induce more IFN-γ producing cells upon ex vivo stimulation of PBMC from HLA-A2+ active TB (ATB) patients as compared to those from healthy controls (HCs). Also, the numbers of LppZp7 and LppZp10-specific IFN-γ producing cells exhibited positive correlations with those of ESAT-6 peptide (E6p) or CFP-10 peptide (C10p) in ATB. Interestingly, stimulation with LppZp7/p10 mixture was able to induce higher intracellular expression of IFN-γ and IL-2 cytokines in CD8+ and CD4+ T cells from ATB as compared to HC, associated with lower expression of TNF-α in both CD8+ and CD4+ T cells. Taken together, HLA-A2-restricted LppZp7 and LppZp10 peptides display high immunoreactivity in HLA-matched ATB patients demonstrated by high responsiveness in both CD8+ and CD4+ T cells. With the ability to induce strong antigen-specific cellular responses, LppZp7 and LppZp10 are of potential value for the future applications in the prevention and control of TB.

1575. Clinical Validation of a Novel ELISpot-based in vitro Diagnostic Assay to Monitor CMV-Specific Cell-Mediated Immunity in SOT and HSCT Immunocompromised Patients

BackgroundImpaired cytomegalovirus (CMV)-specific cell-mediated immunity (CMV-CMI) is a major cause of uncontrolled CMV reactivation and associated complications in both solid-organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT). Reliably assessing CMV-CMI is desirable to individually adjust antiviral and immunosuppressive therapy. We demonstrate here the suitability of a novel IFN-γ ELISpot assay (T-Track® CMV), based on the stimulation of PBMC with pp65 and IE-1 CMV proteins, to monitor CMV-CMI in SOT and HSCT patients.MethodsTwo independent prospective, longitudinal, observational, multicenter studies were conducted: in 86 intermediate-risk (D−/R+, D+/R+) renal transplant recipients (completed), and in 175 intermediate- or high-risk (D+/R+, D+/R−, D−/R+) HSCT recipients (ongoing). In both studies, patients underwent pre-emptive antiviral therapy. CMV-CMI, CMV load and clinical complications were monitored over ~6 months post-transplantation.ResultsIn the kidney transplantation setting, 95% and 88–92% of IFN-γ ELISpot test results were positive pre- and post-transplantation, respectively. CMV-specific response was reduced following immunosuppressive therapy and increased in patients with graft rejection, indicating the ability of the assay to monitor the patients’ immunosuppressive state. Interestingly, median pp65-specific response was 9-fold higher in patients with self-clearing viral load compared with antivirally-treated patients prior to first detection of CMV (MWU; P < 0.001), suggesting that reactivity to pp65 is a potential immunocompetence marker. In HSCT patients, interim data analysis indicates that pp65-specific CMI measured after resolution of a primary CMV reactivation (requiring antiviral treatment) is a fair predictor of occurrence of recurrent CMV reactivation. Out of 71 patients (25 D+/R+, 3 D+/R−, 43 D−/R+) who experienced a primary CMV reactivation, 27 encountered a recurrent CMV reactivation. Interestingly, 39/44 (89%) patients free of recurrent reactivation had a positive pp65-specific test result following primary CMV reactivation.ConclusionAltogether, this novel IFN-γ ELISpot assay is a highly sensitive immune-monitoring tool with a potential use for the risk assessment of CMV-related clinical complications after SOT and HSCT.Disclosures All authors, Lophius Biosciences: Investigator, Research support.

DosR proteins of Mycobacterium tuberculosis upregulate effector T cells and down regulate T regulatory cells in TB patients and their healthy contacts

It is well established that the current problem of tuberculosis (TB) can be combated by overcoming the drawbacks of the currently available BCG vaccine. This would involve incorporation of antigens that can control TB at all stages including the dormant phase which is generally ignored. Hence, DosR regulon proteins, which are expressed in latent infection, could prove to be very good vaccine candidates as they can possibly target the silent but most predominant form of TB infection. In the present study, the immune response to two DosR proteins Rv2627 and Rv2628 has been studied in PBMCs derived from normal individuals, TB patients and healthy contacts of TB patients. It was found that these antigens were capable of stimulating a strong IFN-γ+ T cell response along with accentuation of memory T cells and other protective cytokines such as IL-2 and IL-17. At the same time these proteins decreased the frequencies of immune-suppressor regulatory T cells in in vitro stimulation of PBMC from both patients and their contacts. Considering all these facts together, we suggest Rv2627 and Rv2628 to be one of the extremely promising candidates for incorporation into a post exposure subunit vaccine against TB.

Deep analysis of immune response and metabolic signature in children with food protein induced enterocolitis to cow\u2019s milk

BackgroundFood Protein-Induced Enterocolitis Syndrome (FPIES) is considered to be a non-IgE mediated food allergy. However, its pathogenesis remains poorly understood and biomarkers are lacking. We aimed to perform in-depth characterization of humoral and cellular immune responses in children with cow’s milk (CM)-FPIES and investigated whether there is a FPIES metabolomic signature.MethodsChildren with CM-FPIES and control subjects with an IgE-mediated CM allergy (IgE-CMA), both avoiding CM, were recruited on the day of an oral food challenge. Blood samples were collected before the challenge. Total and specific levels of IgE, IgG1-4, IgA, IgM and IgD to various whey and casein allergens and to their gastroduodenal digestion products were measured in plasma, using plasma from CM-tolerant peanut allergic patients (IgE-PA, not avoiding CM) as additional controls. Cytokine secretion and cellular proliferation were analyzed after stimulation of PBMC with different CM allergens. Metabolomic profiles were obtained for plasma samples using liquid chromatography coupled to high-resolution mass spectrometry.ResultsNine children with CM-FPIES and 12 control subjects (6 IgE-CMA and 6 IgE-PA) were included. In children with CM-FPIES, total Ig concentrations were lower than in control subjects, specific Ig against CM components were weak to undetectable, and no specific IgE against CM digestion products were detected. Moreover, in CM-FPIES patients, we did not find any Th cell proliferation or associated cytokine secretion after allergen reactivation, whereas such responses were clearly found in children with IgE-CMA. Plasma metabolic profiles were different between CM allergic patients, with significantly lower concentrations of various fatty acids and higher concentrations of primary metabolites such as amino acids in CM-FPIES compared to IgE-CMA patients.ConclusionsIn CM-FPIES, both humoral and cellular specific immune responses are weak or absent, and this is not related to CM avoidance. A metabolomic signature was identified in patients with CM-FPIES that may be useful for the diagnosis and management of this disease.

C-C motif chemokine ligand (CCL) production in equine peripheral blood mononuclear cells identified by newly generated monoclonal antibodies

Chemokines are soluble molecules directing immune cell trafficking and homing, mediating inflammation, and initiating immune responses to infection. In horses, the analysis of chemokines has been limited by the lack of specific antibodies. We generated mAbs specific for the equine C-C motif chemokine ligands (CCL) CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES) and CCL11 (eotaxin) using hybridoma technology.Antibody specificity was confirmed by intracellular staining of Chinese Hamster Ovary cells transfected with expression vectors encoding for CCL2, CCL3, CCL5, or CCL11. Transfectants were stained with the anti-CCL mAbs. Flow cytometric analysis confirmed the specificity of the different mAbs for the respective chemokine. In addition, equine PBMC were stained after isolation, culture in medium, or stimulation with LPS, or PMA and ionomycin.CCL2 was detected in few cluster of differentiation (CD)14+ monocytes in PBMC stimulated with PMA and ionomycin for 2 h. CCL3 was produced by CD14+ monocytes after 4–6 h culture in medium. After stimulation with PMA and ionomycin for 12–24 h, CCL3 was also expressed in lymphocytes, mainly in CD4+ T cells. Stimulation with LPS reduced the percentage of CCL3+ monocytes in PBMC. CCL5 was detected in PBMC ex vivo in CD4+ and CD8+ T cells. Culture of PBMC for longer than 6 h or stimulation with PMA and ionomycin reduced the percentage of CCL5+ cells. CCL11 was produced by CD4+ T cells in PBMC after stimulation with PMA and ionomycin for 4–24 h. After LPS stimulation of PBMC, CCL2, CCL5, and CCL11 production were comparable to culture in medium alone.ELISAs for each of the four chemokines were developed using pairs of anti-equine CCL mAbs. Supernatants from PMA and ionomycin stimulated PBMC contained detectable amounts of CCL2, CCL3 and CCL5, while CCL11 secretion could be stimulated from equine tracheal epithelial cells in response to IL-4.The newly generated mAbs for equine CCL chemokines facilitate the quantitative analysis of intracellular chemokine production by flow cytometry and soluble chemokines by ELISA. The CCL mAbs are valuable tools to improve the evaluation of innate immune responses in horses.

PD-1 Expression on Mycobacterium tuberculosis-Specific CD4 T Cells Is Associated With Bacterial Load in Human Tuberculosis.

Persistent antigen stimulation in chronic infections has been associated with antigen-specific T cell dysfunction and upregulation of inhibitory receptors, including programmed cell death protein 1 (PD-1). Pulmonary tuberculosis (TB) disease is characterized by high levels of Mycobacterium tuberculosis (Mtb), yet the relationship between bacterial load, PD-1 expression, and Mtb-specific T cell function in human TB has not been well-defined. Using peripheral blood samples from adults with LTBI and with pulmonary TB disease, we tested the hypothesis that PD-1 expression is associated with bacterial load and functional capacity of Mtb-specific T cell responses. We found that PD-1 was expressed at significantly higher levels on Th1 cytokine-producing Mtb-specific CD4 T cells from patients with smear-positive TB, compared with smear-negative TB and LTBI, which decreased after completion of anti-TB treatment. By contrast, expression of PD-1 on Mtb-specific CD8 T cells was significantly lower than on Mtb-specific CD4 T cells and did not differ by Mtb infection and disease status. In vitro stimulation of PBMC with Mtb antigens demonstrated that PD-1 is induced on proliferating Mtb-specific CD4 T cells and that Th1 cytokine production capacity is preferentially maintained within PD-1+ proliferating CD4 T cells, compared with proliferating Mtb-specific CD4 T cells that lack PD-1 expression. Together, these data indicate that expression of PD-1 on Mtb-specific CD4 T cells is indicative of mycobacterial antigen exposure and identifies a population of effector cells with Th1 cytokine production capacity. These studies provide novel insights into the role of the PD-1 pathway in regulating CD4 and CD8 T cell responses in Mtb infection and provide rationale for future studies to evaluate PD-1 expression on antigen-specific CD4 T cells as a potential biomarker for bacterial load and treatment response in human TB.

Diagnostic performance of GM-CSF and IL-2 in response to long-term specific-antigen cell stimulation in patients with active and latent tuberculosis infection

BackgroundA simple blood test for detecting active tuberculosis (TB) could be key to this epidemic containment, given that a large proportion of patients are unable to produce sputum for testing. Currently available interferon-γ release assays (IGRAs) are inadequate to diagnose active TB, with reported pooled sensitivity and specificity both under 81%. ObjectiveTo explore whether cytokines/chemokines other than interferon-γ in response to long-term cell stimulation could improve the ability to distinguish between different TB infection status. MethodsWe prospectively enrolled subjects with newly diagnosed pulmonary TB and their household contacts in Santiago. All contacts were tested with IGRA. Peripheral blood mononuclear cells were obtained and antigen-specific stimulated for 72 h before collecting their culture supernatants. ResultsSubjects with active TB displayed markedly low cytokines/chemokines secretion upon PBMC stimulation, with lower GM-CSF being the best differentiator from IGRA(+) contacts, with 71% (95% CI 53–85) sensitivity, 86% (95% CI 65–97) specificity and AUC = 0.79 (p = 0.0003). On the other hand, when compared to the uninfected IGRA(−) contacts, higher level of IL-2 secretion was the best indicator of active TB, with 73.5% (95% CI 56–87) sensitivity, 85% (95% CI 66–96) specificity and AUC = 0.79 (p = 0.0001). No single cytokine/chemokine released upon stimulation could accurately differentiate between active TB and all TB contacts grouped together. ConclusionGM-CSF and IL-2 provided the best yield to differentiate active TB from latent TB and from TB uninfected, respectively, with higher specificities than that reported for IGRAs. However, none of both resulted sensitive enough to be used as a stand-alone biomarker for active TB.

Human MAITcells are rapidly activated by Aspergillus spp. in an APC-dependent manner.

Mucosal associated invariant Tcells (MAITcells) are innate-like Tcells (TC) which are known to be activated by several bacteria and viruses. However, activation of MAITcells by moulds, such as the opportunistic human pathogen Aspergillus, is not well described. Stimulation of human PBMC with A. fumigatus, A. flavus, or A. terreus conidia revealed that in contrast to conventional CD4+ and CD8+ TC, MAITcells responded already after 4 h of coincubation with upregulation of CD69. Furthermore, concurrent increase of CD107a expression and reduced intracellular expression of cytolytic proteins like perforin and granzyme indicated degranulation of intracellular vesicles. MAITcell activation only occurred in the presence of APC and was dependent on cell-cell contact as separation of TC and APC abrogated MAITcell activation. Furthermore, we observed that MAITcell activation by moulds requires presentation of riboflavin metabolites and depends on TCR engagement as antibody blocking of MR1, the antigen presenting molecule for MAITcells, prevented upregulation of CD69 and CD107a. In summary, we could demonstrate that MAITcells are activated by Aspergillus conidia in a TCR-dependent manner by APC. These findings reveal MAITcells as an interesting new target in antifungal defense.

Immune Checkpoint Blockade Restores HIV-Specific CD4 T Cell Help for NK Cells.

Immune exhaustion is an important feature of chronic infections, such as HIV, and a barrier to effective immunity against cancer. This dysfunction is in part controlled by inhibitory immune checkpoints. Blockade of the PD-1 or IL-10 pathways can reinvigorate HIV-specific CD4 T cell function in vitro, as measured by cytokine secretion and proliferative responses upon Ag stimulation. However, whether this restoration of HIV-specific CD4 T cells can improve help to other cell subsets impaired in HIV infection remains to be determined. In this study, we examine a cohort of chronically infected subjects prior to initiation of antiretroviral therapy (ART) and individuals with suppressed viral load on ART. We show that IFN-γ induction in NK cells upon PBMC stimulation by HIV Ag varies inversely with viremia and depends on HIV-specific CD4 T cell help. We demonstrate in both untreated and ART-suppressed individuals that dual PD-1 and IL-10 blockade enhances cytokine secretion of NK cells via restored HIV-specific CD4 T cell function, that soluble factors contribute to these immunotherapeutic effects, and that they depend on IL-2 and IL-12 signaling. Importantly, we show that inhibition of the PD-1 and IL-10 pathways also increases NK degranulation and killing of target cells. This study demonstrates a previously underappreciated relationship between CD4 T cell impairment and NK cell exhaustion in HIV infection, provides a proof of principle that reversal of adaptive immunity exhaustion can improve the innate immune response, and suggests that immune checkpoint modulation that improves CD4/NK cell cooperation can be used as adjuvant therapy in HIV infection.

MiR-181c-BRK1 axis plays a key role in actin cytoskeleton-dependent T cell function.

MicroRNAs are short endogenous noncoding RNAs that play pivotal roles in a diverse range of cellular processes. The miR-181 family is important in T cell development, proliferation, and activation. In this study, we have identified BRK1 as a potential target of miR-181c using a dual selection functional assay and have showed that miR-181c regulates BRK1 by translational inhibition. Given the importance of miR-181 in T cell function and the potential role of BRK1 in the involvement of WAVE2 complex and actin polymerization in T cells, we therefore investigated the influence of miR-181c-BRK1 axis in T cell function. Stimulation of PBMC derived CD3+ T cells resulted in reduced miR-181c expression and up-regulation of BRK1 protein expression, suggesting that miR-181c-BRK1 axis is important in T cell activation. We further showed that overexpression of miR-181c or suppression of BRK1 resulted in inhibition of T cell activation and actin polymerization coupled with defective lamellipodia generation and immunological synapse formation. Additionally, we found that BRK1 silencing led to reduced expressions of other proteins in the WAVE2 complex, suggesting that the impairment of T cell actin dynamics was a result of the instability of the WAVE2 complex following BRK1 depletion. Collectively, we demonstrated that miR-181c reduces BRK1 protein expression level and highlighted the important role of miR-181c-BRK1 axis in T cell activation and actin polymerization-mediated T cell functions.

The Act1 D10N missense variant impairs CD40 signaling in human B-cells.

The TRAF3IP2 gene resides within one of at least 63 psoriasis susceptibility loci and encodes Act1, an adapter protein involved in IL-17 receptor and CD40 signaling pathways. TRAF3IP2 is distinctive (among <10% of candidate susceptibility genes) in that a strongly disease-associated variant encodes a missense SNP predicted to be functionally relevant (SNP rs33980500 C/T encoding Act1 pD10N). As assessed by flow cytometry, Act1 protein was expressed at the highest levels in monocytes, with lower levels in T-cells and B-cells. However, monocytes, T-cells and B-cells failed to respond to IL-17A stimulation of PBMC, as measured by flow cytometric determination of NF-κB phospho-p65. As an alternative stimulus, we treated PBMCs with trimerized recombinant human CD40L and assessed p65, p38 and Erk phosphorylation in CD19+ B-cells as a function of D10N genotype. The increase of phosphorylated p65, p38 and Erk was well-correlated across individuals, and CD40L-induced phosphorylation of p65, p38, and Erk was significantly attenuated in B-cells from Act1 D10N homozygotes, compared to heterozygotes and nullizygotes. Our results indicate that the Act1 D10N variant is a relevant genetic determinant of CD40L responsiveness in human B-cells, with the risk allele being associated with lower B-cell responses in an acute signaling context.

Circulating LL37 targets plasma extracellular vesicles to immune cells and intensifies Behçet’s disease severity

ABSTRACTBehçet’s disease (BD) activity is characterised by sustained, over-exuberant immune activation, yet the underlying mechanisms leading to active BD state are poorly defined. Herein, we show that the human cathelicidin derived antimicrobial peptide LL37 associates with and directs plasma extracellular vesicles (EV) to immune cells, thereby leading to enhanced immune activation aggravating BD pathology. Notably, disease activity was correlated with elevated levels of circulating LL37 and EV plasma concentration. Stimulation of healthy PBMC with active BD patient EVs induced heightened IL1β, IFNα, IL6 and IP10 secretion compared to healthy and inactive BD EVs. Remarkably, when mixed with LL37, healthy plasma-EVs triggered a robust immune activation replicating the pathology inducing properties of BD EVs. The findings of this study could be of clinical interest in the management of BD, implicating LL37/EV association as one of the major contributors of BD pathogenesis.Abbreviations: BD: Behçet’s disease; EV: extracellular vesicle; BB: binding buffer; AnV: annexin V; autologEV: autologous extracellular vesicles; alloEV: allogeneic extracellular vesicles

Standardized whole blood stimulation improves immunomonitoring of induced immune responses in multi-center study

Functional immune responses are increasingly important for clinical studies, providing in depth biomarker information to assess immunotherapy or vaccination. Incorporating functional immune assays into routine clinical practice has remained limited due to challenges in standardizing sample preparation. We recently described the use of a whole blood syringe-based system, TruCulture®, which permits point-of-care standardized immune stimulation. Here, we report on a multi-center clinical study in seven FOCIS Centers of Excellence to directly compare TruCulture to conventional PBMC methods. Whole blood and PBMCs from healthy donors were exposed to LPS, anti-CD3 anti-CD28 antibodies, or media alone. 55 protein analytes were analyzed centrally by Luminex multi-analyte profiling in a CLIA-certified laboratory. TruCulture responses showed greater reproducibility and improved the statistical power for monitoring differential immune response activation. The use of TruCulture addresses a major unmet need through a robust and flexible method for immunomonitoring that can be reproducibly applied in multi-center clinical studies. One sentence summaryA multi-center study revealed greater reproducibility from whole blood stimulation systems as compared to PBMC stimulation for studying induced immune responses.

Lymphoproliferative responses to dendritic cell presentation of sensitizing allergens in atopic children with multiple allergies

BackgroundPeripheral blood mononuclear cells (PBMCs) proliferate inconsistently, rendering current lymphoproliferation assays unreliable in diagnosis. ObjectiveTo investigate the utility and nature of proliferation responses in allergy by comparison of the standard lymphoproliferation with a new dendritic cell (DC) stimulated assay. MethodsMonocyte-derived DCs were pulsed with allergens and incubated with autologous T cells for 7 days. DC-stimulated and standard PBMC proliferation responses to 3 common dietary allergens in children with allergy and without atopy were measured by incorporation of tritiated thymidine and reduction of carboxyl fluorescein succinimidyl ester staining. ResultsThe DC presentation of sensitizing allergens induced significantly higher proliferative responses than PBMC stimulation (P = .04) and greater distinction between normal and allergic responses. DC-induced stimulation indices of children without sensitivity and those with allergy were significantly different with all 3 foods (P < .001). All children with allergy presented with peanut allergy and 12 of 14 (86%) β-lactoglobulin–pulsed DC preparations proliferated more than 3.3-fold above un-pulsed cells, but 8 of 18 children (44%) with ovalbumin egg allergy showed proliferation below this level. The stimulation index of DC tritiated thymidine incorporation correlated closely with carboxyl fluorescein succinimidyl ester reduction (P < .001). Sensitivity of detection of peanut, milk, or egg allergy was 100%, 85.7%, or 55.6% and specificity was 60%, 88.9%, or 86.7%, respectively. DC-stimulated T cells expressed increased levels of CD45 RO and CD25 and most produced interferon-γ. DC-stimulated proliferation correlated with total immunoglobulin E and peanut antigen-stimulated proliferation correlated with peanut specific immunoglobulin E (P = .03). ConclusionThe DC-induced lymphoproliferation had higher sensitivity, specificity, and reproducibility than the standard assay and caused increased memory and activated T-cell proliferation in children with food allergy.

An invitro model demonstrates the potential of neoplastic human germ cells to influence the tumour microenvironment.

Testicular germ cell tumours (TGCT) typically contain high numbers of infiltrating immune cells, yet the functional nature and consequences of interactions between GCNIS (germ cell neoplasia insitu) or seminoma cells and immune cells remain unknown. A co-culture model using the seminoma-derived TCam-2 cell line and peripheral blood mononuclear cells (PBMC, n=7 healthy donors) was established to investigate how tumour and immune cells each contribute to the cytokine microenvironment associated with TGCT. Three different co-culture approaches were employed: direct contact during culture to simulate insitu cellular interactions occurring within seminomas (n=9); indirect contact using well inserts to mimic GCNIS, in which a basement membrane separates the neoplastic germ cells and immune cells (n=3); and PBMC stimulation prior to direct contact during culture to overcome the potential lack of immune cell activation (n=3). Transcript levels for key cytokines in PBMC and TCam-2 cell fractions were determined using RT-qPCR. TCam-2 cell fractions showed an immediate increase (within 24h) in several cytokine mRNAs after direct contact with PBMC, whereas immune cell fractions did not. The high levels of interleukin-6 (IL6) mRNA and protein associated with TCam-2 cells implicate this cytokine as important to seminoma physiology. Use of PBMCs from different donors revealed a robust, repeatable pattern of changes in TCam-2 and PBMC cytokine mRNAs, independent of potential inter-donor variation in immune cell responsiveness. This invitro model recapitulated previous data from clinical TGCT biopsies, revealing similar cytokine expression profiles and indicating its suitability for exploring the invivo circumstances of TGCT. Despite the limitations of using a cell line to mimic invivo events, these results indicate how neoplastic germ cells can directly shape the surrounding tumour microenvironment, including by influencing local immune responses. IL6 production by seminoma cells may be a practical target for early diagnosis and/or treatment of TGCT.

Early life allergen and air pollutant exposures alter longitudinal blood immune profiles in infant rhesus monkeys

Early life is a critical period for the progressive establishment of immunity in response to environmental stimuli; the impact of airborne challenges on this process is not well defined. In a longitudinal fashion, we determined the effect of episodic house dust mite (HDM) aerosol and ozone inhalation, both separately and combined, on peripheral blood immune cell phenotypes and cytokine expression from 4 to 25weeks of age in an infant rhesus monkey model of childhood development. Immune profiles in peripheral blood were compared with lung lavage at 25weeks of age. Independent of exposure, peripheral blood cell counts fluctuated with chronologic age of animals, while IFNγ and IL-4 mRNA levels increased over time in a linear fashion. At 12weeks of age, total WBC, lymphocyte numbers, FoxP3 mRNA and IL-12 mRNA were dramatically reduced relative to earlier time points, but increased to a steady state with age. Exposure effects were observed for monocyte numbers, as well as CCR3, FoxP3, and IL-12 mRNA levels in peripheral blood. Significant differences in cell surface marker and cytokine expression were detected following in vitro HDM or PMA/ionomycin stimulation of PBMC isolated from animals exposed to either HDM or ozone. Lavage revealed a mixed immune phenotype of FoxP3, IFNγ and eosinophilia in association with combined HDM plus ozone exposure, which was not observed in blood. Collectively, our findings show that airborne challenges during postnatal development elicit measureable cell and cytokine changes in peripheral blood over time, but exposure-induced immune profiles are not mirrored in the lung.

Dengue Virus Induces NK Cell Activation through TRAIL Expression during Infection.

Dengue is an acute febrile illness with a wide spectrum of signs and symptoms ranging from mild to severe forms characterized by plasma leakage that can be fatal. NK cells are one of the main effectors in early infection and may play an important role in dengue pathogenesis. We investigated NK cell involvement during dengue infection. A higher frequency of NK cell subsets and TRAIL+NK cells was found in mild DF cases when compared to that in severe cases or healthy donors. NK activation markers such as CD107a and TLR3 were upregulated in patients' cells compared to those in healthy donors. In addition, IL12 related to NK cell activation were upregulated in mild DF cases. In vitro PBMC culture models show that DENV-stimulated and IFNα-stimulated NK cells were able to express TRAIL, suggesting an indirect activation of cells, regarding TRAIL expression. Type I IFN receptor blockage on DENV-stimulated PBMCs showed TRAIL expression on NK cells is partially IFNα dependent. In addition, during PBMC stimulation, TRAIL expression on NK cells was inversely correlated with DENV-positive monocytes. Therefore, we observed DENV-induced activation of NK cell populations. A higher activation of NK cells would promote limited viral spread, resulting in decreased inflammatory response, contributing to protection against dengue severity.

Stimulation of PBMC and Monocyte-Derived Macrophages via Toll-Like Receptor Activates Innate Immune Pathways in HIV-Infected Patients on Virally Suppressive Combination Antiretroviral Therapy.

In HIV-infected, combination antiretroviral therapy (cART)-treated patients, immune activation and microbial translocation persist and associate with inadequate CD4 recovery and morbidity/mortality. We analyzed whether alterations in the toll-like receptor (TLR) pathway could be responsible for the immune hyperactivation seen in these patients. PBMC/monocyte-derived macrophages (MDMs) of 28 HIV+ untreated and 35 cART-treated patients with HIV-RNA < 40 cp/mL [20 Full Responders (FRs): CD4 ≥ 350; 15 Immunological Non-Responders (INRs): CD4 < 350], as well as of 16 healthy controls were stimulated with a panel of TLR agonists. We measured: CD4/CD8/CD14/CD38/HLA-DR/Ki67/AnnexinV/CD69/TLR4/8 (Flow Cytometry); PBMC expression of 84 TLR pathway genes (qPCR); PBMC/MDM cytokine release (Multiplex); and plasma lipopolysaccharide (LPS)/sCD14 (LAL/ELISA). PBMC/MDM from cART patients responded weakly to LPS stimulation but released high amounts of pro-inflammatory cytokines. MDM from these patients were characterized by a reduced expression of HLA-DR+ MDM and failed to expand activated HLA-DR+ CD38+ T-lymphocytes. PBMC/MDM from cART patients responded more robustly to ssRNA stimulation; this resulted in a significant expansion of activated CD38 + CD8 and the release of amounts of pro-inflammatory cytokines comparable to those seen in untreated viremic patients. Despite greater constitutive TLR pathway gene expression, PBMC from INRs seemed to upregulate only type I IFN genes following TLR stimulation, whereas PBMC from full responders showed a broader response. Systemic exposure to microbial antigens drives immune activation during cART by triggering TLRs. Bacterial stimulation modifies MDM function/pro-inflammatory profile in cART patients without affecting T-lymphocytes; this suggests translocating bacteria as selective stimulus to chronic innate activation during cART. High constitutive TLR activation is seen in patients lacking CD4 recovery, suggesting an exhausted immune milieu, anergic to further antigen encounters.

Safety Data from a First-in-Human Phase 1 Trial of NKG2D Chimeric Antigen Receptor-T Cells in AML/MDS and Multiple Myeloma

Introduction: Conventional CAR-T cells express a single chain antibody variable fragment that restricts recognition to one tumor antigen and a limited set of cancers. This study employs a novel CAR fusing full-length human NKG2D with the CD3z signaling domain. In autologous transduced CM-CS1 T cells, NKG2D CAR receives endogenous costimulation via DAP10 to target multiple NKG2D-ligands that are upregulated in solid and hematologic malignancies but absent or poorly expressed on healthy tissues. Methods: A phase 1 dose-escalation study to establish safety and feasibility of a single infusion of CM-CS1 T cells without lymphodepleting conditioning enrolled subjects with AML/MDS-RAEB or relapsed/refractory progressive multiple myeloma (MM) without standard therapy options (NCT02203825). Eligibility criteria included suitable organ function, no CNS disease, no prior allogeneic SCT or adoptive T-cell therapy, no therapy within 3 weeks prior to infusion, no immune suppression, and no uncontrolled infection. Dose-escalation spanned 4 cohorts [half-log increments from 1x106 to 3x107 CM-CS1 T cells] according to a 3+3 design. DLTs included ≥ Grade 3 non-hematologic toxicity or ≥ Grade 2 autoimmune toxicity related to CAR T cells. Initial assessment was at 28 days. At least 1 AML/MDS and 1 MM subject were mandated in each dose level. Manufacturing included PBMC stimulation with OKT3 and IL-2 followed by 2 rounds of retroviral transduction at DFCI9s Cell Manipulation Core Facility. Vector copy number (VCN) and replication-competent retrovirus (RCR) testing were performed on whole blood and PBMCs, respectively, using quantitative PCR. Results: From April 2015 to July 2016, 11 subjects were infused, and 10 completed the DLT period. Eight of 11 were male, 6 had AML/MDS, and median age was 70 (range 44 to 79) (Panel A). Median WBC was 2.3 (range 0.7 to 7.2 K/uL); median ALC was 0.74 (range 0.09-2.37 K/uL). Five had cells manufactured from peripheral blood; 6 underwent apheresis. Median percentage of blasts in bone marrow for AML/MDS patients was 50% (range 4-68%). All myeloma patients had undergone ≥ 5 therapies including ≥1 autologous SCT. Four of the 6 AML/MDS patients had secondary disease, 3 had complex cytogenetics, 3 had p53 mutations, and 1 had a FLT3-ITD mutation. Dose-escalation proceeded from 1x106 to 3x107 CM-CS1 T cells. All 11 products passed release criteria, and there were no infusion reactions. Products consisted of median 97.2% CD3+ cells and 31.0% CD8+ cells, with vector-specific NKG2D expression on median 74.6% of CD3+ and 66.3% of CD8+ cells (Panel B). The first 10 subjects completed their 28 day evaluation period without DLTs. There were no cases of cytokine release syndrome, cell-related neurotoxicity, auto-immunity, or CAR T-related death. SAEs included a Grade 4 intracochlear bleed and an episode each of grade 4 neutropenia and thrombocytopenia deemed related to disease progression. Forty percent of patients experienced some Grade 3 toxicity, all related to underlying disease or a complication thereof (Panel C). At these initial cell doses, no patient to date has had objective tumor response at the 28 day evaluation mark. Nine initiated subsequent therapies; there have been 4 deaths secondary to disease or complications of subsequent therapies. However, cases of unexpected survival without further therapy and responses to subsequent treatments were noted. For example a patient with p53-mutated AML survived 4 months despite 50% blasts at infusion, and another entered PR at 6+months after cells on an IDH-1 inhibitor with Conclusion: In the first 3+ dose-escalation cohorts of patients with AML/MDS and myeloma, a single dose of CM-CS1 T cells without lymphodepletion was feasible and well-tolerated, with no DLTs. CAR T cells generally have not persisted beyond 1 week, consistent with pre-clinical models. Correlative analyses including post-infusion immunophenotyping are in process. Future studies of multiple infusions of NKG2D CAR T cells in both hematologic malignancies and solid tumors at the higher cell doses associated with efficacy in pre-clinical models are in planning. Disclosures Murad:Celdara Medical, LLC: Employment. Reder:Celdara Medical, LLC: Employment. Sentman:Celdara Medical, LLC: Membership on an entity9s Board of Directors or advisory committees, Other: Holds patents on this technology. Wade:Celdara Medical, LLC: Employment. Schmucker:Celdara Medical, LLC: Employment. Lehmann:Celyad, SA: Employment. Snykers:Celyad, SA: Employment. Allen:Celyad, SA: Employment. Stone:Celator: Consultancy; Jansen: Consultancy; Novartis: Consultancy; Merck: Consultancy; ONO: Consultancy; Sunesis Pharmaceuticals: Consultancy; Roche: Consultancy; Celgene: Consultancy, Membership on an entity9s Board of Directors or advisory committees; Juno Therapeutics: Consultancy; Xenetic Biosciences: Consultancy; Agios: Consultancy; Abbvie: Consultancy, Membership on an entity9s Board of Directors or advisory committees; Seattle Genetics: Consultancy; Pfizer: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy. Soiffer:Kiadis: Membership on an entity9s Board of Directors or advisory committees; Juno: Consultancy. Dranoff:Novartis: Employment. Ritz:Kiadis: Membership on an entity9s Board of Directors or advisory committees.

IRES-dependent translation of the long non coding RNA meloe in melanoma cells produces the most immunogenic MELOE antigens.

MELOE-1 and MELOE-2, two highly specific melanoma antigens involved in T cell immunosurveillance are produced by IRES-dependent translation of the long « non coding » and polycistronic RNA, meloe. In the present study, we document the expression of an additional ORF, MELOE-3, located in the 5′ region of meloe. Data from in vitro translation experiments and transfection of melanoma cells with bicistronic vectors documented that MELOE-3 is exclusively translated by the classical cap-dependent pathway. Using a sensitive tandem mass spectrometry technique, we detected the presence of MELOE-3 in total lysates of both melanoma cells and normal melanocytes. This contrasts with our previous observation of the melanoma-restricted expression of MELOE-1 and MELOE-2. Furthermore, in vitro stimulation of PBMC from 6 healthy donors with overlapping peptides from MELOE-1 or MELOE-3 revealed a very scarce MELOE-3 specific T cell repertoire as compared to the abundant repertoire observed against MELOE-1. The poor immunogenicity of MELOE-3 and its expression in melanocytes is consistent with an immune tolerance towards a physiologically expressed protein. In contrast, melanoma-restricted expression of IRES-dependent MELOE-1 may explain its high immunogenicity. In conclusion, within the MELOE family, IRES-dependent antigens represent the best T cell targets for immunotherapy of melanoma.