scholarly journals Breast cancer-induced immune suppression in the sentinel lymph node is effectively countered by CpG-B in conjunction with inhibition of the JAK2/STAT3 pathway

2020 ◽  
Vol 8 (2) ◽  
pp. e000761 ◽  
Author(s):  
Kim M van Pul ◽  
Ronald J C L M Vuylsteke ◽  
Monique T A de Beijer ◽  
Rieneke van de Ven ◽  
M Petrousjka van den Tol ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Lymph Node ◽  
T Cell ◽  
Sentinel Lymph Node ◽  
Immune Suppression ◽  
Ex Vivo ◽  
T Cell Response ◽  
Cell Reactivity ◽  
T Cell Reactivity ◽  
Stat3 Inhibition

BackgroundWe previously showed selectively hampered activation of lymph node-resident (LNR) dendritic cell (DC) subsets in the breast cancer (BrC) sentinel lymph node (SLN) to precede a state of profound T cell anergy. Reactivating these DC subsets by intratumoral delivery of the Toll-like receptor-9 (TLR9) agonist CpG-B could potentially offer a promising immune therapeutic strategy to combat this immune suppression and prevent disease spread. Unfortunately, CpG-B can limit its own immune stimulatory activity through direct TLR9-mediated activation of signal transducer and activator of transcription 3 (STAT3), pinpointed as a key regulator of immune suppression in the tumor microenvironment. Here, we have investigated whether in vitro exposure to CpG-B, with or without simultaneous inhibition of STAT3 signaling, could overcome immune suppression in BrC SLN.MethodsImmune modulatory effects of CpG-B (CPG7909) with or without the JAK2/STAT3 inhibitor (STAT3i) AG490 were assessed in ex vivo cultured BrC SLN-derived single-cell suspensions (N=29). Multiparameter flow cytometric analyses were conducted for DC and T cell subset characterization and assessment of (intracellular) cytokine profiles. T cell reactivity against the BrC-associated antigen Mammaglobin-A was determined by means of interferon-γ ELISPOT assay.ResultsAlthough CpG-B alone induced activation of all DC subsets, combined inhibition of the JAK2/STAT3 pathway resulted in superior DC maturation (ie, increased CD83 expression), with most profound activation and maturation of LNR DC subsets. Furthermore, combined CpG-B and JAK2/STAT3 inhibition promoted Th1 skewing by counterbalancing the CpG-induced Th2/regulatory T cell response and significantly enhanced Mammaglobin-A specific T cell reactivity.ConclusionEx vivo immune modulation of the SLN by CpG-B and simultaneous JAK2/STAT3 inhibition can effectively overcome BrC-induced immune suppression by preferential activation of LNR DC, ultimately restoring type 1-mediated antitumor immunity, thereby securing a BrC-specific T cell response. These findings provide a clear rationale for clinical exploration of SLN-immune potentiation through local CpG/STAT3i administration in patients with BrC.

scholarly journals P09.02 Mapping and tackling tumor and chemotherapy-induced immune suppression in breast cancer sentinel lymph nodes

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A52-A53
Author(s):  
N Prokopi ◽  
M Heeren ◽  
I Milenova ◽  
K van Pul ◽  
T Muijlwijk ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Lymph Node ◽  
T Cell ◽  
High Risk ◽  
Immune Suppression ◽  
Ex Vivo ◽  
T Cell Responses ◽  
List Type ◽  
Color Flow ◽  
Cell Responses

BackgroundBreast cancer (BrC) is the most prevalent cancer in women worldwide. Unfortunately, still limited treatment options are available for the most aggressive subtypes (i.e. hormone receptor [HR] negative). The response to neoadjuvant chemotherapy (NACT) in patients with HR-negative BrC can in part be influenced by an effective anti-tumor immune response. The sentinel lymph node (SLN) is the first site where BrC-specific T cell priming will occur but unfortunately it is also a major target of BrC-induced immune suppression. Lymph-node resident dendritic cells (LNR-DC) were found to be suppressed in metastatic SLN.1 In addition, this tumor-mediated immune suppression of LNR-DC is related to high-risk triple-negative BrC and may be a negative predictor for prognosis1. Preliminary data showed that NACT further reduced the activation status of LNR-DC. The goal of this study is to identify immune-enhancing agents that can counteract the tumor-mediated immune suppression of LNR-DC and promote tumor-specific T cell responses in order to improve therapy outcome in BrC patients upon NACT.Materials and MethodsPhenotypic analyses were performed on immune-cell subsets in human BrC SLN using multi-color flow cytometry. In addition, ex-vivo cultures with human BrC SLN-derived cells and in vivo mouse experiments were performed to study the therapeutic efficacy of Toll-like receptor (TLR)-ligands (R848 and CpG) and a STING-ligand (STING-L; 2’3’-c-di-AM(PS)2(Rp,Rp)).ResultsHigher rates of LNR-DCs, but with an apparently reduced activation state, were found in SLN of NACT-treated patients compared to patients treated with surgery only. A comparative ex-vivo study with SLN cultures on the effects of R848, CpG-B and STING-L showed R848 to be superior in terms of LNR-DC activation. In a Krt14 (K14)-cre;Cdh1F/F;Trp53F/F (KEP) BrC mouse model, the effects of intratumoral administration of TLR- and STING-L were determined in combination with doxorubicin. STING-L outperformed R848 and CpG-B in terms of controlling primary tumor growth. Of note, in human ex-vivo cultures CpG-B proved effective in LNR-DC activation when combined with a STAT3 inhibitor, leading to the boosting of mammaglobin-specific T cell responses, Th1 skewing, and a drop in CpG-induced Treg levels.ConclusionsIn summary, intratumoral delivery of TLR- and STING-ligands in combination with NACT might be an interesting therapeutic approach in patients with high-risk HR-negative BrC, leading to SLN potentiation and enhanced antitumor T cell immunity. Future clinical studies should demonstrate the therapeutic benefit of this approach.Referencevan Pul, et al. 2019, Journal for ImmunoTherapy of Cancer.Disclosure InformationN. Prokopi: None. M. Heeren: None. I. Milenova: None. K. van Pul: None. T. Muijlwijk: None. M. Arends: None. S. van der Velde: None. K. Vrijland: None. A. van Weverwijk: None. K. de Visser: None. R. van de Ven: None. T. de Gruijl: None.

Use of tumor exome analysis to reveal neo-antigen-specific T-cell reactivity in ipilimumab-responsive melanoma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 9085-9085 ◽  
Author(s):  
Nienke van Rooij ◽  
John B. A. G. Haanen ◽  
Marit van Buren ◽  
Daisy Philips ◽  
Mireille Toebes ◽  
...  
Keyword(s):  
T Cell ◽  
Human Cancer ◽  
T Cell Response ◽  
Patient Specific ◽  
Product Analysis ◽  
Cell Reactivity ◽  
Synonymous Mutations ◽  
Genome Data ◽  
T Cell Reactivity ◽  
Peptide Exchange

9085 Background: Evidence for T cell mediated regression of human cancer in particular melanoma following immunotherapy is strong. Anti-CTLA4 treatment has been approved for treatment of metastatic melanoma and blockade of PD-1 has shown encouraging results. However, it is unknown which T cell reactivities are involved in cancer regression. Reactivity against non-mutated tumor self-antigens has been analyzed in patients treated with Ipilimumab or with autologous TILs, but the size of these responses are modest. Therefore, T cell recognition of patient-specific mutant epitopes may be a potentially important component. Animal model data recently suggested that analysis of T cell reactivity against patient-specific neo-antigens may be feasible through exploitation of cancer genome data. However, human data have thus far been lacking. Methods: To address this we have used MHC class I peptide exchange technology allowing production of very large collections of pMHC complexes, together with a pMHC "combinatorial coding" strategy for parallel detection of dozens of different T cell populations within a single sample. Results: From a melanoma patient responding to ipilimumab treatment, we identified tumor specific mutations via exome sequencing of tumor material. The exome contained 1,075 non-synonymous mutations. Possible MHC epitopes covering these mutations were predicted based on; 1) predicted to bind the patient’s MHC; 2) predicted to be cleaved by the proteasome; 3) genes of which the mutated peptides arose had evidence of RNA expression. The analysis yielded 1,952 epitopes restricted to the HLA-A and HLA-B. To screen for T cell reactivity against these epitopes we used the pMHC combinatorial coding approach. We found T cell reactivity against 2 neo-antigens, including a dominant T cell response against a mutant epitope of the ATR gene product. Analysis of PBMC samples collected before and during Ipilimumab therapy showed that this particular response increased strongly after treatment from 0.06% to 0.28% of CD8 T cells after being stable in magnitude for 10 months. Conclusions: These data provide the first demonstration of cancer exome-guided analysis to dissect the effects of melanoma immunotherapy.

scholarly journals Profound CD8 T cell responses towards the SARS-CoV-2 ORF1ab in COVID-19 patients

2020 ◽  
Author(s):  
Anastasia Gangaev ◽  
Steven L. C. Ketelaars ◽  
Sanne Patiwael ◽  
Anna Dopler ◽  
Olga I. Isaeva ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Clinical Activity ◽  
Cell Reactivity ◽  
T Cell Recognition ◽  
Cell Responses ◽  
T Cell Reactivity ◽  
Very High

Abstract A large global effort is currently ongoing to develop vaccines against SARS-CoV-2, the causative agent of COVID-19. While there is accumulating evidence on the antibody response against SARS-CoV-2, little is known about the SARS-CoV-2 antigens that are targeted by CD8 T cells. To address this issue, we have analyzed samples from 20 COVID-19 patients for T cell recognition of 500 predicted MHC class I epitopes. CD8 T cell reactivity against SARS-CoV- 2 was common. Remarkably, a substantial fraction of the observed CD8 T cell responses were directed towards the ORF1ab polyprotein 1ab, and these CD8 T cell responses were frequently of a very high magnitude. The fact that a major part of the SARS-CoV-2 specific CD8 T cell response is directed against a part of the viral genome that is not included in the majority of vaccine candidates currently in development may potentially influence their clinical activity and toxicity profile.

scholarly journals Automatic Generation of Validated Specific Epitope Sets

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Sebastian Carrasco Pro ◽  
John Sidney ◽  
Sinu Paul ◽  
Cecilia Lindestam Arlehamn ◽  
Daniela Weiskopf ◽  
...  
Keyword(s):  
T Cell ◽  
Web Application ◽  
Ex Vivo ◽  
Automatic Generation ◽  
Cell Reactivity ◽  
Cell Responses ◽  
Novel Method ◽  
T Cell Reactivity ◽  
Categorization System ◽  
Immune Epitope Database

Accurate measurement of B and T cell responses is a valuable tool to study autoimmunity, allergies, immunity to pathogens, and host-pathogen interactions and assist in the design and evaluation of T cell vaccines and immunotherapies. In this context, it is desirable to elucidate a method to select validated reference sets of epitopes to allow detection of T and B cells. However, the ever-growing information contained in the Immune Epitope Database (IEDB) and the differences in quality and subjects studied between epitope assays make this task complicated. In this study, we develop a novel method to automatically select reference epitope sets according to a categorization system employed by the IEDB. From the sets generated, three epitope sets (EBV, mycobacteria and dengue) were experimentally validated by detection of T cell reactivityex vivofrom human donors. Furthermore, a web application that will potentially be implemented in the IEDB was created to allow users the capacity to generate customized epitope sets.

scholarly journals Redefining Criteria to Ensure Adequate Sentinel Lymph Node Biopsy With Dual Tracer for Breast Cancer

2020 ◽  
Vol 10 ◽  
Author(s):  
Li Xu ◽  
Jiqiao Yang ◽  
Zhenggui Du ◽  
Faqing Liang ◽  
Yanyan Xie ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Lymph Node ◽  
Sentinel Lymph Node ◽  
Sentinel Lymph Node Biopsy ◽  
Ex Vivo ◽  
Lymph Node Biopsy ◽  
Blue Staining ◽  
Blue Dye ◽  
Node Biopsy ◽  
Dual Tracer

BackgroundFor sentinel lymph node biopsy (SLNB) in patients with breast cancer, the dual tracer of blue dye and radioisotope with the 10% rule that all nodes with radioactive count of 10% or more of the hottest node ex vivo should be removed is widely accepted. However, the cut-off point of radioactivity is being questioned for possibly excessive removal of negative nodes.MethodsTo compare different percentile rules and optimize the criteria for identifying SLNs, we established a database which prospectively collected the radioactivity, status of blue dye and the pathological results of each SLN in breast cancer patients who successfully underwent SLNB with a combination of methylene blue and radioisotope.ResultsA total of 2,529 SLNs from 1,039 patients were identified from August 2010 to August 2019. 16.4% (414/2,529) positive nodes were removed at a cost of 83.6% (2115/2,529) negative nodes removed excessively. Up to 17.9% (375/2,115) negative nodes were removed as radioactively hot nodes without blue staining. By gradually increasing the threshold by each 10%, the number of negative nodes identified reduced by 18.2% (385/2,115) with only three node-positive patients (1.0%) missed to be identified using the “40% + blue” rule. In patients with ≥ 2 SLNs removed, 12.3% (238/1,942) negative nodes avoided unnecessary removal with only 0.8% (2/239) positive patients missed with the “hottest two + blue” rule.ConclusionsOur data indicated that the “40% + blue” rule or the “hottest two + blue” rule for SLNB with the dual tracer of blue dye and radioisotope may be considered as a potential alternative rule to minimize extra nodes resected. Nonetheless, it should be validated by prospective trials with long-term follow-up.

scholarly journals CD4+ CD25+ T Cells Regulate Vaccine-Generated Primary and Memory CD8+ T-Cell Responses against Herpes Simplex Virus Type 1

2004 ◽  
Vol 78 (23) ◽  
pp. 13082-13089 ◽  
Author(s):  
Felix N. Toka ◽  
Susmit Suvas ◽  
Barry T. Rouse
Keyword(s):  
T Cells ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
T Cell ◽  
Cell Response ◽  
Cell Activity ◽  
T Cell Response ◽  
Cell Reactivity ◽  
T Cell Reactivity ◽  
Simplex Virus

ABSTRACT It has become evident that naturally occurring CD25+ regulatory T cells (Treg cells) not only influence self-antigen specific immune response but also dampen foreign antigen specific immunity. This report extends our previous findings by demonstrating that immunity to certain herpes simplex virus (HSV) vaccines is significantly elevated and more effective if Treg cell response is curtailed during either primary or recall immunization. The data presented here show that removal of CD25+ Treg cells prior to SSIEFARL-CpG or gB-DNA immunization significantly enhanced the resultant CD8+ T-cell response to the immunodominant SSIEFARL peptide. The enhanced CD8+ T-cell reactivity in Treg cell-depleted animals was between two- and threefold and evident in both acute and memory stages. Interestingly, removal of CD25+ Treg cells during the memory recall response to plasmid immunization resulted in a twofold increase in CD8+ T-cell memory pool. Moreover, in the challenge experiments, memory CD8+ T cells generated with plasmid DNA in the absence of Treg cells cleared the virus more effectively compared with control groups. We conclude that CD25+ Treg cells quantitatively as well as qualitatively affect the memory CD8+ T-cell response generated by gB-DNA vaccination against HSV. However, it remains to be seen if all types of vaccines against HSV are similarly affected by CD25+ Treg cells and if it is possible to devise means of limiting Treg cell activity to enhance vaccine efficacy.

scholarly journals Development and Validation of a Bordetella pertussis Whole-Genome Screening Strategy

2020 ◽  
Vol 2020 ◽  
pp. 1-11 ◽  
Author(s):  
Ricardo da Silva Antunes ◽  
Lorenzo G. Quiambao ◽  
Aaron Sutherland ◽  
Ferran Soldevila ◽  
Sandeep Kumar Dhanda ◽  
...  
Keyword(s):  
T Cell ◽  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Ex Vivo ◽  
Screening Strategy ◽  
Cell Reactivity ◽  
Genome Screening ◽  
T Cell Reactivity ◽  
Development And Validation ◽  
Genomic Map

The immune response elicited by the protective whole-cell pertussis (wP) versus the less-protective acellular pertussis (aP) vaccine has been well characterized; however, important clinical problems remain unsolved, as the inability of the currently administered aP vaccine is resulting in the reemergence of clinical disease (i.e., whooping cough). Strong evidence has shown that original, childhood aP and wP priming vaccines provide a long-lasting imprint on the CD4+ T cells that impacts protective immunity. However, aP vaccination might prevent disease but not infection, which might also affect the breadth of responses to Bordetella pertussis (BP) antigens. Thus, characterizing and defining novel targets associated with T cell reactivity are of considerable interest. Here, we compare the T cell reactivity of original aP and wP priming for different antigens contained or not contained in the aP vaccine and define the basis of a full-scale genomic map of memory T cell reactivity to BP antigens in humans. Our data show that the original priming after birth with aP vaccines has higher T cell reactivity than originally expected against a variety of BP antigens and that the genome-wide mapping of BP using an ex vivo screening methodology is feasible, unbiased, and reproducible. This could provide invaluable knowledge towards the direction of a new and improved pertussis vaccine design.

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