scholarly journals Immune Profiling of Responses to Influenza Vaccination in Patients with Myeloproliferative Neoplasms

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3631-3631
Author(s):  
Joan How ◽  
Petra Bachanova ◽  
Maia Pavlovic ◽  
Patrick M. Reeves ◽  
Gabriela Hobbs
Keyword(s):  
T Cell ◽  
Influenza Vaccination ◽  
Peripheral Blood ◽  
Research Funding ◽  
Immune Cell ◽  
Myeloproliferative Neoplasms ◽  
Tissue Bank ◽  
Healthy Controls ◽  
Cell Responses ◽  
Healthy Donors

Abstract Myeloproliferative neoplasms (MPNs) are clonal stem cell neoplasms that include polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). MPN patients are immunocompromised due to immune dysregulation from clonal hematopoiesis, heightened inflammation, and immunosuppressive treatments. Infection is a leading cause of morbidity and mortality in MPNs, and as a result annual influenza vaccination is a critical component of care. However, efficacy of the influenza vaccine in MPN patients is unclear. This study sought to identify differences between MPN patients and healthy donors in their immune response to influenza vaccination and subsequent in vitro stimulation. The Massachusetts General Hospital (MGH) maintains a tissue bank of peripheral blood and bone marrow samples collected in consented patients with hematologic malignancy, including MPNs. The tissue bank was interrogated for MPN patients with peripheral blood samples banked 1-6 months after yearly trivalent/quadrivalent influenza vaccination. A total of 13 MPN patients were included, with a median age of 70 (32-86). Five patients had MF, 3 patients had PV, 4 patients had ET, and 1 patient had pre-fibrotic MF. Five patients were on treatment with ruxolitinib, 4 patients were on hydroxyurea (HU), and 4 patients were without treatment. Peripheral blood mononuclear cells (PBMCs) from 11 healthy donors 3-6 months post-vaccination, with a median age of 50 (28-56), were sourced commercially as controls. Cryopreserved samples were thawed and PBMC samples divided equally prior to incubation for 18 hours with peptides derived from Influenza A or sham. Subsequently samples were labeled with a 41-antibody panel and analyzed by mass cytometry. Manual gating together with FlowSOM clustering followed by EdgeR using T-test and FDR to correct for multiple comparisons were used to identify differentially abundant immune cell clusters between cohorts and treatment conditions. Without stimulation, MPN patients were decreased in B cell and memory CD4+ T cell populations as compared to healthy donors (p<0.05), in agreement with a previous report. MPN patients also exhibited an increase in a subset of mature B cells (p<0.05) and lineage negative CD123+ cells (p<0.05) as compared to controls. Stimulation included in this study aimed to expand on these distinctions and identified that baseline differences were retained with additional increases in CD4+ and CD8+ T cell responses in healthy donors as compared to MPN patients. Conversely, stimulation increased the frequency of inflammatory monocytic cells in MPN patients compared to healthy controls (p<0.05). In healthy donors stimulation induced several alterations in, most notably an increase in CD4+ effector memory cells (p<0.03), as compared to matched unstimulated controls. In contrast, comparison of pre- and post-stimulation within the MPN cohort revealed no significant differences in immune cell populations apart from a decrease in naïve CD4+ T cells (p<0.05). Patients receiving HU were enriched for naïve CD4+ and effector CD8+ T cells as compared to patients receiving ruxolitinib (p<0.05); however, these differences were diminished following stimulation. Within MPN patients, pre-stimulation samples in MF patients were characterized by increased abundance of activated monocyte/macrophage clusters and a lineage-negative CD123+ population; there were no significant differences in post-stimulation samples between MF and ET/PV/pre-fibrotic MF patients. We evaluated immunologic responses to influenza vaccination, with and without peptide stimulation, in MPN patients and compared to healthy controls. Overall, MPN patients demonstrated less robust B- and T-cell responses and a muted response to stimulation as compared to healthy controls, which may indicate reduced cellular responses following influenza vaccination. Ongoing analysis will interrogate specific immune populations and subsets in detail to refine current observations and will evaluate associations between immunologic cell clusters with serologic responses and additional patient characteristics. The results of our study highlight important alterations in the immune response to influenza vaccination and muted response to stimulation in MPN patients and will inform a future prospective study of vaccine responsiveness and immune dysfunction in patients with MPNs. Figure 1 Figure 1. Disclosures Reeves: PR Fludigm: Honoraria; Immunoscape: Consultancy. Hobbs: Merck: Research Funding; Incyte Corporation: Research Funding; AbbVie.: Consultancy; Novartis: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; Constellation Pharmaceuticals: Consultancy, Research Funding; Bayer: Research Funding.

scholarly journals Antibody and T-Cell Responses to COVID-19 Vaccination in Myeloproliferative Neoplasm Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 316-316
Author(s):  
Joan How ◽  
Kathleen M.E. Gallagher ◽  
Yiwen Liu ◽  
Ashley DeMato ◽  
Katelin Katsis ◽  
...  
Keyword(s):  
T Cell ◽  
Whole Blood ◽  
Stock Options ◽  
Research Funding ◽  
T Cell Responses ◽  
Spike Protein ◽  
Healthy Controls ◽  
Whole Blood Assay ◽  
Post Vaccination ◽  
Cell Responses

Abstract The efficacy of COVID-19 vaccines in cancer populations remain unknown. Myeloproliferative neoplasms (MPNs), including chronic myeloid leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) remain a vulnerable patient population and are immunocompromised due to impaired innate and adaptive immunity, heightened inflammation, and effects of ongoing treatment. We evaluate antibody and T-cell responses in MPN patients following completion of the BNT162b2 (Pfizer/BioNTech) and mRNA-1273 (Moderna) COVID-19 vaccine series. Patients with a known diagnosis of MPN presenting at Massachusetts General Hospital and eligible for COVID-19 vaccination were recruited. All participants gave informed consent and the study protocol was approved by the Institutional Review Board. 33 MPN patients were enrolled and 23 patients completed vaccination. Baseline and post-vaccination peripheral blood samples were collected and peripheral blood mononuclear cells (PBMCs) isolated. 26 vaccinated participants with no history of malignancy were included as healthy controls (PMID 33972942). Baseline characteristics are tabled below. Qualitative ELISA for human IgG/A/M against SARS-CoV-2 spike protein using donor serum was performed per manufacturer instructions. Seroconversion occurred in 22/23 (96%) of MPN patients and 25/26 (96%) of healthy controls (Figure). To measure SARS-CoV-2 T-cell immunity, an IFNγ ELISpot assay previously developed in convalescent and vaccinated healthy individuals was used. Freshly isolated PBMCs from patients were stimulated with commercially available overlapping 15mer peptide pools spanning the SARS-CoV-2 spike and nucleocapsid proteins. Given its size, the spike protein was split into two pools (Spike A or B). IFNγ-producing T-cells were quantified by counting the median spot forming units (SFU) per 2.5x10 5 PBMCs from duplicate wells. A positive threshold was defined as >6 SFUs per 2.5x10 5 PBMCs to either Spike A or B after subtraction of background, based on prior receiver operator curve (ROC) analysis of ELISpot responses (sensitivity 90% specificity 92%). Post-vaccination ELISpot responses occurred in 21/23 (91%) of MPN patients and 26/26 (100%) of healthy controls (p=0.99) (Figure). The median SFU to total spike protein (Spike A+B) increased after vaccination in both MPN patients (0 to 38, p=0.02) and healthy controls (6 to 134, p=0.002). MPN patients had significantly lower median SFU's on post-vaccination ELISpot compared to healthy controls (38 vs 134, p=0.044), although this was not significant after adjusting for age in multivariable logistic regression. MF patients had the lowest seroconversion and ELISpot response rates, and lowest median post-vaccination SFUs, although this was not significant. There were no other differences in post-vaccination SFUs with regards to gender, vaccine type, number of days post-vaccine, treatment, and absolute lymphocyte count. Whole-blood assay based on the in vitro diagnostic QuantiFERON TB Gold Plus assay was also used to assess T-cell response. Heparinized whole blood from donors was stimulated with S1 and S2 subdomains for the SARS-CoV-2 spike protein, with measurement of IFNγ released into plasma with the QuantiFERON ELISA. IFNγ release of >0.3 IU/mL was considered a positive threshold, based on prior ROC analysis (sensitivity and specificity 100%). MPN patients had significantly lower IFNγ response rates compared to healthy controls (57% versus 100%, p=0.003) (Figure). Our findings demonstrate robust antibody and T-cell responses to BNT162b2 and mRNA-1273 vaccination in MPN patients, with >90% serologic and ELISpot responder rates. We detected subtle differences in T-cell responses in MPN patients compared to healthy controls. MPN patients had lower median post-vaccination ELISpot SFUs and lower rates of T-cell responses on IFNγ-whole blood assay compared to healthy controls. As the whole blood assay uses whole protein antigen rather than peptide pools, differences from ELISpot testing may reflect deficiencies in antigen processing and presentation. It is unclear whether these subtle differences translate into less clinical protection from COVID-19, or to what extent our results are confounded by the older age of MPN patients. Further evaluation of B and T-cell responses to COVID-19 vaccination in a larger sample size of MPN patients is warranted. Figure 1 Figure 1. Disclosures Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Other: Stock ownership. Maus: Atara: Consultancy; Bayer: Consultancy; BMS: Consultancy; Cabaletta Bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; In8bio (SAB): Consultancy; Intellia: Consultancy; GSK: Consultancy; Kite Pharma: Consultancy, Research Funding; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Novartis: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; WindMIL: Consultancy; AstraZeneca: Consultancy; Agenus: Consultancy; Arcellx: Consultancy; Astellas: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company. Hobbs: AbbVie.: Consultancy; Incyte Corporation: Research Funding; Novartis: Consultancy; Bayer: Research Funding; Merck: Research Funding; Constellation Pharmaceuticals: Consultancy, Research Funding; Celgene/Bristol Myers Squibb: Consultancy.

scholarly journals Comprehensive Analysis of Cell Population Dynamics and Related Core Genes During Vitiligo Development

2021 ◽  
Vol 12 ◽  
Author(s):  
Jingzhan Zhang ◽  
Shirong Yu ◽  
Wen Hu ◽  
Man Wang ◽  
Dilinuer Abudoureyimu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Immune Cell ◽  
Cd8 T Cell ◽  
Cd4 T Cell ◽  
Healthy Controls ◽  
Cell Abundance

Vitiligo is a common immune-related depigmentation condition, and its pathogenesis remains unclear. This study used a combination of bioinformatics methods and expression analysis techniques to explore the relationship between immune cell infiltration and gene expression in vitiligo. Previously reported gene expression microarray data from the skin (GSE53146 and GSE75819) and peripheral blood (GSE80009 and GSE90880) of vitiligo patients and healthy controls was used in the analysis. R software was used to filter the differentially expressed genes (DEGs) in each dataset, and the KOBAS 2.0 server was used to perform functional enrichment analysis. Compared with healthy controls, the upregulated genes in skin lesions and peripheral blood leukocytes of vitiligo patents were highly enriched in immune response pathways and inflammatory response signaling pathways. Immunedeconv software and the EPIC method were used to analyze the expression levels of marker genes to obtain the immune cell population in the samples. In the lesional skin of vitiligo patients, the proportions of macrophages, B cells and NK cells were increased compared with healthy controls. In the peripheral blood of vitiligo patients, CD8+ T cells and macrophages were significantly increased. A coexpression analysis of the cell populations and DEGs showed that differentially expressed immune and inflammation response genes had a strong positive correlation with macrophages. The TLR4 receptor pathway, interferon gamma-mediated signaling pathway and lipopolysaccharide-related pathway were positively correlated with CD4+ T cells. Regarding immune response-related genes, the overexpression of IFITM2, TNFSF10, GZMA, ADAMDEC1, NCF2, ADAR, SIGLEC16, and WIPF2 were related to macrophage abundance, while the overexpression of ICOS, GPR183, RGS1, ILF2 and CD28 were related to CD4+ T cell abundance. GZMA and CXCL10 expression were associated with CD8+ T cell abundance. Regarding inflammatory response-related genes, the overexpression of CEBPB, ADAM8, CXCR3, and TNIP3 promoted macrophage infiltration. Only ADORA1 expression was associated with CD4+ T cell infiltration. ADAM8 and CXCL10 expression were associated with CD8+ T cell abundance. The overexpression of CCL18, CXCL10, FOS, NLRC4, LY96, HCK, MYD88, and KLRG1, which are related to inflammation and immune responses, were associated with macrophage abundance. We also found that immune cells infiltration in vitiligo was associated with antigen presentation-related genes expression. The genes and pathways identified in this study may point to new directions for vitiligo treatment.

scholarly journals High Immunogenicity to Influenza Vaccination in Crohn’s Disease Patients Treated with Ustekinumab

Vaccines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 455
Author(s):  
Laura Doornekamp ◽  
Rogier L. Goetgebuer ◽  
Katharina S. Schmitz ◽  
Marco Goeijenbier ◽  
C. Janneke van der Woude ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
T Cell ◽  
Crohn's Disease ◽  
Immune Responses ◽  
Influenza Vaccination ◽  
Seasonal Influenza ◽  
T Cell Responses ◽  
Healthy Controls ◽  
Post Vaccination ◽  
Cell Responses

Influenza vaccination can be less effective in patients treated with immunosuppressive therapy. However, little is known about the effects of ustekinumab; an anti-IL-12/23 agent used to treat Crohn’s disease (CD), on vaccination response. In this prospective study, we assessed immune responses to seasonal influenza vaccination in CD patients treated with ustekinumab compared to CD patients treated with anti-TNFα therapy (adalimumab) and healthy controls. Humoral responses were assessed with hemagglutinin inhibition (HI) assays. Influenza-specific total CD3+, CD3+CD4+, and CD3+CD8+ T-cell responses were measured with flow cytometry. Fifteen patients treated with ustekinumab; 12 with adalimumab and 20 healthy controls were vaccinated for seasonal influenza in September 2018. Seroprotection rates against all vaccine strains in the ustekinumab group were high and comparable to healthy controls. Seroconversion rates were comparable, and for A/H3N2 highest in the ustekinumab group. HI titers were significantly higher in the ustekinumab group and healthy controls than in the adalimumab group for the B/Victoria strain. Post-vaccination T-cell responses in the ustekinumab group were similar to healthy controls. One-month post-vaccination proliferation of CD3+CD8+ T-cells was highest in the ustekinumab group. In conclusion, ustekinumab does not impair immune responses to inactivated influenza vaccination. Therefore, CD patients treated with ustekinumab can be effectively vaccinated for seasonal influenza.

scholarly journals Using peripheral blood immune signatures to stratify patients with adult and juvenile inflammatory myopathies

Rheumatology ◽  
2019 ◽  
Author(s):  
Meredyth G Ll Wilkinson ◽  
Anna Radziszewska ◽  
Chris Wincup ◽  
Yiannis Ioannou ◽  
David A Isenberg ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Disease Activity ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Assessment Tool ◽  
Immune Cell ◽  
Memory B Cells ◽  
Healthy Controls ◽  
Immune Signatures

AbstractObjectiveThe inflammatory idiopathic myopathies (IIM) are a group of rare autoimmune diseases defined by muscle weakness and characterized by pro-inflammatory infiltrates in muscle. Little is known about the immunological profile in peripheral blood of these patients and how this relates to IIM subtypes. This study aimed to stratify adult and juvenile-onset IIM patients according to immune cell profile.MethodsPeripheral blood mononuclear cells from 44 patients with adult myositis (AM), 15 adolescent-onset juvenile dermatomyositis (a-JDM), and 40 age-matched healthy controls were analysed by flow cytometry to quantify 33 immune cell subsets. Adult myositis patients were grouped according to myositis subtype; DM and polymyositis; and also autoantibody specificity. Disease activity was determined by the myositis disease activity assessment tool and clinicians’ decision on treatment.ResultsUnique immune signatures were identified for DM, polymyositis and a-JDM compared with healthy controls. DM patients had a T-cell signature comprising increased CD4+ and TH17 cell frequencies and increased immune cell expression of IL-6. Polymyositis patients had a B-cell signature with reduced memory B cells. A-JDM had decreased naïve B cells and increased CD4+T cells. All patient groups had decreased CD8+central memory T-cell frequencies. The distinct immune signatures were also seen when adult myositis patients were stratified according to auto-antibody expression; patients with anti-synthetase-antibodies had reduced memory B cells and patients with autoimmune rheumatic disease overlap had an elevated Th17 profile.ConclusionUnique immune signatures were associated with adult vs juvenile disease. The Th17 signature in DM patients supports the potential use of IL-17 inhibitors in treatment of IIMs.

Regulatory B Cell Defects In Patients with Immune Thrombocytopenia (ITP)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 379-379 ◽  
Author(s):  
Xiaojuan Li ◽  
Hui Zhong ◽  
Weili Bao ◽  
Nayla Boulad ◽  
James B. Bussel ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
T Cell Responses ◽  
Healthy Controls ◽  
Advisory Committees ◽  
Cell Responses ◽  
Regulatory Capacity

Abstract Abstract 379 B lymphocytes participate in immune responses through production of antibodies, antigen presentation to T cells, and cytokine secretion. In ITP, B cells that produce platelet-specific autoantibodies play a major role in the pathogenesis of disease. Recent data from mouse models of autoimmune and inflammatory diseases suggest that B cells have regulatory functions mediated by the production of regulatory cytokines such as IL-10 and/or through inhibitory interactions with effector T cells. In humans, CD19+CD24hiCD38hi B cells, originally identified as immature transitional B cells, were recently shown to possess regulatory capacity mediated in part by IL-10. Given that ITP pathogenesis is in part related to defective T helper functions and since Bregs are important for controlling CD4+ T cell responses, we initiated studies to characterize the CD19+CD24hiCD38hi B cell compartment in ITP patients. Phenotypic analysis in patients with ITP showed statistically significant increases in the frequency of CD19+CD24hiCD38hi B cells (13.2± 1.5% versus 7.7±0.6%, p=0.0015) as well as in the percentage of CD19+CD24hiCD38int mature B cells (72.0± 1.7% versus 62.3±1.6%, p=0.0003). In contrast there was a pronounced decrease in CD19+CD24hiCD38− memory B cells (9.3± 2% versus 26.2±2%, p<0.0001 as confirmed by expression of CD27 memory marker) in patients (n=23) compared to healthy controls (n=23). The suppressive capacity of human CD19+CD24hiCD38hi Bregs involves CD80. We found a statistically significant decrease in the overall frequency of CD80 expressing CD19+ B cells in ITP patients compared to healthy controls (12.40± 1.3% versus 21.8±2.1%, p=0.002), indicating that despite the increase in the Breg frequency, their regulatory capacity may be impaired. To determine the functional activity of the Bregs, PBMCs from 4 patients with ITP were depleted of CD19+CD24hiCD38hi B cells by cell sorting and levels of cytokine production in the CD4+ T cell population following stimulation with plate-bound anti-CD3 for 72h was evaluated. As has been reported previously, we observed a >30% increase in the frequency of CD4+IFN-g+ in CD19+CD24hiCD38hi B cell-depleted compared with non-depleted PBMCs from healthy controls. However, depletion of CD19+CD24hiCD38hi B cells from PBMCs of ITP patients did not alter CD4+IFN-g+ production in 3 /4 patients, indicating a possible defect in the suppressive activity of Bregs in those 3 patients. IL-10 secretion following CD40 engagement in human Bregs is pivotal in mediating their suppressive activity. Given that platelets express CD40L, we tested the ability of Bregs to respond to platelets. We found two-fold lower IL-10 production in CD19+CD24hiCD38hi Bregs (1.9±2.1%, versus 5.0± 1.0%, p<0.05), but not in CD19+CD24hiCD38− B cells (p=0.9) from patients (n=3, different from above) compared to controls (n=5) when PBMCs were stimulated with acid-treated platelets (to remove HLA molecules) from normal controls, indicating a defect in IL-10 production in the Breg population in patients with ITP. Altogether, our in vitro studies of circulating Bregs suggest that these cells may be functionally compromised in some patients with ITP patients as indicated by reduced IL-10 production and indirectly by Breg depletion studies showing inability to dampen effector T cell responses. Given the important role of Bregs in controlling CD4+T responses, the data implicates defective Bregs as an additional mechanism to explain increased T cell responses in patients with ITP. The impaired Breg activity may also explain the variability in response to treatment with anti-CD20 in patients with ITP. It may be that B cell depletion therapy in patients who have defective Breg activity will result in the removal of primarily pathogenic B cells and therefore these patients will have a good response to rituximab treatment. However, in patients with intact Bregs, the same treatment will deplete both pathogenic and regulatory B cells, causing a less effective response. Ongoing studies to test patients' platelets as well as CD40-specific Breg responsiveness are currently underway to further explore the role of Bregs in the pathogenesis of ITP. Disclosures: Bussel: Portola: Consultancy; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai, Inc.: Membership on an entity's Board of Directors or advisory committees; Cangene: Research Funding; Genzyme: Research Funding.

scholarly journals CTIM-08. COMBINATION OF THE IMA950/POLY-ICLC MULTIPEPTIDE VACCINE WITH PEMBROLIZUMAB IN RELAPSING GLIOBLASTOMA PATIENTS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii34-ii34
Author(s):  
Valérie Dutoit ◽  
Eliana Marinari ◽  
Pierre-Yves Dietrich ◽  
Denis Migliorini
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
Immune Modulation ◽  
Immune Cell ◽  
Peptide Vaccine ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Blood Analysis ◽  
Tumor Site ◽  
Cell Responses

Abstract The IMA950 peptide vaccine is composed of 9 HLA-A2-restricted peptides eluted from the surface of GBM samples and of two HLA class II-binding peptides1. It was tested in combination with poly-ICLC in patients with newly diagnosed GBM, demonstrating safety. The vaccine was able to elicit CD4 and CD8 T cell responses in the peripheral blood in the majority of patients, with however an overall low magnitude of T cell responses and suboptimal migration of elicited T cells to the brain, probably limiting clinical efficacy2,3. With the aim to improve homing of vaccine-specific T cell to the tumor, we are conducting a phase II clinical trial in patients with recurrent GBM testing the IMA950/poly-ICLC multipeptide vaccine with or without the anti-PD1 antibody pembrolizumab (NCT03665545). 24 patients will be included (12 patients in each arm) and pre- and post-vaccination tumor sample will be available, allowing assessing effect of the vaccine at the tumor site. The primary objective of this trial is safety of IMA950/poly-ICLC given together with pembrolizumab. Secondary objectives include (i) estimation of 6, 9 and 12-month progression-free survival (PFS), (ii) overall survival, (iii) analysis of patient quality of life and (iv) of the synergy/immunogenicity of IMA950/poly-ICLC and pembrolizumab. Immunomonitoring will include measure of vaccine-induced immune responses, IHC for immune cell markers, RNA/TCR sequencing and methylome analysis, to assess vaccine-induced T cell responses, immune modulation and potential signatures predictive of response. Thus far, 6 patients have been included (3 in each arm). Preliminary results show CD4 and CD8 T cell responses to the vaccine are detected in both groups in the peripheral blood. Analysis at the tumor site and comparison between arms will be performed once all patients have been included. 1. Dutoit, V. et al. Brain (2012); 2. Migliorini, D. et al. Neuro Oncol (2019); 3. Rampling, R. et al. Clin Cancer Res(2016)

scholarly journals Somatic Mutations in CD8+ T Cells in Patients with Chronic Immune Thrombocytopenia Are Associated with Increased Clonality and Cytotoxic Phenotype of CD8+ T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 131-131
Author(s):  
Hanna Rajala ◽  
Paula Savola ◽  
Sofie Lundgren ◽  
Samuli Eldfors ◽  
Tiina Kelkka ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Deep Sequencing ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Somatic Mutations ◽  
Healthy Controls ◽  
Variant Allele Frequency

Abstract Background The role of self-antigen-targeting T cells is well established in multiple autoimmune disorders. In immune thrombocytopenia (ITP), CD8+ cytotoxic T cells target both peripheral blood platelets and bone marrow megakaryocytes. In addition, CD4+ helper T cell imbalance and cytokine secretion leads to the release of autoantibodies by B cells favoring the destruction of platelets. In order to understand if somatic mutations play a role and drive the aberrant immune responses in ITP, we analyzed sorted CD4+ and CD8+ T cells with deep sequencing panel. Methods The study population consisted of 13 adult patients diagnosed with chronic ITP (median age 47 years) at least one year before the first sampling. The median number of lines of therapy was 4, (range 0-8). In addition, 11 healthy control samples were collected for T cell phenotyping and TCR analysis (median age 63 years). After separation of peripheral blood mononuclear cells, immunophenotyping by flow cytometry was performed for CD4+ and CD8+ T cells and both fractions were sorted using magnetic beads. The CD4+ and CD8+ clonality was analyzed by deep sequencing of TCRB CDR3 using Adaptive platform. Custom-made deep sequencing gene panel covering 2433 genes related to immunity and cancer was used to analyze somatic mutations from 11 ITP patients. Mean target coverage was 567X (range 405-757X). Somatic variants with over 2% variant allele frequency (VAF) were called using Varscan2-based bioinformatics tool and CD4+ and CD8+ fractions were used as each other's germline control. Candidate somatic variants were visually validated with Integrative Genomics Viewer. Results ITP patients harbored clonal TCR rearrangements both in CD8+ and CD4+ fractions. The median percentage of the largest T cell clone of all TCR rearrangements in an individual sample was 9.5% (range 1.8%-31.9%) in CD8+ cells and 7.1% (range 0.9-13.6%) in CD4+. The median clone size or clonality index did not differ between ITP patients and healthy controls. Age correlated with the largest rearrangement and clonality index both in CD4+ and CD8+ fractions in ITP patients (r=0.70, p=0.015 and r=0.60, p=0.043 for CD4+ and r=0.63, p=0.033 and r=0.77, p=0.0050 for CD8+), but no correlation was observed in healthy controls. The size of the maximum CD8+ TCR rearrangement and clonality index correlated positively with the percentage of highly cytotoxic CD8+CD57+ (r=0.80, p=0.0029 and r=0.90, p=0.0002) and terminally differentiated CD8+ effector memory with CD45RA (TEMRA) T cells (r=0.66, p=0.022 and r=0.78, p=0.0038). In healthy controls positive correlation was observed only between CD8+ clonality index and CD8+CD57+ T cells, but not with TEMRAs. Somatic mutations in CD8+ T cells were detected in 7/11 (64%) of ITP patients. Somatic variants were distinct between individuals and their mean variant allele frequency (VAF) was 4.5% (range 2.0-13.6%). The mutated genes included those related to IFNg-signaling pathway (STAT1, NLRP4, CARD6, and EBF1), apoptosis (CASP6), and inflammation and cancer (ATM, EGR1, NOD1). ITP patients with CD8+ mutations had larger immunodominant CD8+ TCR clone (Figure 1A) and higher CD8+ clonality index (Figure 1B). The patients with CD8+ mutations had more cytotoxic CD8+CD57+ T cells than mutation-negative cases, but CD8+TEMRAs did not differ between the two groups (Figure 1C). The mutation profile of the CD4+ samples was different: 3/11 (27%) of the ITP patients had mutations in the genes connected to clonal hematopoiesis of indeterminate potential (CHIP). One patient with a maximum of 8% clone in CD4+ T cells harbored mutations both in TET2 (VAF 13.5%) and PPM1D (VAF 12.8%): the VAFs suggest that the mutation was not restricted to the largest CD4+ clone. The second patient had a different mutation in PPM1D gene (VAF 2.8%) and the third one had a PPM1B-mutation both in CD4+ (VAF 18.0%) and CD8+ (VAF 11.1%) T cells. Conclusions To our knowledge, this is the first report of somatic variants in T cells in patients with ITP. Discovered somatic mutations in CD8+ T cells were associated with increased clonality and highly cytotoxic phenotype of CD8+ T cells. Interestingly, three patients harbored mutations in CHIP-associated genes in CD4+ and also in CD8+ fraction, with recurrent PPM1D-mutations. Further studies and screening of a larger ITP patient cohort is warranted to understand the meaning and functional consequence of somatic mutations in the pathogenesis of chronic ITP. Disclosures Ebeling: Boehringer Ingelheim: Consultancy; Celgene: Speakers Bureau; Otsuka Pharma Scandinavia AB: Consultancy. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Research Funding; Celgene: Honoraria; Pfizer: Honoraria, Research Funding.

scholarly journals Evidence for Epitope-Specific T Cell Responses in HIV-Associated Non Neoplastic Lymphadenopathy: High-Throughput Immunogenetic Evidence

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1117-1117
Author(s):  
Katerina Gemenetzi ◽  
Evangelia Stalika ◽  
Andreas Agathangelidis ◽  
Fotis Psomopoulos ◽  
Elisavet Vlachonikola ◽  
...  
Keyword(s):  
Lymph Node ◽  
T Cell ◽  
Research Funding ◽  
T Cell Responses ◽  
Gene Repertoire ◽  
Healthy Controls ◽  
Lymphoid Follicles ◽  
Cell Responses ◽  
P24 Protein ◽  
Trbv Gene

Abstract Non-neoplastic lymphadenopathy (NNL) associated with the human immunodeficiency virus (HIV) infection may develop concurrently with the onset of HIV viremia (acute retroviral syndrome) that can persist beyond the acute phase. Histopathological findings at this early phase mainly pertain to hyperplastic changes with large lymphoid follicles; with time, the number of lymphoid follicles diminishes, while plasma cells increase; at the extreme is a pattern characterized by sclerosis of the germinal centers in the residual follicles. HIV-specific CD8+ T cell responses have been reported and certain viral protein epitopes have been identified e.g. the p24 protein, a component of the HIV particle capsid. Overall, these findings reflect an ongoing immune response that is still incompletely characterized at the molecular level, particularly as it concerns the composition of the T cell receptor (TR) gene repertoire. In order to obtain a comprehensive view into the role of antigen selection in shaping T cell responses in HIV-associated NNL [HIV(+) NNL], we studied in-depth the TR repertoire in: (i) lymph node biopsy samples from 12 patients with HIV(+) NNL, (ii) lymph node samples from 5 non-HIV patients with reactive lymphadenopathy [HIV(-) RL]; and, (iii) peripheral blood samples from 4 healthy, HIV-seronegative individuals without lymphadenopathy [healthy controls, HIV(-) HC]. Genomic DNA was isolated from either paraffin-embedded lymph nodes (for patients with lymphadenopathy) or blood mononuclear cells (for healthy individuals). TRBV-TRBD-TRBJ gene rearrangements were amplified according to the BIOMED2 protocol. PCR products were subjected to next generation sequencing (NGS) on the MiSeq Illumina Platform. NGS data analysis, interpretation and visualization was performed by a validated, in-house bioinformatics pipeline. Overall, we obtained: (i) 1,440,305 (mean: 120,025) productive rearrangement sequences in the HIV(+) NNL group; (ii) 702,533 (mean: 140,506) productive sequences in the HIV(-) RL group; and, (iii) 539,981 (mean: 134,995) productive sequences in HIV(-) HC cases. Rearrangements with identical TRBV gene usage and CDR3 sequence were defined as clonotypes. In total, we identified 15,553 unique clonotypes in patients with HIV(+) NNL (mean: 1,296, range: 337-6,212), 53,874 in HIV(-) RL (mean: 10,774, range: 3,336-16,304) and 220,069 clonotypes in HIV(-) HC cases (mean; 55,017, range: 35,430-68,916), indicating significant repertoire restriction in the former group. Indeed, this group was characterized by an increased level of oligoclonality compared to the other two groups: the mean values of the sum of relative frequencies for the 10 most frequent clonotypes were 80%, 19.6% and 16.5%, respectively. Seven of 12 HIV(+) NNL cases carried the same dominant clonotype (TRBV29-1, SVDPSGTGGEGYT) that was also found in the remaining 5 patients of this group, albeit at lower frequencies; in contrast, it was completely absent in the HIV(-) RL and HIV(-) HC groups. Regarding the TRBV gene repertoire, the TRBV29-1 gene was overrepresented (p<0.005) in the HIV(+) NNL group, whereas the TRBV6-5 and TRBV19 genes were frequent in both groups of patients with lymphadenopathy (HIV-associated or not); finally, the TRBV5-1 was underrepresented (p<0.005) in patients with lymphadenopathy (HIV-associated or not) compared to HIV(-) HC cases. Comparison of the present TR gene sequence dataset against public databases identified 2 clonotypes with an established reactivity against the p24 protein that were present in 2 different patients with HIV(+) NNL of the present cohort. In conclusion, the TR gene repertoire of patients with HIV(+) NNL displays increased level of clonality, distinct TRBV gene repertoire as well as a widely shared, specific dominant clonotype compared to HIV(-) RL cases or HIV(-) healthy controls. These findings allude to an antigen-driven, HIV-specific immune process, a claim also supported by the detection of clonotypes with established anti-HIVp24 reactivity in at least a fraction of the analyzed patients. Disclosures Gemenetzi: Gilead: Research Funding. Agathangelidis:Gilead: Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Hadzidimitriou:Gilead: Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Research Funding.

scholarly journals Mass Cytometry Reveals Heterogeneity in the Exhaustion Profile of T-Cells in Chronic Lymphocytic Leukemia and the CD4:CD8 Ratio May be a Reliable Predictor of CD8+ T Cell Compartment "Fitness"

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 353-353 ◽  
Author(s):  
Muharrem Muftuoglu ◽  
Li Li ◽  
Han Chen ◽  
Duncan Mak ◽  
Elif Gokdemir ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Healthy Controls ◽  
Inhibitory Receptors ◽  
Cell Exhaustion ◽  
Healthy Donors ◽  
Checkpoint Receptors ◽  
Inhibitory Molecules

Abstract T cell exhaustion is characterized by coordinated expression of a series of negative checkpoint receptors such as programmed death-1 (PD-1), 2B4, CD160 and TIGIT, resulting in T cell dysfunction and immune evasion. Under physiological states, these inhibitory molecules maintain self-tolerance and prevent autoimmunity by applying a break on cytotoxic T cells. In cancer, T-cells exhibit features of T-cell exhaustion including increased expression of PD-1, 2B4 and CD160, coupled with reduced T cell proliferation, altered synapse formation and impaired cytotoxicity. Although the role of the PD-1/PD-L1 axis in mediating T cell defects in chronic lymphocytic leukemia (CLL) is well-studied, the contribution of other checkpoint molecules such as 2B4, CD160 and TIGIT in mediating tumor-induced immune dysfunction remains to be determined. Checkpoint inhibitors have provided a paradigm-shifting approach to cancer treatment. We hypothesized that the expression levels of checkpoint receptors on T-cells, as well as the "fitness" of the T cell compartment may provide a prognostic stratification system to predict response to checkpoint inhibitors in CLL. To determine if the number of inhibitory receptors per cell and their expression level may identify patient-to-patient differences that may not be easily deciphered using conventional research tools, we performed a detailed single-cell analysis of the T-cell repertoire, using 40-parameter mass cytometry (CyTOF) in 12 untreated CLL and 12 healthy controls. Consistent with previous reports, we found that expression of 2B4 (43.7% vs 30.8%), PD1 (28.8% vs 21%) and CD160 (17% vs. 9.7%) was significantly higher on CLL CD8+ T cells compared to healthy controls. In addition, CD8+T cells in CLL expressed higher levels of TIGIT (48.2% vs 25.2%), CD57 (43.9% vs 17.9%) and KLRG1 (49.5% vs. 29.7%). We clearly distinguished 2 patterns of exhaustion marker distribution in CLL. In one group of patients, the expression of checkpoint receptors was similar to that seen in healthy controls, whereas in the second group, CD8+ T-cells expressed higher levels of PD1, 2B4, TIGIT, CD160 as well as markers of terminal differentiation such as CD57 and KLRG1. Compared to healthy donors, CLL was characterized by an inversion in the CD4:CD8 ratio. Interestingly, CD8+ T cells in patients with a low CD4:CD8 ratio (defined as <2.5) expressed significantly higher levels of 2B4 (56.6% vs 31.25%), TIGIT (62.9% vs 37%), CD160 (22.8% vs 12.6%), CD57 (57% vs 28.7), PD-1 (34.6% vs 24.5%) and KLRG1 (62.3% vs 36.3%). In contrast, the expression levels of PD-1, 2B4 and CD160 in CLL patients with a CD4:CD8 ratio of ≥2.5 were similar to that seen in healthy controls, suggesting that the CD4:CD8 ratio may be a valuable marker of T cell exhaustion in CLL. Next, we compared the number of checkpoint molecules expressed per CD8+ T-cells in CLL patients versus healthy donors. Whereas a similar proportion of CD8+ T-cells in CLL (mean 19.56%, range 18.34-31.73%) and healthy donors (mean 22.13%, 14.17-41.19%) expressed one inhibitory receptor, a significantly higher proportion of CLL patients expressed 2 and more inhibitory receptors (mean 28.4, range 10.52-48.78%) compared to healthy controls (mean 15.38%, range 9.67-21.94%). PD-1 was mostly co-expressed with TIGIT, although TIGIT+PD-1+CD4+ and CD8+ T-cells were higher in CLL compared to healthy controls (12.9% vs 7.1%). Interestingly the predominant population of PD-1+CD8+ T cells in CLL was also positive for 2B4 and TIGIT, whereas expression of TIGIT was more diverse and was seen in association with PD-1, 2B4, KLRG1 or CD57. Taken together, our findings indicate a remarkable heterogeneity in the expression patterns of inhibitory molecules on CD8+ and CD4+ T-cells in CLL. While CLL patients with a normal CD4:CD8 ratio expressed comparable levels of inhibitory molecules to that seen in healthy controls, a low CD4:CD8 ratio was indicative of higher expression of checkpoint molecules. On a per cell basis, CLL CD8+ T cells expressed more inhibitory receptors compared to healthy controls, suggesting that certain patients may benefit from combinational use of checkpoint molecules. A more detailed data and analysis, including transcription and functional profile of exhausted CLL T cells, will be presented in the meeting. Disclosures Wierda: Abbvie: Research Funding; Novartis: Research Funding; Acerta: Research Funding; Gilead: Research Funding; Genentech: Research Funding. Jain:Incyte: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Abbvie: Research Funding; Infinity: Research Funding; BMS: Research Funding; Genentech: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Seattle Genetics: Research Funding; Celgene: Research Funding; Servier: Consultancy, Honoraria; Novimmune: Consultancy, Honoraria.

758 Identification of tumor antigen-specific T cells in the peripheral blood of colorectal cancer patients

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A806-A806
Author(s):  
Emilie Picard ◽  
Alexandrine Martel ◽  
Alain Simard ◽  
Hoang-Thanh Le ◽  
Chris Verschoor ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Healthy Controls ◽  
Ifn Gamma ◽  
Cell Responses ◽  
Healthy Control

BackgroundInteractions between the immune system and the tumor are now recognized as key determinants of clinical outcome in colorectal cancer (CRC). Immune landscapes have been extensively studied within resected primary tumors and immune markers, such as T cells, have been found to be associated with CRC patients‘ survival. Little is known about the immune profile of cells in peripheral blood. We hypothesize that the functional status of T cells, characterized by their response to CRC tumor-associated antigens (TAAs), can be monitored in the peripheral blood of patients and that they have prognostic relevance in CRC.MethodsIn vitro T cell responses to pools of overlapping peptides representing the TAAs MUC-1, hTERT, NY-ESO-1 and CEA were assessed by analyzing IFN-gamma and TNF-alpha production by CD8+ T cells using flow cytometry, in 5 stage II-III CRC patients just prior to surgical resection and 3 healthy age- and sex-matched controls.ResultsT cells responding to MUC-1, hTERT, NY-ESO-1 and CEA were present in 3, 3, 1 and 5 CRC patients, respectively, whereas only one response to TAAs (MUC-1) was found in one healthy control (figure 1). When TAA responses were pooled together, 83.3% of responders were patients (n=5) and 100% of non-responders were healthy controls (n=2).Abstract 758 Figure 1CRC TAA-specific CD8 T cell responses in CRC patieDot plot of one representative healthy control (#3) and one representative patient (#2) showing IFN-γ production by CD8 T cells in response to (A) MUC-1, hTERT, NY-ESO-1 and CEA stimulations or without any restimulation (negative control) and (B) positive controls (CEF and PMA/ionomycin). Percentages of IFN-gamma+ CD8 T cells are displayed in each plot. C Frequency of MUC-1-, hTERT-, NY-ESO-1-, CEA- and pooled TAA-specific CD8 T cell responses in patients (grey) and healthy controls (black). Fisher exact test, * p=0.0179; ns=not significant.ConclusionsThe presence of circulating T cells responding to CEA in all 5 patients, but also to MUC-1 and hTERT in 3 patients suggests that these TAAs may be good targets for immunotherapy in CRC. Our findings also provide a rationale to investigate the prognostic value of CEA-, MUC-1- and hTERT-specific T cells in the peripheral blood of CRC patients and to consider vaccination with these antigens to boost or induce responses to control residual tumor post-surgery.Ethics ApprovalThis study was approved by Health Sciences North’s Research Ethics Board; approval number 18- 104.

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