Invitro Stimulation Research Articles

Cigarette Smoking Is Associated With Lower Chance of Hepatitis B Surface Antigen Seroclearance and Altered Host Immunity.

Cigarette smoking is associated with worse clinical outcomes in patients with chronic hepatitis B (CHB) infection, but the effects on hepatitis B surface antigen (HBsAg) seroclearance are unclear. This study aimed to investigate the effect of active smoking on HBsAg seroclearance (SC) and its impact on peripheral blood lymphocytes in patients with CHB infection. Longitudinal follow-up data was retrieved in 7833 antiviral-treated CHB subjects identified from a centralised electronic patient record database (Part 1). Phenotypic analysis of peripheral blood mononuclear cells (PBMCs) from 27 CHB-infected patients (6 active smokers; 13 with SC) was performed by flow cytometry to assess programmed death-1 (PD-1) expression and proportion of regulatory T cells (CD4+CD25+CD127lo). Effector function of HBV-specific T cells was examined by comparing granzyme B (GZMB) and transforming growth factor beta (TGFβ) production in undepleted PBMCs and Treg-depleted PBMCs after 7 days invitro stimulation with HBV envelope protein overlapping peptides (Part 2). Over a median follow-up of 5 years, smoking was associated with lower probability of SC (aHR 0.70, 95% CI 0.57-0.87). PD-1 expression was increased in CD4+T cells, CD8+T cells and CD20+B cells among smokers compared to non-smokers and positively correlated with pack years (all p < 0.05). Treg depletion led to partial functional recovery of HBV-specific T cells, with significantly bigger magnitude in smokers (p = 0.0451, mean difference = 4.68%) than non-smokers (p = 0.012, mean difference = 4.2%). Cigarette smoking is associated with lower chance of HBsAg seroclearance, higher PD-1 expression on lymphocytes, and impairment of effector functions of HBV-specific T cells in CHB.

Expression conditions and characterization of a novelly constructed lipoprotein intended as a vaccine to prevent human Haemophilus influenzae infections

Bacterial lipoproteins are structurally divided into two groups, based on their lipid moieties: diacylated (present in Gram-positive bacteria) and triacylated (present in some Gram-positive and most Gram-negative bacteria). Diacylated and triacylated lipid moieties differ by a single amide-linked fatty acid chain. Lipoproteins induce host innate immune responses by the mammalian Toll-like receptor 2 (TLR2). In this study, we added a lipid moiety to recombinant OMP26, a native nonlipidated (NL) membrane protein of Haemophilus influenzae, and characterized it extensively under different expression conditions using flow cytometry, LC/MS, and MALDI-TOF. We also investigated the ability of NL and lipidated (L) OMP26 to induce invitro stimulation of HEK Blue-hTLR2-TR1 and hTLR-TLR6 cells. Our L-OMP26 was predominantly expressed in diacylated form, so we employed an additional gene copy of apolipoprotein N-acetyltransferase enzyme (Lnt)-rich Escherichia coli strain that further acylates the diacyl lipoproteins to enhance the production of triacylated L-OMP26. The diacyl and triacyl versions of L-OMP26, intended as a vaccine for use in humans, were characterized and evaluated as protein vaccine components in a mouse model. We found that the diacyl and triacyl L-OMP26 protein formulations differed markedly in their immune-stimulatory activity, with diacylated L-OMP26 stimulating higher adaptive immune responses compared with triacylated L-OMP26 and both stimulating higher adaptive immune response compared to NL-OMP26. We also constructed and characterized an L-OMP26φNL-P6 fusion protein, where NL-P6 protein (a commonly studied H.influenzae vaccine candidate) was recombinantly fused to L-OMP26. We observed a similar pattern of lipidation (predominantly diacylated) in the L-OMP26φNL-P6 fusion protein.

Human skin-resident CD8+ T cells require RUNX2 and RUNX3 for induction of cytotoxicity and expression of the integrin CD49a

The integrin CD49a marks highly cytotoxic epidermal-tissue-resident memory (TRM) cells, but their differentiation from circulating populations remains poorly defined. We demonstrate enrichment of RUNT family transcription-factor-binding motifs in human epidermal CD8+CD103+CD49a+ TRM cells, paralleled by high RUNX2 and RUNX3 protein expression. Sequencing of paired skin and blood samples revealed clonal overlap between epidermal CD8+CD103+CD49a+ TRM cells and circulating memory CD8+CD45RA-CD62L+ Tcells. Invitro stimulation of circulating CD8+CD45RA-CD62L+ Tcells with IL-15 and TGF-β induced CD49a expression and cytotoxic transcriptional profiles in a RUNX2- and RUNX3-dependent manner. We therefore identified a reservoir of circulating cells with cytotoxic TRM potential. In melanoma patients, high RUNX2, but not RUNX3, transcription correlated with a cytotoxic CD8+CD103+CD49a+ TRM cell signature and improved patient survival. Together, our results indicate that combined RUNX2 and RUNX3 activity promotes the differentiation of cytotoxic CD8+CD103+CD49a+ TRM cells, providing immunosurveillance of infected and malignant cells.

Revealing viral and cellular dynamics of HIV-1 at the single-cell level during early treatment periods

While combination therapy completely suppresses HIV-1 replication in blood, functional virus persists in CD4+ Tcell subsets in non-peripheral compartments that are not easily accessible. To fill this gap, we investigated tissue-homing properties of cells that transiently appear in the circulating blood. Through cell separation and invitro stimulation, the HIV-1 "Gag and Envelope reactivation co-detection assay" (GERDA) enables sensitive detection of Gag+/Env+ protein-expressing cells down to about one cell per million using flow cytometry. By associating GERDA with proviral DNA and polyA-RNA transcripts, we corroborate the presence and functionality of HIV-1 in critical body compartments utilizing t-distributed stochastic neighbor embedding (tSNE) and density-based spatial clustering of applications with noise (DBSCAN) clustering with low viral activity in circulating cells early after diagnosis. We demonstrate transcriptional HIV-1 reactivation at any time, potentially giving rise to intact, infectious particles. With single-cell level resolution, GERDA attributes virus production to lymph-node-homing cells with central memory Tcells (TCMs) as main players, critical for HIV-1 reservoir eradication.

Abstract CT274: Individualized APC targeting VB10.NEO cancer vaccines induce broad neoepitope-specific CD8 T cell responses in patients with advanced or metastatic solid tumors: interim results from a phase 1/2a trial

Abstract Background: VB N-01 is an open label phase 1/2a basket trial to evaluate safety, feasibility, and immunogenicity of a therapeutic DNA cancer vaccine VB10.NEO in patients with locally advanced or metastatic solid cancers. Each VB10.NEO vaccine contains up to 20 neoepitopes selected by the proprietary AI platform NeoSELECT and is designed to target antigen presenting cells using Nykode’ s modular vaccine platform known as Vaccibody™. Patients and Methods: The trial enrolled patients with locally advanced or metastatic solid cancers (renal cell carcinoma, urothelial cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, and melanoma), who did not obtain complete responses on immune checkpoint inhibitor therapy (CPI). Up to 14 VB10.NEO doses (3 mg per dose) were administered i.m. by PharmaJet Stratis® for up to 1 year in combination with standard of care (CPI and/or other anti-cancer therapies). Blood samples and tumor biopsies were collected at baseline and during treatment for evaluation of immune responses. Results: At cut-off of May 2022, 41 patients had received at least one vaccination with VB10.NEO. The vaccine was safe and well-tolerated with no new or additional toxicity reported beyond that expected for CPIs alone. All patients displayed immune responses to a minimum of 3 neoepitopes (average 53% of vaccine neoepitopes), assessed by in vitro stimulation (IVS) interferon-gamma ELISpot. T cell responses were elicited in both high and low tumor mutational burden patients (TMB range 2-69mut/Mb). Multiple vaccinations increased the breadth and magnitude of the immune responses with vaccine-induced T cell responses (de novo and/or amplified pre-existing) measured in 95% of eligible patients. IVS intracellular cytokine staining of selected patients demonstrated that the majority of neoepitopes induced polyfunctional CD8 T cells. TCR sequencing of baseline and on-treatment samples (6 patients) showed expansion of T cell clones in both blood and tumor with selected clones being expanded in both compartments. Conclusions: VB10.NEO was generally well tolerated in patients with various pre-treated and advanced cancers. Assessment of neoepitope-specific T cell reactivity demonstrated VB10.NEO-induced broad and long-lasting T cell responses, and the majority of tested neoepitopes activated polyfunctional CD8 T cells. Pre- and post-vaccination TCRseq analysis of blood and tumor samples demonstrated the presence of blood-expanded clones in the on-treatment tumor sample potentially indicating trafficking of VB10.NEO-expanded T cells to the tumor site. Citation Format: Jürgen Krauss, Angela Krakhardt, Stephan Eisenmann, Sebastian Ochsenreither, Kaja C.G Berg, Kushi Kushekhar, Lars-Egil Fallang, Mikkel W. Pedersen, Siri Torhaug, Karsten B. Slot, Karoline Schjetne. Individualized APC targeting VB10.NEO cancer vaccines induce broad neoepitope-specific CD8 T cell responses in patients with advanced or metastatic solid tumors: interim results from a phase 1/2a trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT274.

IL-12 and IL-27 Promote CD39 Expression on CD8+ T Cells and Differentially Regulate the CD39+CD8+ T Cell Phenotype.

Tumor-specific CD8+ T cells are critical components of antitumor immunity; however, factors that modulate their phenotype and function have not been completely elucidated. Cytokines IL-12 and IL-27 have recognized roles in promoting CD8+ T cells' effector function and mediated antitumor responses. Tumor-specific CD8+ tumor-infiltrating lymphocytes (TILs) can be identified based on surface expression of CD39, whereas bystander CD8+ TILs do not express this enzyme. It is currently unclear how and why tumor-specific CD8+ T cells uniquely express CD39. Given the important roles of IL-12 and IL-27 in promoting CD8+ T cell functionality, we investigated whether these cytokines could modulate CD39 expression on these cells. Using invitro stimulation assays, we identified that murine splenic CD8+ T cells differentially upregulate CD39 in the presence of IL-12 and IL-27. Subsequently, we assessed the exhaustion profile of IL-12- and IL-27-induced CD39+CD8+ T cells. Despite the greatest frequency of exhausted CD39+CD8+ T cells after activation with IL-12, as demonstrated by the coexpression of TIM-3+PD-1+LAG-3+ and reduced degranulation capacity, these cells retained the ability to produce IFN-γ. IL-27-induced CD39+CD8+ T cells expressed PD-1 but did not exhibit a terminally exhausted phenotype. IL-27 was able to attenuate IL-12-mediated inhibitory receptor expression on CD39+CD8+ T cells but did not rescue degranulation ability. Using an immunogenic neuro-2a mouse model, inhibiting IL-12 activity reduced CD39+CD8+ TIL frequency compared with controls without changing the overall CD8+ TIL frequency. These results provide insight into immune regulators of CD39 expression on CD8+ T cells and further highlight the differential impact of CD39-inducing factors on the phenotype and effector functions of CD8+ T cells.

Abstract 3576: Identification of T-cell receptors targeting RAS hotspot mutations using TIL IVS in human cancers for use in cell-based immunotherapy

Abstract Immunotherapies mediate the regression of human tumors through the recognition of tumor antigens by immune cells, which triggers an immune response. Mutations in the RAS oncogene occur across various cancer histologies and are found in about 30% of all cancer patients. These mutations play an essential role in tumor establishment and survival and are most commonly found in hotspots - specifically at amino acid positions 12, 13, and 61. We have recently treated a patient who underwent a durable objective response after receiving tumor-infiltrating lymphocytes (TILs) that recognized a KRAS G12D mutation. T cell receptors (TCR) that recognize shared mutated RAS antigens presented on major histocompatibility complex (MHC) class I and class II molecules are thus promising reagents for “off-the-shelf” adoptive cell therapies (ACT) following insertion of the TCR into lymphocytes. We recently published a paper describing the discovery of anti-mutated RAS reactivities by using screening TIL and by performing in-vitro stimulation (IVS) of peripheral blood lymphocytes (PBLs). Interestingly by those two methods, we sometimes found reactivity or TCR in a patient in one method but not with the other method. In this work, we described the discovery of anti-mutated RAS TCRs by TIL IVS, which combines the best of the two described methods: using TILs coming from the tumor and therefore assumed to have a higher proportion of anti-tumor-reactive cells and stimulate them, which could enrich smaller populations. The specific anti-mutated RAS CD4 and CD8 TCRs were isolated by coculture of the reactive T cells with dendritic cells (DC) loaded with peptides or transfected with mRNA encoding RAS mutated antigens. The reactive T cells were subjected to single-cell sequencing to identify TCRs. The TCRs were reconstructed as a viral plasmid and retrovirally transduced into PBLs. The transduced TCRs were tested for avidity, MHC restriction, and specific recognition of the mutation. In the same patients tested by both methods, by TIL IVS, identified 12 anti-mutated RAS TCRs in addition the 9 TCRs that we could find by TIL screening. Overall, we present here the discovery of multiple mutation-specific anti-RAS TCRs and its MHC class I and class II restriction elements involved in tumor recognition. Each of the newly discovered TCRs recognized mutated RAS with high avidity and specificity and low to no reactivity against wild-type RAS. Testing of the TCR transduced cells to recognize tumor cell lines in-vitro and in-vivo demonstrated tumor cell line recognition. The anti-RAS mutated TCRs we discovered by all of our methods can potentially be used as an “off the shelf” treatment for the evaluation of TCR-based ACT in 45-60% of patients were tumor bears RAS mutations. Citation Format: Noam Levin, Frank J. Lowery, Maria R. Parkhurst, Zhiya Yu, Nolan R. Vale, Sanghyun P. Kim, Steven A. Rosenberg. Identification of T-cell receptors targeting RAS hotspot mutations using TIL IVS in human cancers for use in cell-based immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3576.

Blockade of tumor necrosis factor superfamily members CD30 and OX40 abrogates disease activity in murine immune-mediated glomerulonephritis

Co-stimulation is a prerequisite for pathogenic activity in T cell-mediated diseases and has been demonstrated to achieve tolerance in organ-specific autoimmunity as a therapeutic target. Here, we evaluated the involvement of the tumor necrosis factor family members CD30 and OX40 in immune-complex mediated kidney disease. Invitro stimulation and proliferation studies were performed with CD4+ cells from wild type and CD30/OX40 double knock-out (CD30OX40-/-) mice. Invivo studies were performed by induction of nephrotoxic serum nephritis in wild type, CD30OX40- /- , CD30-/-, OX40-/-, reconstituted Rag1-/- and C57Bl/6J mice treated with αCD30L αOX40L antibodies. CD30, OX40 and their ligands were upregulated on various leukocytes in nephrotoxic serum nephritis. CD30OX40-/- mice, but not CD30-/- or OX40-/- mice were protected from nephrotoxic serum nephritis. Similar protection was found in Rag1-/- mice injected with CD4+ T cells from CD30OX40-/- mice compared to Rag1-/- mice injected with CD4+ T cells from wild type mice. Furthermore, CD4+ T cells deficient in CD30OX40-/- displayed decreased expression of CCR6 invivo. CD30OX40-/- cells were fully capable of differentiating into disease mediating T helper cell subsets, but showed significantly decreased levels of proliferation invivo and invitro compared to wild type cells. Blocking antibodies against CD30L and OX40L ameliorated nephrotoxic serum nephritis without affecting pan-effector or memory T cell populations. Thus, our results indicate disease promotion via CD30 and OX40 signaling due to facilitation of exaggerated T cell proliferation and migration of T helper 17 cells in nephrotoxic serum nephritis. Hence, co-stimulation blockade targeting the CD30 and OX40 signaling pathways may provide a novel therapeutic strategy in autoimmune kidney disease.

Antigen Experienced T Cells from Peripheral Blood Recognize p53 Neoantigens.

The purpose of this study was to evaluate antigen experienced T cells in peripheral blood lymphocytes (PBL) for responses to p53 neoantigens. PBLs from patients with a mutated TP53 tumor were sorted for antigen-experienced T cells and in vitro stimulation (IVS) was performed with p53 neoantigens. The IVS cultures were stimulated with antigen-presenting cells expressing p53 neoantigens, enriched for 41BB/OX40 and grown with rapid expansion protocol. T-cell responses were not observed in the PBLs of 4 patients who did not have tumor-infiltrating lymphocyte (TIL) responses to mutated TP53. In contrast, 5 patients with TIL responses to mutated TP53 also had similar T-cell responses in their PBLs, indicating that the PBLs and TILs were congruent in p53 neoantigen reactivity. CD4+ and CD8+ T cells were specific for p53R175H, p53Y220C, or p53R248W neoantigens, including a 78% reactive T-cell culture against p53R175H and HLA-A*02:01. Tracking TCRB clonotypes (clonality, top ranked, and TP53 mutation-specific) supported the enrichment of p53 neoantigen-reactive T cells from PBLs. The same T-cell receptor (TCR) from the TIL was found in the IVS cultures in three cases and multiple unique TCRs were found in another patient. TP53 mutation-specific T cells also recognized tumor cell lines bearing the appropriate human leukocyte antigen restriction element and TP53 mutation, indicating these T cells could recognize processed and presented p53 neoantigens. PBL was a noninvasive source of T cells targeting TP53 mutations for cell therapy and can provide a window into intratumoral p53 neoantigen immune responses.See related commentary by Olivera et al., p. 1203.

1178PD - A phase I trial of GEN-009, a neoantigen vaccine using ATLAS™, an autologous immune assay, to identify immunogenic and inhibitory tumour mutations

1178PD - A phase I trial of GEN-009, a neoantigen vaccine using ATLAS™, an autologous immune assay, to identify immunogenic and inhibitory tumour mutations

Glucocorticoid Receptor-α and MKP-1 as Candidate Biomarkers for Treatment Response and Disease Activity in Vogt-Koyanagi-Harada Disease

To investigate the potential of utilizing the expression of genes for glucocorticoid receptor (GR) and mitogen-activated protein kinase phosphatase-1 (MKP-1) as biomarkers of corticosteroid (CS) refractoriness and disease activity in patients with Vogt-Koyanagi-Harada (VKH) disease. Prospective cohort study. Twenty VKH patients receiving their first cycle of CS treatment in the absence of additional systemic immunosuppressive therapy and a control group of fifteen healthy volunteers were recruited from the University of Chile (Santiago, Chile) and US National Institutes of Health (Bethesda, United States). Intraocular inflammation was clinically quantified at enrolment and all follow-up visits. CS refractoriness was defined as an ocular reactivation of VKH upon CS withdrawal at a daily oral prednisone dose of 10mg or more. Quantitative Reverse transcription polymerase chain reaction (qRT-PCR) was performed to measure the mRNA levels of the alpha (α) and beta (β) isoforms of GR and MKP-1 in peripheral blood mononuclear cells (PBMC) after invitro stimulation with either anti-CD3/anti-CD28 antibodies, lipopolysaccharide (LPS), or phytohemagglutinin (PHA), in the presence or absence of dexamethasone (Dex). After 6 hours of stimulation in the presence of Dex, PBMC from CS-refractory VKH patients had an impaired elevation in GRα expression (P= .03). Furthermore, inactive patients showed a significant Dex-induced upregulation of MKP-1 (P= .005). In this pilot study, the expression of GR isoforms and MKP-1 corresponded with patients' clinical response to systemic CS treatment and disease activity, respectively. Hence, these candidate biomarkers have potential clinical utility in the early identification of CS refractoriness and subclinical inflammation in patients with VKH disease.

Abstract 4059: Identification of pre-existing neoantigen-specific T cells in patients receiving checkpoint inhibitor therapy using a deep learning antigen prediction model

Abstract Immune Checkpoint Blockade (ICB) has revolutionized the treatment of patients with cancer but provides durable clinical benefit to only a minority of patients. Neoantigens, antigens derived from tumor mutations, are emerging as key targets of T cells in patients with clinical responses to ICB whose tumors exhibit high mutational burden. T cells specific to some neoantigens appear to be naturally generated in these patients and can be further expanded upon ICB. Accurate neoantigen identification may fuel therapeutic advances by driving potent anti-neoantigen immune responses through therapeutic immunization or adoptive cell therapy. However, identification of neoantigens and neoantigen-reactive T cells remains a significant challenge. Current methods are time consuming and labor intensive and require non-routine clinical specimen collections. Here, we apply deep learning to a large HLA peptide and genomic dataset from NSCLC patients to create a computational model of minimal epitope presentation for neoantigen prediction. We demonstrate that our model allows rapid identification of neoantigens and neoantigen reactive T cells using routine clinical biopsies and blood specimens. This methodology does not require HLA-specific reagents and enables identification of T cell responses for every patient. To identify neoantigen-specific T cells, selected peptides, prioritized by our prediction model, were synthesized as minimal epitopes and added to short-term in vitro stimulation (IVS) cultures to expand neoantigen-reactive T cells. The presence of antigen-specific T cells was assessed two weeks later using IFN-gamma (IFN-g) ELISpot against pools of the prioritized neoantigens. Patient-specific peptide pools and IFN-g ELISpot enabled identification of neoantigen-reactive T cells in 5/9 (56%) patients on ICB. Deconvolution yielded an average of 2 recognized neoantigens per patient, with increased responses over time observed in one patient where multiple time points were available. Neoantigen responses were confirmed in repeat IVS cultures where additional samples were available. Importantly, expansion of HLA-matched healthy controls in the presence of patient peptide pools did not result in any responses by IFN-g ELISpot. In addition, antigen-reactive T cells were sorted for single cell sequencing on the 10x platform using CD137 as an HLA-independent T-cell activation marker. In summary, pre-existing neoantigen specific T cells and TCR clones can be rapidly identified utilizing routine clinical specimens, through (i) a powerful neoantigen prediction model, (ii) short-term in vitro T-cell expansion followed by IFN-gamma ELISpot, and (iii) HLA-independent selection and single cell sequencing. This methodology may allow rapid design of neoantigen-targeting vaccines or selection of TCRs for cell therapeutic approaches. Citation Format: Christine D. Palmer, Brendan Bulik, Aaron Yang, Meghan Hart, Matthew Davis, Joshua Francis, Fujiko Duke, Rita Zhou, Jennifer Busby, Tyler Murphy, Andrew Clark, Lauren Young, Mike Zhong, Kamilah Caldwell, Jesse Abhyankar, Alison Cook, Tommy Boucher, Raphaël Rousseau, Cynthia Voong, Naiyer Rizvi, Mark Frattini, Roman Yelensky, Karin Jooss, Mojca Skoberne. Identification of pre-existing neoantigen-specific T cells in patients receiving checkpoint inhibitor therapy using a deep learning antigen prediction model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4059.

A multipeptide vaccine plus toll-like receptor agonists LPS or polyICLC in combination with incomplete Freund\u2019s adjuvant in melanoma patients

BackgroundCancer vaccines require adjuvants to induce effective immune responses; however, there is no consensus on optimal adjuvants. We hypothesized that toll-like receptor (TLR)3 agonist polyICLC or TLR4 agonist lipopolysaccharide (LPS), combined with CD4 T cell activation, would support strong and durable CD8+ T cell responses, whereas addition of an incomplete Freund’s adjuvant (IFA) would reduce magnitude and persistence of immune responses.Patients and methodsParticipants with resected stage IIB-IV melanoma received a vaccine comprised of 12 melanoma peptides restricted by Class I MHC (12MP), plus a tetanus helper peptide (Tet). Participants were randomly assigned 2:1 to cohort 1 (LPS dose-escalation) or cohort 2 (polyICLC). Each cohort included 3 subgroups (a-c), receiving 12MP + Tet + TLR agonist without IFA (0), or with IFA in vaccine one (V1), or all six vaccines (V6). Toxicities were recorded (CTCAE v4). T cell responses were measured with IFNγ ELIspot assay ex vivo or after one in vitro stimulation (IVS).ResultsFifty-three eligible patients were enrolled, of which fifty-one were treated. Treatment-related dose-limiting toxicities (DLTs) were observed in 0/33 patients in cohort 1 and in 2/18 patients in cohort 2 (11%). CD8 T cell responses to 12MP were detected ex vivo in cohort 1 (42%) and in cohort 2 (56%) and in 18, 50, and 72% for subgroups V0, V1, and V6, respectively. T cell responses to melanoma peptides were more durable and of highest magnitude for IFA V6.ConclusionsLPS and polyICLC are safe and effective vaccine adjuvants when combined with IFA. Contrary to the central hypothesis, IFA enhanced T cell responses to peptide vaccines when added to TLR agonists. Future studies will aim to understand mechanisms underlying the favorable effects with IFA.Trial registrationThe clinical trial Mel58 was performed with IRB (#15781) and FDA approval and is registered with Clinicaltrials.gov on April 25, 2012 (NCT01585350). Patients provided written informed consent to participate. Enrollment started on June 24, 2012.

Siglec-7 on peripheral blood eosinophils: Surface expression and function.

Siglec-7 is an inhibitory receptor (IR) expressed on human blood eosinophils. Whereas activation of other IRs, including Siglec-8 and CD300a, has been shown to downregulate eosinophil function, little is known about the role of Siglec-7 on human eosinophils. To examine Siglec-7 expression and function in eosinophils from normal (ND) and eosinophilic (EO) donors. Eosinophil expression of Siglec-7 was quantified by flow cytometry and quantitative PCR. Soluble Siglec-7 (sSiglec-7) levels were measured by ELISA in serum. The effect of Siglec-7 on eosinophil viability and degranulation was assessed invitro by AnnexinV-FITC/7-AAD staining and by measuring GM-CSF-induced mediator release in culture supernatants. Signal transduction was studied by Western blot. Siglec-7 was expressed exvivo on blood eosinophils from all eosinophilic and normal individuals studied. Siglec-7 surface, but not SIGLEC-7mRNA expression, was correlated with absolute eosinophil count (AEC). Siglec-7 was upregulated on purified eosinophils after invitro stimulation with GM-CSF or IL-5. Serum sSiglec-7 was detectable in 133/144 subjects tested and correlated with AEC. Siglec-7 cross-linking inhibited GM-CSF-induced release of eosinophil peroxidase, TNF-α, and IL-8 (n=7-8) but did not promote eosinophil apoptosis (n=5). Finally, Siglec-7 cross-linking on GM-CSF-activated eosinophils induced phosphorylation of SHP-1 and de-phosphorylation of ERK1/2 and p38. Siglec-7 is constitutively expressed on human eosinophils and downmodulates eosinophil activation. Targeting of Siglec-7 on eosinophils might enhance treatment efficacy in eosinophil-driven disorders. Conversely, therapeutic interventions that inhibit Siglec-7 could have unanticipated consequences and promote eosinophilic inflammation.

Gene expression profiles of CD4/CD8 double-positive T cells in porcine peripheral blood.

A characteristic subset of T cells, known as double positive T cells (DPTC) and expressing both cluster of differentiation 4 (CD4) and CD8, is observed in porcine peripheral blood. Previous studies suggested that DPTC might be memory cells. However, detailed phenotypes and functions of DPTC are yet to be fully elucidated and thus, the relatedness of DPTC with memory phenotypes remains unclear. In this study, DPTC gene expression profiles in peripheral blood were analyzed by DNA microarray in Experiment 1 and compared with those of CD4 single positive T cells (4SPTC) and CD8 single positive T cells (8SPTC). Expressions of IFNG, CCL5, NCK2, CCR2 and ITGB1 were higher than that of 4SPTC and 8SPTC. In contrast, expressions of CCR7 and SELL were lower than that of 4SPTC and 8SPTC. These results suggested that DPTC were either effector T cells or effector memory T cells (TEM ). Next, to determine whether DPTC were effector T cells or TEM , differences in the response of DPTC and 8SPTC against immunized/primed antigens were compared (Experiment 2). While DPTC showed quick elevation of IL2 and CD25 gene expressions against invitro stimulation of primed/immunized antigens, 8SPTC did not. These results suggest that at least some DPTC likely belong to TEM .

Alternative Th17 and CD4+ CD25+ FoxP3+ cell frequencies increase and correlate with worse cardiac function in Chagas cardiomyopathy.

Immune homeostasis has been suggested to play an important role in the clinical evolution of chronic Chagas disease; however, the immunopathologic factors involved have not been fully elucidated. Therefore, our study aimed to analyse the frequency of CD4+ CD25+ FoxP3+ cells, classic Th17 cells, alternative Th17 cells and IL-17+ B cells from peripheral blood of chronic cardiac patients after invitro stimulation with Trypanosoma cruzi soluble EPI antigen. Patients were selected and classified according to clinical evaluation of cardiac involvement: mild, B1 (CARD1) (n=20) and severe, C (CARD2) (n=11). Patients with the indeterminate form of CD were included as the control group A (IND) (n=17). Blood samples were collected and cultured in the presence of EPI antigen. Cells frequency and median fluorescence intensity (MFI) were obtained by flow cytometry. Our results showed that only CD4+ CD25+ FoxP3+ , CD4+ CD25high FoxP3+ , CD4+ IL-17+ IFN-γ- and CD4+ IL-17+ IFN-γ+ cells are more frequent in patients with severe cardiac disease and correlate with worse global cardiac function. However, while indeterminate patients demonstrated a positive correlation between CD4+ CD25+ FoxP3+ and CD4+ IL-17+ IFN-γ- Th17 cells, this relationship was not observed in cardiac patients. IL-17 expression by Th17 cells and B cells correlated with disease progression. Altogether our results suggest that the clinical progression of Chagas cardiomyopathy involves worsening of inflammation and impairment of immunoregulatory mechanisms.

Bone marrow type 2 innate lymphoid cells: a local source of interleukin-5 in interleukin-33-driven eosinophilia.

T helper type 2 (Th2) cells, type 2 innate lymphoid cells (ILC2s) and eosinophil progenitors have previously been described to produce interleukin-5 (IL-5) in the airways upon allergen provocation or by direct administration of IL-33. Eosinophilic airway inflammation is known to be associated with IL-5-dependent eosinophil development in the bone marrow, however, the source of IL-5 remains unclear. T helper cells, ILC2s and CD34+ progenitors have been proposed to be involved in this process, therefore, we investigated whether these cells are taking part in eosinophilopoiesis by producing IL-5 locally in the bone marrow in IL-33-driven inflammation. Airway exposure with IL-33 led to eosinophil infiltration in airways and elevated eotaxin-2/CCL24. Importantly, IL-5 production as well as expression of the IL-33 receptor increased in ILC2s in the bone marrow under this treatment. A small but significant induction of IL-5 was also found in CD34+ progenitors but not in T helper cells. Similar results were obtained by invitro stimulation with IL-33 where ILC2s rapidly produced large amounts of IL-5, which coincided with the induction of eosinophil hematopoiesis. IL-33-mediated eosinophil production was indeed dependent on IL-5 as both airway and bone marrow eosinophils decreased in mice treated with anti-IL-5 in combination with IL-33. Interestingly, the responsiveness of ILC2s to IL-33 as well as IL-33-induced eotaxin-2/CCL24 were independent of the levels of IL-5. In summary, we demonstrate for the first time that IL-33 acts directly on bone marrow ILC2s, making them an early source of IL-5 and part of a process that is central in IL-33-driven eosinophilia.

Plasma cell deficiency in human subjects with heterozygous mutations in Sec61 translocon alpha 1 subunit (SEC61A1)

Primary antibody deficiencies (PADs) are the most frequent primary immunodeficiencies in human subjects. The genetic causes of PADs are largely unknown. Sec61 translocon alpha 1 subunit (SEC61A1) is the major subunit of the Sec61 complex, which is the main polypeptide-conducting channel in the endoplasmic reticulum membrane. SEC61A1 is a target gene of spliced X-box binding protein 1 and strongly induced during plasma cell (PC) differentiation. We identified a novel genetic defect and studied its pathologic mechanism in 11 patients from 2 unrelated families with PADs. Whole-exome and targeted sequencing were conducted to identify novel genetic mutations. Functional studies were carried out exvivo in primary cells of patients and invitro in different cell lines to assess the effect of SEC61A1 mutations on B-cell differentiation and survival. We investigated 2 families with patients with hypogammaglobulinemia, severe recurrent respiratory tract infections, and normal peripheral B- and T-cell subpopulations. On invitro stimulation, B cells showed an intrinsic deficiency to develop into PCs. Genetic analysis and targeted sequencing identified novel heterozygous missense (c.254T>A, p.V85D) and nonsense (c.1325G>T, p.E381*) mutations in SEC61A1, segregating with the disease phenotype. SEC61A1-V85D was deficient in cotranslational protein translocation, and it disturbed the cellular calcium homeostasis in HeLa cells. Moreover, SEC61A1-V85D triggered the terminal unfolded protein response in multiple myeloma cell lines. We describe a monogenic defect leading to a specific PC deficiency in human subjects, expanding ourknowledge about the pathogenesis of antibody deficiencies.

Electrical preconditioning of stem cells with a conductive polymer scaffold enhances stroke recovery.

Exogenous human neural progenitor cells (hNPCs) are promising stroke therapeutics, but optimal delivery conditions and exact recovery mechanisms remain elusive. To further elucidate repair processes and improve stroke outcomes, we developed an electrically conductive, polymer scaffold for hNPC delivery. Electrical stimulation of hNPCs alters their transcriptome including changes to the VEGF-A pathway and genes involved in cell survival, inflammatory response, and synaptic remodeling. In our experiments, exogenous hNPCs were electrically stimulated (electrically preconditioned) via the scaffold 1 day prior to implantation. After invitro stimulation, hNPCs on the scaffold are transplanted intracranially in a distal middle cerebral artery occlusion rat model. Electrically preconditioned hNPCs improved functional outcomes compared to unstimulated hNPCs or hNPCs where VEGF-A was blocked during invitro electrical preconditioning. The ability to manipulate hNPCs via a conductive scaffold creates a new approach to optimize stem cell-based therapy and determine which factors (such as VEGF-A) are essential for stroke recovery.

Sialylated Fetuin-A as a candidate predictive biomarker for successful grass pollen allergen immunotherapy

Eligibility to immunotherapy is based on the determination of IgE reactivity to a specific allergen by means of skin prick or invitro testing. Biomarkers predicting the likelihood of clinical improvement during immunotherapy would significantly improve patient selection. Proteins were differentially assessed by using 2-dimensional differential gel electrophoresis and label-free mass spectrometry in pretreatment sera obtained from clinical responders and nonresponders within a cohort of 82 patients with grass pollen allergy receiving sublingual immunotherapy or placebo. Functional studies of Fetuin-A (FetA) were conducted by using gene silencing in a mouse asthma model, human dendritic cell invitro stimulation assays, and surface plasmon resonance. Analysis by using quantitative proteomics of pretreatment sera from patients with grass pollen allergy reveals that high levels of O-glycosylated sialylated FetA isoforms are found in patients exhibiting a strong decrease in rhinoconjunctivitis symptoms after sublingual immunotherapy. Although FetA is involved in numerous inflammatory conditions, its potential role in allergy is unknown. Invivo silencing of the FETUA gene in BALB/c mice results in a dramatic upregulation of airway hyperresponsiveness, lung resistance, and TH2 responses after allergic sensitization to ovalbumin. Both sialylated and nonsialytated FetA bind to LPS, but only the former synergizes with LPS and grass pollen or mite allergens to enhance the Toll-like receptor 4-mediated proallergic properties of human dendritic cells. As a reflection of the patient's inflammatory status, pretreatment levels of sialylated FetA in the blood are indicative of the likelihood of clinical responses during grasspollen immunotherapy.