HLA Ligandome Profiling Identifies a Novel Category of Pathophysiologically Relevant CLL Associated Antigens

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 715-715
Author(s):  
Daniel J. Kowalewski ◽  
Heiko Schuster ◽  
Claudia Berlin ◽  
Stefan Kahn ◽  
Linus Backert ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Conflicts Of Interest ◽  
Peptide Vaccine ◽  
Rational Approach ◽  
Tumor Control ◽  
Immune Recognition ◽  
Hla Ligands ◽  
Hla Ligandome

Abstract The breakthrough clinical success of immune checkpoint inhibition with PD-1 and CTLA-4 antibodies illustrates the potential of T cell based immunotherapy to effectively treat malignancies. However, a remaining major challenge is to increase the specificity and frequency of anti-cancer immune responses. A rational approach to achieve this goal is therapeutic vaccination, as highlighted by a recent phase II vaccination study using naturally presented HLA ligands, which induced vaccine-specific immune responses that were associated with improved clinical outcome (Walter et al., Nat. Med. 2012). This underscores the potential of identifying patho-physiologically relevant targets by direct differential analysis of the entire landscape of HLA-presented peptides, termed the HLA ligandome. Here we applied this approach to chronic lymphocytic leukemia (CLL) with the aim of developing a CLL-specific multi-peptide vaccine. Using cohorts of 30 patients and 30 healthy volunteers, we comparatively and extensively mapped the HLA ligandome landscape of CLL and identified more than 20,000 different HLA class I and II ligands representing 7,377 source proteins, attaining >95% of the maximum attainable coverage (as extrapolated from saturation analysis). A set of 49 HLA ligand source proteins (225 different HLA ligands) showed CLL-exclusive representation in >20% of CLL patients, thereby constituting the novel category of ligandome-derived tumor associated antigens (LiTAAs). These LiTAAs were validated to be broadly and frequently represented across different stages and mutational subtypes of CLL and found to be robustly represented in HLA ligandomes of patients undergoing standard chemo-/immunotherapy. Functional characterization by IFNγ-ELISPOT revealed peptide-specific immune recognition of 14/15 (93.3%) corresponding HLA ligands (LiTAPs) exclusively in CLL patients. These immune responses were strictly CLL (LiTAP-) directed and mediated by functional patient-derived CD8+ T cells. Notably, for immunoreactive LITAPs, a linear correlation between frequencies of representation in the CLL ligandomes and immune recognition by CLL patient PBMC was observed (R²=0.59), suggesting that LiTAP presentation on cancer cells is a direct prerequisite for the presence of anti-LiTAP immune responses. Moreover, we investigated the prognostic relevance of LiTAP-specific immune responses: Retrospective survival analysis of 45 CLL patients analyzed by IFNγ-ELISPOT assays comparing cases with T cell responses specific for 0-1 LiTAPs (n=32) versus >1 LiTAPs (n=13) suggests a prolonged overall survival in the multiple-response cohort as compared to the single/non-responders (P=0.0695, log-rank test, Fig. 1). Taken together these data provide clear evidence for tumor-dependent priming of LiTAP-specific T cells in CLL patients and indicate their involvement in tumor control in vivo. This directly implies these non-mutant self-peptides as pathophysiologically relevant tumor antigens in CLL thereby validating our target identification approach and encouraging future evaluation in clinical vaccination trials. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Ten Year Survival in Patients with Myeloma Is Characterized by the Persistence of Immunological Control Which Is Detected by Immunological Biomarkers

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1818-1818
Author(s):  
Ross D Brown ◽  
Hayley Suen ◽  
Christian E Bryant ◽  
Shihong Yang ◽  
James Favaloro ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Cytotoxic T Cells ◽  
Interferon Γ ◽  
Tumor Control ◽  
T Cell Clones ◽  
Tumour Control ◽  
Cell Clones ◽  
Split Anergy

Abstract Abstract 1818 There is clinical evidence for the presence of a limited degree of host-tumor control in patients with multiple myeloma (MM) but the exact mechanisms involved are not known. Patients who survive for more than ten years are likely to have the most active immunological host-tumour control and are an ideal cohort to study. We now add to our preliminary observations using a range of immunological biomarkers in the 29 of these patients who attend our clinic. 51% of MM patients (n=264) had expanded CD3+CD8+ TCRVβ+ CD57+T cell clones detected by TCRVβ analysis (Beckman, BetaMark). Clonality was confirmed by IgH CDR3 sequencing. These clones accounted for 14.3% (median) of the CD3 cells (range 4–49%). CFSE tracking demonstrated the anergic nature of these clonal T cells (median 6% proliferation) compared with other CD8 cells (70% proliferation) while Geneset analysis of mRNA microarrays (Affymetrix U133) identified that anergy was caused by upregulated RAS, CSK, TOB and suppressed ERK pathways. Unlike recent reports for T-LGL, microarray analysis suggested that there was no evidence of STAT3 upregulation in the MM T cell clones. Functional studies suggested that these non-proliferating clones have split anergy as interferon-γ production was normal. In contrast, all ten year survivors had expanded T cell clones and serial studies demonstrated a >8 year persistence of the same clone in 8 out of 12 patients studied. More importantly, unlike the other MM patients, the T cell clones in 19 out of 21 of the ten year survivors studied were not anergic. The Treg/Th17 ratio in the ten year survivors was significantly lower than other MM patients (median 1.9 vs 12.0; p<0.0002) and even lower than age matched normals (median 5.6; p<0.006). This difference in the ten year survivors was due to an increased absolute Th17 cell (p<0.005) number and a reduced absolute Tregnumber (p<0.05). MM patients had a reduced number of absolute 6-Sulfo-LacNAc (SLAN)-DCs, a major source of IL-12, compared to both age matched controls and long term survivors (p < 0.01). Allo SLAN-DCs stimulated a higher proliferative response by MM T cells than could be achieved with mononuclear preparations. In conclusion, MM patients have expanded clones of cytotoxic T cells that exhibit split anergy and these cells are potential candidates for restoring immunological control of MM and other cancers. The normalised Treg/Th17 ratio suggests that the induced tolerance associated with increased Treg cell numbers is also absent in these patients. Our observations that the ten year MM survivors do not have anergy in their clonal T cells and have less Treg suppression offers a unique cohort for future studies. Disclosures: No relevant conflicts of interest to declare.

HLA Class I Ligandome Analysis In Acute Myeloid Leukemia – Novel T-Cell Epitopes For Peptide-Based Immunotherapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5431-5431
Author(s):  
Stickel S. Juliane ◽  
Claudia Berlin ◽  
Daniel J. Kowalewski ◽  
Heiko Schuster ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
T Cell ◽  
Conflicts Of Interest ◽  
Peptide Vaccine ◽  
T Cell Responses ◽  
Hla Class I ◽  
Class I ◽  
Isolation And Identification ◽  
Cell Responses ◽  
Healthy Donors ◽  
Hla Ligands

Abstract Data regarding the graft-versus-leukemia (GVL) effect after allogeneic stem cell transplantation (SCT) and donor lymphocyte infusion strongly suggest that T lymphocytes play a major role in the rejection of leukemic cells. Immunotherapy directed against leukemia- associated antigens might elicit specific immune responses that may serve to eliminate minimal residual disease after chemotherapy, or enhance the GVL effect after SCT. To achieve this goal there is need to identify appropriate leukemia associated HLA ligands, which are able to induce specific T cell responses. We here aimed to characterize the HLA class I ligandome in AML patients to provide novel tumor associated antigens (TAA) for peptide-based immunotherapy employing our recently implemented approach of direct isolation and identification of naturally presented HLA ligands by affinity chromatography and mass spectrometry (LC-MS/MS) in AML (Stickel et.al., abstract in Blood 2012). Absolute HLA surface expression on AML cells and autologous monocytes and granulocytes was quantified by flow cytometry. HLA class I ligands were isolated from AML cells as well as bone marrow and peripheral blood mononuclear cell (BMNCs/PBMCs) of healthy donors. LC-MS/MS peptide analysis provided qualitative and semi-quantitative information regarding the composition of the respective ligandomes. Comparative analysis of malignant and benign samples served to identify ligandome-derived TAA (LiTAA) and to select peptide vaccine candidates. The most abundantly detected peptide candidates were checked for immunogenicity by ELISpot and confirmed by intracellular interferon-g staining of CD8+ T-cells. Meanwhile 15 AML patients (8 FLT3-ITD mutant) and 35 healthy donors were analyzed. We observed overexpression of HLA class I and II on AML cells as compared to autologous monocytes and granulocytes, with the level of significance reached for HLA class II (p=0,04). A total of more than 12,000 AML derived HLA ligands representing >6,000 different source proteins were identified; of which 2,220 were exclusively represented in AML, but not in healthy PBMC/BMNC. Data mining for broadly represented LiTAA pinpointed 98 TAA as most promising targets. HLA ligands derived from these TAA were presented exclusively on more than 33% of all analyzed AML samples, amongst them already described TAA (e.g. JUP, FAF1) as well as several new leukemia-associated proteins (e.g. MTCH2, METTL7A). Subset analysis of the FLT3-ITD positive AML cohort revealed 21 LiTAA presented exclusively on more than 50% of FLT3-ITD positive AML cases. Additional screening for HLA ligands derived from described leukemia associated antigens revealed overrepresentation for e.g. FLT3, NUSAP, RHAMM and RGS5. Specific CD8+ T cell responses were detected against two A*03 epitope pools (pool 1: APLP2, DKGZ, FAF1, MTCH2; pool 2: KLF2, METTL7A, VCIP1, WIPI1) in AML patients. Notably, the chosen A*03 epitope pools did not elicit specific responses of CTL from healthy donors. Taken together, our HLA class I ligandome analysis in AML for the first time identified naturally presented HLA ligands from patients including a vast array of new leukemia associated antigens representing promising targets for a multipeptide-based immunotherapy approach in AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Profiling for Monocyte-Regulated T-Cell Immune Responses in Human PBMC Samples: Simultaneous Assessments of Cell Surface Markers and Cytokine Secretion

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5730-5730 ◽  
Author(s):  
Zhaoping Liu
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cell Viability ◽  
Dose Response ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Cytokine Secretion ◽  
Activation Markers ◽  
Specific Patient

Abstract Advances in immuno-oncology have accelerated the need for higher content cell analysis tools that are capable of reducing the time needed to actionable results. The complexity of immune responses necessitates that data should yield insight into both the context of mechanism (cell activation markers) and pathways (signaling molecules) for how patients would respond to a certain treatments, or which therapies are most effective for a specific patient. Traditionally, cellular endpoints and secreted cytokines have been measured using two separate assays on two different analysis platforms: cellular endpoints measured by flow cytometry or imaging, and secreted cytokines measured by plate-based or bead-based ELISA. IntelliCyt's iQue Screener PLUS with 3 lasers and 13 fluorescent channels, was built as an immunology profiling tool and can measure cellular activation markers and secreted cytokine levels in a single assay in under 20 minutes for a 384-well plate. In a highlighted example showing monocyte regulation of T cells, we profiled the immune responses of three different patients across seven different cell activation markers, cell viability, and secretion of IFNγ, TNF, and IL-10 in response to several immunogenic compounds. PBMCs from 3 individual healthy donors were treated with the monocyte activator lipopolysaccharide (LPS) and/or with T cell activator staphylococcal enterotoxin B (SEB) in duplicate dose response. Cell and compounds were incubated for 24 hours, then analyzed for viability, surface expression of CD3, CD4, CD8, CD14, CD25, CD45 and CD127, and cytokine secretion using the QBeads reagent kits (IntelliCyt Corp). ForeCyt software was utilized for data acquisition and analysis, where sequential gates were used to individually identify cells and beads from the samples based on size. The cells were further analyzed for viability and cell marker expression, and QBeads were further segregated into the individual detection beads for each cytokine. The media concentration of each cytokine was calculated via the use of a standard curve. Utilizing the built-in plate level analytics of ForeCyt, we generated 27 unique cytokine curves from a single 384 well plate, and were able to assess the differences in cytokine secretion for each of the 3 cytokines, across the 3 treatments (LPS alone, SEB alone, LPS+SEB), for all 3 donors. The cell-based measurements yielded 54 dose-response curves detailing the percentage of regulatory T-cells, T-helper cells, cytotoxic T-cells, monocytes, lymphocytes, and cell viability for each treatment/patient. The ability to simultaneously measure cell and bead based endpoints from a single sample reduces assay-to-assay variability and conserves the amount of patient cells required for testing. Taken together, these results highlight the power of the iQue Screener PLUS platform as an immunological profiling tool with not only the speed of sampling required for large-scale combinatorial studies, but the analysis power to quickly transform raw data into clinically relevant results for individual patients. Disclosures No relevant conflicts of interest to declare.

Human Double-Negative Regulatory T Cells Selectively Suppress mTOR Signaling and Metabolic Reprogramming of Conventional T Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3694-3694
Author(s):  
Tabea Haug ◽  
Heiko Bruns ◽  
Michael Aigner ◽  
Andreas Mackensen ◽  
Simon Voelkl
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Mtor Signaling ◽  
Metabolic Reprogramming ◽  
Double Negative ◽  
Hematopoietic Stem ◽  
The Impact

Abstract Regulatory T (Treg) cells have been shown to be involved in downregulating immune responses in autoimmunity, transplant rejection, and graft-versus-host disease (GvHD). The subpopulation of TCRαβ+ CD4- CD8- (double-negative, DN) T cells has been described to suppress immune responses in both mice and humans. Of particular interest, infusion and/or activation of murine DN T cells specifically suppressed alloreactive T cells and prevented development of GvHD after allogeneic hematopoietic stem cell transplantation (SCT). Moreover, clinical studies in patients after SCT revealed an inverse correlation between the frequency of circulating DN T cells and the severity of GvHD, suggesting a therapeutic potential of human DN T cells. However, the molecular mechanism of suppression still remains unclear. To gain a better understanding of DN T-cell functionality, we investigated whether human DN T cells modulate distinct TCR signaling processes in conventional T cells. We found that DN T cells selectively inhibit the mechanistic target of rapamycin (mTOR) signaling pathway but not activation of mitogen-activated protein kinases (MAPK). The crucial function of mTOR signaling was confirmed by treating effector T cells with a chemical activator of protein kinase Akt, which induces mTOR phosphorylation. Indeed, enforced activation of the mTOR pathway rendered conventional T cells unsusceptible to DN T cell-mediated suppression. Given that mTOR is a critical regulator of cellular metabolism, we further determined the impact of DN T cells on the metabolic framework of conventional T cells. Intriguingly, DN T cells significantly diminished upregulation of the glycolytic machinery, expression of glucose transporters and glucose uptake in conventional T cells. These findings indicate that DN T cells inhibit metabolic reprogramming of conventional T cells by abrogating mTOR signaling, thereby inducing a quiescent phenotype. Further understanding of the mechanisms involved in human DN T-cell suppression may have important implications for using them as a cellular-based therapy to limit alloreactive immune responses. Disclosures No relevant conflicts of interest to declare.

scholarly journals Radiofrequency Ablation of Liver Metastases from Colorectal Cancer Induces a Cancer-Specific T Cell Repertoire in Humans

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1120-1120
Author(s):  
Markus W. Loeffler ◽  
Bianca Nussbaum ◽  
Philipp S. Jurmeister ◽  
Jan Budczies ◽  
Daniel Johannes Kowalewski ◽  
...  
Keyword(s):  
T Cells ◽  
Radiofrequency Ablation ◽  
T Cell ◽  
Immune Responses ◽  
Tumor Antigens ◽  
Immune Cell ◽  
Cell Responses ◽  
Hla Ligand ◽  
Hla Ligands ◽  
Immune Cell Infiltrates

Abstract Introduction: For patients with otherwise non-resectable primary and secondary cancer manifestations within the liver or contraindications against surgery, radiofrequency ablation (RFA) is a potentially curative treatment option. Heat causes coagulative necrosis and breaks local immune tolerance with subsequent release of cellular content like soluble danger signals which may reshape adaptive anti-tumor immune responses. The induction of effective functional tumor-specific T cells and in situ tumor-recognition remain a major challenge for immunotherapy, frequently limiting therapeutic effects. In this study, the induction of antigen-specific T cells against patient-individual, naturally presented tumor antigens and the infiltration of immune cells into distant liver metastasis after RFA were evaluated. Methods: Six patients suffering from at least two metastatic sites in different liver lobes derived from colorectal carcinoma (CRC) were enrolled. As an initial step, RFA was performed on those manifestations that were inaccessible by surgery. Patients subsequently underwent liver surgery of the residual metastases. These specimen were used for HLA-ligandome analyses of tumor and corresponding non-malignant liver (NML) tissues by uHPLC-coupled tandem mass spectrometry (MS) following HLA-immunoprecipitation. The identified naturally presented HLA-ligands were screening against an extensive HLA-ligand database containing data from non-malignant tissues from different origins to assess tumor specificity of single peptides. Further, whole transcriptome sequencing (WTS) was performed and correlated with MS data selecting HLA-ligands of interest. Functional T cell reactivity against selected antigens was analyzed by intracellular cytokine staining (ICS). Immune cell infiltrates of surgically resected liver metastases were assessed by immunohistochemistry staining (IHC including antibodies specific for CD3, CD4, CD8, CD56, HLA class I and II, and heat shock protein 70) in 11 and 5 patients undergoing hemihepatectomy with or without previous RFA, respectively. Results: 32 tumor-exclusive T cell epitope candidates were selected based on WTS and ligandome analyses. Antigen-specific T cells were detected in all included patients at least at one timepoint (including boosting of preexisting immune responses and de novo induction). Of note, one patient exhibited RFA-mediated induction of antigen-specific CD4+ T cells against one mutation-derived HLA-class II peptide. This epitope was predicted on the basis of the WST data, but could not be corroborated in MS data as a naturally presented HLA-ligand. Immunohistochemistry of immune cell infiltrates in the lesions resected after RFA did not reveal any significantly increased immune infiltrates in RFA pre-treated patients as compared to the control group. Conclusions: Specific T cell responses directed against tumor-antigens were detected in every patient investigated. Most notably, T cell responses were boosted or induced in 3 out of 6 patients following RFA, including in one patient against a tumor-specific neoantigen. Still, this tumor-specific immunity per se is probably insufficient to eradicate established tumors. This is underlined by the observation that no increased immune infiltrates in distant metastases were shown. Therefore, our findings support the clinical development of combinatorial therapies, combining RFA with suitable immune stimulatory agents. Disclosures Kowalewski: Immatics Biotechnologies GmbH: Employment.

Three Steps to Breaking Immune Tolerance: A Microparticle Approach

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4504-4504 ◽  
Author(s):  
Amani Makkouk ◽  
Vijaya B Joshi ◽  
Caitlin D Lemke ◽  
Amaraporn Wongrakpanich ◽  
Alicia K Olivier ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Tumor Cells ◽  
Tumor Antigens ◽  
Conflicts Of Interest ◽  
Optimal Size ◽  
Step Therapy ◽  
Three Components

Abstract Lymphoma immunotherapy can result in durable immune and clinical responses. Nevertheless, the clinical impact of such therapy remains suboptimal and there is still a need to apply our growing understanding of cancer immunity to the design and testing of rational combination approaches to lymphoma immunotherapy. This includes consideration of the three steps necessary to generate an antitumor response: (1) providing tumor antigens to dendritic cells (DCs) in a manner that enhances their ability to process and present antigens to T cells; (2) enhancing T cell activation; and (3) overcoming immunosuppression so that activated anti-lymphoma T cells can proceed unrestrained. In situ immunization generates systemic immune responses through local injection of agents into the tumor, thus providing DCs with the patient’s own tumor antigens. We evaluated a three-step approach to in situ immunization against lymphoma using Doxorubicin, anti-CTLA-4 and anti-OX40: (1) Doxorubicin (Dox) to induce the expression of “eat-me” signals by dying tumor cells, facilitating their phagocytosis by DCs; (2) anti-OX40 antibody to augment OX40-mediated stimulation of T cells; (3) anti-CTLA-4 antibody to block immunosuppression imposed by CTLA-4 on T cells. While Dox is highly effective as a systemic anti-lymphoma agent and has been reported to induce immunogenic cell death, intratumoral injection of soluble Dox is not clinically feasible due to its strong vesicant effect. Poly(lactide-co-glycolide) (PLGA) is an FDA-approved polymer that is used in biodegradable surgical sutures and microparticles (MPs) with sustained-release properties. Thus, PLGA MPs loaded with Dox (Dox MPs) represent a clinically translatable approach for delivering Dox, as its slow release would decrease likelihood of vesication. In addition, PLGA MPs at an optimal size of 1-µm enhance antigen-specific immune responses by activating the NALP3 inflammasome in DCs. While both tumor cells and DCs are exposed to Dox released by MPs in the tumor, we found in vitro that Dox MPs (1-µm) are less cytotoxic to DCs than to A20 B-lymphoma cells and do not require internalization for their cytotoxic activity. Dox MPs significantly enhanced phagocytosis of A20 by DCs as compared to soluble Dox. In vivo, we used the two-tumor mouse model to assess immune responses. This allowed us to monitor the local effect (on the tumor injected with MP) and the systemic effect (on the distant tumor that was not injected with MP) of therapy. Dox MPs injected intratumorally do not induce vesication even at doses as high as 100 µg Dox. Using a low dose of Dox MPs (2 µg Dox) and of antibody to limit systemic toxicity, we found that three-step therapy induced CD4- and CD8- T cell-dependent systemic immune responses that enhanced T cell infiltration into distant tumors. This led to their eradication and significantly improved survival as compared to antibody-only therapy (87% of mice treated with all three components became tumor-free). Moreover, all three components were required for maximum efficacy (Figure 1). These results demonstrate that systemic antitumor immune responses can be generated locally by three-step therapy. The potential value of this approach to immunotherapy is not limited to lymphoma and merits further evaluation in lymphoma and other cancers. Figure 1. Mice challenged with two A20 tumors were treated with PBS (control) or Dox MP into one tumor and antibodies (anti-CTLA-4 and/or anti-OX40) given systemically. Figure 1. Mice challenged with two A20 tumors were treated with PBS (control) or Dox MP into one tumor and antibodies (anti-CTLA-4 and/or anti-OX40) given systemically. Disclosures No relevant conflicts of interest to declare.

scholarly journals HLA ligandome analysis identifies the underlying specificities of spontaneous antileukemia immune responses in chronic lymphocytic leukemia (CLL)

2014 ◽  
Vol 112 (2) ◽  
pp. E166-E175 ◽  
Author(s):  
Daniel J. Kowalewski ◽  
Heiko Schuster ◽  
Linus Backert ◽  
Claudia Berlin ◽  
Stefan Kahn ◽  
...  
Keyword(s):  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Immune Responses ◽  
Tumor Antigens ◽  
Checkpoint Inhibitors ◽  
Hla Class I ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Immune Recognition ◽  
Hla Ligandome

The breakthrough development of clinically effective immune checkpoint inhibitors illustrates the potential of T-cell–based immunotherapy to effectively treat malignancies. A remaining challenge is to increase and guide the specificities of anticancer immune responses, e.g., by therapeutic vaccination or by adoptive T-cell transfer. By analyzing the landscape of naturally presented HLA class I and II ligands of primary chronic lymphocytic leukemia (CLL), we delineated a novel category of tumor-associated T-cell antigens based on their exclusive and frequent representation in the HLA ligandome of leukemic cells. These antigens were validated across different stages and mutational subtypes of CLL and found to be robustly represented in HLA ligandomes of patients undergoing standard chemo-/immunotherapy. We demonstrate specific immune recognition of these antigens exclusively in CLL patients, with the frequencies of representation in CLL ligandomes correlating with the frequencies of immune recognition by patient T cells. Moreover, retrospective survival analysis revealed survival benefits for patients displaying immune responses to these antigens. These results directly imply these nonmutant self-peptides as pathophysiologically relevant tumor antigens and encourages their implementation for cancer immunotherapy.

scholarly journals Listeria monocytogenes personalized cancer vaccines drive therapeutic immune responses to cancer derived neoantigens

2020 ◽  
Author(s):  
Brandon Coder ◽  
Olga Pryshchep ◽  
Dipti Kelkar ◽  
Elena Filippova ◽  
Xiaoming Ju ◽  
...  
Keyword(s):  
T Cells ◽  
Listeria Monocytogenes ◽  
T Cell ◽  
Immune Responses ◽  
Tumor Growth ◽  
T Cell Responses ◽  
Absolute Number ◽  
Tumor Control ◽  
Mouse Tumor ◽  
Cell Responses

AbstractBackgroundRecent advances in the field of cancer immunotherapy have identified CD8+ T cell responses against tumor-specific mutations as a key driver of tumor regression and overall survival. ADXS-NEO is a personalized Listeria monocytogenes (Lm)-based immunotherapy designed to target a patient’s mutation-derived tumor-specific neoantigens. The objective of this study is to demonstrate the feasibility of using the ADXS-NEO platform to target tumor-specific point mutations and control tumor growth by generating neoantigen-specific T cell responses using a pre-clinical mouse tumor model.MethodsWhole-exome sequencing of the MC38 mouse tumor cell line identified 2870 unique non-synonymous mutations. The netMHCcons algorithm was used to predict 137 potential neoantigens. We validated 20 immunogenic neoantigens either by peptide immunization followed by ELISPOT or by the presence of CD8+ T cells recognizing the neoantigen peptide following checkpoint inhibitor treatment. Two ADXS-NEO vectors were constructed; Lm20, targeting 20 validated immunogenic neoantigens, and Lm19, targeting most of the non-validated NSMs.ResultsBoth Lm19 & Lm20 significantly slowed tumor growth in C57BL/6 mice compared to control. An accumulation of ADXS-NEO-specific TILs was observed in tumor bearing mice treated with either Lm19 or Lm20. Examination of the tumor microenvironment in Lm19 or Lm20 treated mice revealed a decrease in the frequency and absolute number of Tregs, TAMs, MDSCs, and PD1high exhausted CD8+ T cells as well as an increase in the frequency and absolute number of effector CD8+ T cells, relative to control.ConclusionADXS-NEO is a potent immunotherapy capable of driving immune responses against tumor-specific mutations and leading to tumor control in mice.

scholarly journals Super Cross-Presentation of Tumor Antigens to Elicit Anti-Lymphoma Immunity By Synthetic Design of an Anti-Phosphatidylserine Bridge Protein

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1844-1844
Author(s):  
Daniel Corey ◽  
Irving L Weissman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Mhc Class Ii ◽  
Tumor Antigens ◽  
Conflicts Of Interest ◽  
Checkpoint Inhibitors ◽  
Apoptotic Cells ◽  
Lymphoma Cells ◽  
Cross Presentation

Abstract Cell loss by apoptosis is a common feature in tumors. Dying tumor cells induce immune tolerance within the tumor microenvironment largely through highly conserved homeostatic clearance programs that restore tissue immune homeostasis and contribute to the formation of an immunosuppressive niche. The translocation of phosphatidylserine (PS) on cellular membranes, during the initial phases of apoptosis, functions as a recognition and removal signal that limits the immunogenicity of cell death. We examined whether altering clearance of dying cancer cells to elicit inflammatory turnover potentiates immune responses against lymphoma cells. To remove inhibitory signals in the homeostatic clearance pathway we utilized a molecular bridge scaffold to engineer a modified phosphatidylserine bridge protein (FA58C2-hIgG1 or C2-hIgG1) that works as a bridge between apoptotic cells expressing aminophospholipids and phagocytes bearing Fc receptors. In vitro administration of C2-hIgG1 to murine bone marrow derived macrophages promotes engulfment of apoptotic murine lymphoma cells (38C13 cell line) and ablates the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10) and suppression of pro-inflammatory cytokines tumor necrosis factor (TNF-α) and IL-12p40 to the presence of apoptotic cells. Similarly, uptake of C2-hIgG1 treated lymphoma cells triggers upregulation of the costimulatory markers CD80, CD86, and MHC class II on macrophages and promotes secretion of Th1-recruiting lymphocyte chemokines CXCL9, CXCL10, and CCL5. Accordingly, in vivo administration of C2-hIgG1 partially restores immune responses to dead lymphoma cells in antigen cross presentation assays and promotes recruitment and retention of tumor antigen specific CD8+ T cells, dendritic cells, and natural killer cells into tumors. These effects combine to elicit anti-lymphoma immunity, improve responses to immune checkpoint inhibitors, and enhance the effectiveness of adoptive T cell transfers using engineered T Cell Receptors (TCRs) but not CD19-directed chimeric antigen receptor engineered (CAR-T) T cells. Disclosures No relevant conflicts of interest to declare.

Characterization of the Immunoregulatory Function of Human TCRαβ+ CD4-CD8- Double-Negative T Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2776-2776
Author(s):  
Simon Voelkl ◽  
Regina Gary ◽  
Andreas Mackensen
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Conflicts Of Interest ◽  
T Cell Responses ◽  
Treg Cells ◽  
Cell Contact ◽  
Double Negative ◽  
Suppressive Activity ◽  
Cell Responses

Abstract Abstract 2776 Regulatory T (Treg) cells have been shown to be involved in downregulating immune responses in autoimmunity, transplant rejection, and graft-versus-host disease. Moreover, the important role of Treg cells in tumor progression has also been extensively investigated and Treg-mediated immune suppression has emerged as a crucial mechanism of tumor evasion. Recently, a novel subset of TCRαβ+ CD4−CD8− (double-negative, DN) T cells has been characterized to specifically suppress immune responses in mice. Here we demonstrate that human DN T cells belong to the family of inducible Treg cells with potent suppressor activity towards CD4+ and CD8+ T-cell responses. Resting DN T cells failed to suppress responder cells, whereas antigen presenting cell (APC)-stimulated DN T-cells and freshly isolated CD4+CD25+ Treg cells revealed a strong suppressive activity. Of importance, when more potent stimulators such as allogeneic dendritic cells were used for activation of responder T cells, CD4+CD25+ Treg cells were unable to mediate any suppressor function, while APC-primed DN T cells were still able to suppress. The suppressive activity of DN T cells is neither mediated indirectly by modulation of APCs nor by competition for T-cell growth factors. Furthermore, DN T-cell mediated suppression towards responder T cells requires cell-cell contact and is TCR dependent. Based on the importance of Treg cells in tumor immunity, we determined the frequency of circulating DN T cells in patients with malignant diseases. Of interest, the DN T-cell pool revealed an increased frequency in cancer patients compared to healthy controls, indicating that these suppressor cells might limit tumor-specific T-cell responses and thereby impair tumor immunosurveillance. Taken together, our results demonstrate that human DN T cells are a new subset of inducible Treg cells exerting a very potent suppressive activity towards cellular immune responses. Further understanding of the mechanisms involved in human DN T-cell suppression may have important implications for novel immunotherapies. Disclosures: No relevant conflicts of interest to declare.

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