scholarly journals IFN-γ treatment protocol for MHC-Ilo/PD-L1+ pancreatic tumor cells selectively restores their TAP-mediated presentation competence and CD8 T-cell priming potential

2020 ◽  
Vol 8 (2) ◽  
pp. e000692
Author(s):  
Katja Stifter ◽  
Jana Krieger ◽  
Leonie Ruths ◽  
Johann Gout ◽  
Medhanie Mulaw ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Cells ◽  
Cd8 T Cells ◽  
Antigen Processing ◽  
Treatment Protocol ◽  
Cd8 T Cell ◽  
Mhc I ◽  
Ifn Γ ◽  
Deficient Mice

BackgroundMany cancer cells express a major histocompatibility complex class I low/ programmed cell death 1 ligand 1 positive (MHC-Ilo/PD-L1+) cell surface profile. For immunotherapy, there is, thus, an urgent need to restore presentation competence of cancer cells with defects in MHC-I processing/presentation combined with immune interventions that tackle the tumor-initiated PD-L1/PD-1 signaling axis. Using pancreatic ductal adenocarcinoma cells (PDACCs) as a model, we here explored if (and how) expression/processing of tumor antigens via transporters associated with antigen processing (TAP) affects priming of CD8 T cells in PD-1/PD-L1-competent/-deficient mice.MethodsWe generated tumor antigen-expressing vectors, immunized TAP-competent/-deficient mice and determined de novo primed CD8 T-cell frequencies by flow cytometry. Similarly, we explored the antigenicity and PD-L1/PD-1 sensitivity of PDACCs versus interferon-γ (IFN-γ)-treated PDACCs in PD-1/PD-L1-competent/deficient mice. The IFN-γ-induced effects on gene and cell surface expression profiles were determined by microarrays and flow cytometry.ResultsWe identified two antigens (cripto-1 and an endogenous leukemia virus-derived gp70) that were expressed in the Endoplasmic Reticulum (ER) of PDACCs and induced CD8 T-cell responses either independent (Cripto-1:Kb/Cr16-24) or dependent (gp70:Kb/p15E) on TAP by DNA immunization. IFN-γ-treatment of PDACCs in vitro upregulated MHC-I- and TAP- but also PD-L1-expression. Mechanistically, PD-L1/PD-1 signaling was superior to the reconstitution of MHC-I presentation competence, as subcutaneously transplanted IFN-γ-treated PDACCs developed tumors in C57BL/6J and PD-L1-/- but not in PD-1-/- mice. Using PDACCs, irradiated at day 3 post-IFN-γ-treatment or PD-L1 knockout PDACCs as vaccines, we could selectively bypass upregulation of PD-L1, preferentially induce TAP-dependent gp70:Kb/p15E-specific CD8 T cells associated with a weakened PD-1+ exhaustion phenotype and reject consecutively injected tumor transplants in C57BL/6J mice.ConclusionsThe IFN-γ-treatment protocol is attractive for cell-based immunotherapies, because it restores TAP-dependent antigen processing in cancer cells, facilitates priming of TAP-dependent effector CD8 T-cell responses without additional check point inhibitors and could be combined with genetic vaccines that complement priming of TAP-independent CD8 T cells.

scholarly journals Cross-presentation of a TAP-independent signal peptide induces CD8 T immunity to escaped cancers but necessitates anchor replacement

2021 ◽  
Author(s):  
Koen A. Marijt ◽  
Lisa Griffioen ◽  
Laura Blijleven ◽  
Sjoerd. H. van der Burg ◽  
Thorbald van Hall
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Cells ◽  
Cd8 T Cells ◽  
Antigen Processing ◽  
Signal Sequence ◽  
Cell Repertoire ◽  
Cross Presentation ◽  
Normal Tissues ◽  
Cell Immunity

AbstractCancer cells frequently display defects in their antigen-processing pathway and thereby evade CD8 T cell immunity. We described a novel category of cancer antigens, named TEIPP, that emerge on cancers with functional loss of the peptide pump TAP. TEIPPs are non-mutated neoantigens despite their ‘self’ origin by virtue of their absence on normal tissues. Here, we describe the development of a synthetic long peptide (SLP) vaccine for the most immunogenic TEIPP antigen identified thus far, derived from the TAP-independent LRPAP1 signal sequence. LRPAP121–30-specific CD8 T cells were present in blood of all tested healthy donors as well as patients with non-small cell lung adenocarcinoma. SLPs with natural flanking, however, failed to be cross-presented by monocyte-derived dendritic cells. Since the C-terminus of LRPAP121–30 is an unconventional and weakly binding serine (S), we investigated if replacement of this anchor would result in efficient cross-presentation. Exchange into a valine (V) resulted in higher HLA-A2 binding affinity and enhanced T cell stimulation. Importantly, CD8 T cells isolated using the V-variant were able to bind tetramers with the natural S-variant and respond to TAP-deficient cancer cells. A functional screen with an array of N-terminal and C-terminal extended SLPs pointed at the 24-mer V-SLP, elongated at the N-terminus, as most optimal vaccine candidate. This SLP was efficiently cross-presented and consistently induced a strong polyclonal LRPAP121–30-specific CD8 T cells from the endogenous T cell repertoire. Thus, we designed a TEIPP SLP vaccine from the LRPAP1 signal sequence ready for validation in clinical trials.

scholarly journals Virally Activated CD8 T Cells Home to Mycobacterium bovis BCG-Induced Granulomas but Enhance Antimycobacterial Protection Only in Immunodeficient Mice

2006 ◽  
Vol 75 (3) ◽  
pp. 1154-1166 ◽  
Author(s):  
Laura H. Hogan ◽  
Dominic O. Co ◽  
Jozsef Karman ◽  
Erika Heninger ◽  
M. Suresh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mycobacterium Bovis ◽  
Cd8 T Cells ◽  
Bacterial Load ◽  
Granulomatous Inflammation ◽  
Cd8 T Cell ◽  
Activated T Cells ◽  
Immunodeficient Mice ◽  
Ifn Γ

ABSTRACT The effect of secondary infections on CD4 T-cell-regulated chronic granulomatous inflammation is not well understood. Here, we have investigated the effect of an acute viral infection on the cellular composition and bacterial protection in Mycobacterium bovis strain bacille Calmette-Guérin (BCG)-induced granulomas using an immunocompetent and a partially immunodeficient murine model. Acute lymphocytic choriomeningitis virus (LCMV) coinfection of C57BL/6 mice led to substantial accumulation of gamma interferon (IFN-γ)-producing LCMV-specific T cells in liver granulomas and increased local IFN-γ. Despite traffic of activated T cells that resulted in a CD8 T-cell-dominated granuloma, the BCG liver organ load was unaltered from control levels. In OT-1 T-cell-receptor (TCR) transgenic mice, ovalbumin (OVA) immunization or LCMV coinfection of BCG-infected mice induced CD8 T-cell-dominated granulomas containing large numbers of non-BCG-specific activated T cells. The higher baseline BCG organ load in this CD8 TCR transgenic animal allowed us to demonstrate that OVA immunization and LCMV coinfection increased anti-BCG protection. The bacterial load remained substantially higher than in mice with a more complete TCR repertoire. Overall, the present study suggests that peripherally activated CD8 T cells can be recruited to chronic inflammatory sites, but their contribution to protective immunity is limited to conditions of underlying immunodeficiency.

148 Identification of prostate-restricted epithelial antigens for transgenic T cell adoptive therapy against prostate cancer

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A161-A161
Author(s):  
Diana DeLucia ◽  
Tiffany Pariva ◽  
Roland Strong ◽  
Owen Witte ◽  
John Lee
Keyword(s):  
Prostate Cancer ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Adoptive Therapy ◽  
Mhc I ◽  
Epithelial Antigens ◽  
Ifn Γ ◽  
Stimulation Of

BackgroundIn advanced prostate cancer (PCa), progression to castration-resistant PCa (CRPC) is inevitable and novel therapies for CRPC are needed. Adoptive transfer of T cells targeting tumor antigens is a promising approach in the cancer field. Unfortunately, identifying antigens expressed exclusively in prostate tumor cells has been challenging. Since the prostate is not an essential organ, we alternatively selected prostate-restricted epithelial antigens (PREAs) expressed in both malignant and normal prostate tissue for transgenic T cell studies.MethodsRNA-seq data sets identifying genes enriched in PCa were cross-referenced with the NIH Genotype-Expression database to identify PREAs. Using a novel molecular immunology approach, select PREAs and major histocompatibility complex class I (MHC-I) molecules were co-expressed in HEK293F cells, from which MHC–peptide complexes were efficiently isolated. Peptides were eluted and sequenced by mass spectrometry. Peptide–MHC binding was validated with a T2 stabilization assay and peptide immunodominance was determined using an interferon-γ (IFN-γ) ELISpot assay following stimulation of healthy HLA-A2+ peripheral blood mononuclear cells (PBMC) with peptide pools. Following peptide stimulation, CD8+ T cells with peptide-specific T cell receptors (TCR) were enriched by peptide–MHC-I dextramer labeling and fluorescence activated cell sorting for single cell TCR α/β chain sequencing.ResultsWe identified 11 A2+ peptides (8 previously unpublished) from prostatic acid phosphatase (ACPP), solute carrier family 45 member 3 (SLC45A3), and NK3 homeobox 1 (NKX3.1) that bound to HLA-A2 with varying affinities. Extended culture stimulation of PBMC with peptide pools from each PREA, compared to the standard overnight culture, revealed a greater number of IFN-γ producing cells overall and a greater breadth of response across all the peptides. Antigen specific CD8+ T cells were detectable at low frequencies in both male and female healthy PBMC for 7 of the 11 peptides. Dextramer-sorted antigen-specific cells were used for single-cell paired TCR αβ sequencing and transgenic T cell development.ConclusionsThrough this work we identified HLA-A2-presented antigenic peptides from the PREAs ACPP, SLC45A3, and NKX3.1 that can induce the expansion of IFN-γ producing CD8+ T cells. Through peptide–MHC-I dextramer labeling, we isolated PREA-specific CD8+ T cells and characterized TCR αβ sequences with potential anti-tumor functionality. Our results highlight a rapid and directed platform for the development of MHC-I-restricted transgenic CD8+ T cells targeting lineage-specific proteins expressed in prostate epithelia for adoptive therapy of advanced PCa.

scholarly journals 677 Reverse abscopal effect: intertumoral heterogeneity suppresses systemic CD8 T cell-mediated antitumor immunity and confers PD-1 inhibitor resistance in synchronous melanoma

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A705-A705
Author(s):  
Shuyang Qin ◽  
Booyeon Han ◽  
Alexander Chacon ◽  
Alexa Melucci ◽  
Alyssa Williams ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cd8 T Cells ◽  
Antitumor Immunity ◽  
Immune Escape ◽  
Cd8 T Cell ◽  
Abscopal Effect ◽  
Immunogenic Tumor ◽  
Ifn Γ

BackgroundDespite recent advancements in systemic therapy, only a minority of metastatic patients develop meaningful clinical responses to immune checkpoint inhibitors. Inherent genetic instability of melanoma generates genomically and microenvironmentally distinct metastases. These different tumor microenvironments (TMEs) contain numerous T cell suppression mechanisms, such as upregulation of the PD-1/PD-L1 exhaustion pathway. However, as synchronous metastases share one host immune system, intertumoral heterogeneity may result in increasing cross-talk between metastases that impairs systemic antitumor immunity and promotes PD-1 immunotherapy resistance.MethodsYUMM 1.7 (less immunogenic) and YUMMER 1.7 (more immunogenic cell line derived from YUMM following UVB irradiation) melanoma cell lines were simultaneously injected into opposite flanks of the same mice as a model of synchronous melanoma. We assessed tumor growth in wildtype, interferon-gamma (IFN-γ) knockout, and CD8-depleted mice as well as in response to PD-1 inhibitor. We characterized the TME with flow cytometry and performed TCR sequencing on tumor-infiltrating CD8 T cells.ResultsDistinct TMEs were observed for YUMM and YUMMER tumors simultaneously grown in the same mouse. The presence of the less immunogenic YUMM tumor allows the more immunogenic YUMMER tumors to escape IFN-γ and CD8 T cell-mediated rejection, despite abundant tumor-infiltrating, clonally expanded CD8 T cells. Identical immunodominant CD8 T cell clones were found in both YUMM and YUMMER tumors within the same mouse. Synchronous YUMMER-infiltrating CD8 T cells exhibit suppressed phenotypes, including increased persistence of surface PD-1 and decreased surface CD107a expressions. Simultaneously, these synchronous YUMMER tumors additionally upregulate macrophage surface PD-L1 expression, which potentially contributes to tumor immune escape. Lastly, synchronous YUMMER tumors become resistant to PD-1 inhibition, in direct contrast to control YUMMER tumors.ConclusionsIn a host with multiple melanoma lesions, immunogenicity of all tumors contribute to the systemic antitumor immune response. We show that two synchronous tumors with synonymous mutations (<40%), as is the case with metastatic patients, lead to skewed CD8 T cell expansion of the same clones in both tumors. The presence of a less immunogenic tumor prevents CD8 and IFN-γ mediated rejection of the more immunogenic tumor. Furthermore, CD8 T cells in the more immunogenic tumor exhibit decreased effector function and increased resistance to PD-1 blockade, as tumor-infiltrating macrophages concurrently become more immunosuppressive. These results are highly suggestive of a “reverse abscopal effect,” by which immunologically “cold” tumors generate systemic immunosuppression that facilitate PD-1 immunotherapy resistance and immune escape of all other tumors in synchronous metastatic melanoma patients.AcknowledgementsWe would like to thank Dr. Marcus Bosenberg from the Department of Dermatology at Yale University for kindly gifting us with the YUMMER 1.7 murine melanoma cell line.Ethics ApprovalAnimal experiments were approved by the University Committee on Animal Resources and performed in accordance with University of Rochester approved guidelines.

scholarly journals Combination Treatment of Topical Imiquimod Plus Anti-PD-1 Antibody Exerts Significantly Potent Antitumor Effect

Cancers ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 3948
Author(s):  
Kazumasa Oya ◽  
Yoshiyuki Nakamura ◽  
Zhu Zhenjie ◽  
Ryota Tanaka ◽  
Naoko Okiyama ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Combination Therapy ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Antitumor Effect ◽  
Skin Lesions ◽  
Ifn Γ ◽  
Deficient Mice

The exact mechanisms of the imiquimod (IMQ)-induced antitumor effect have not been fully understood. Although both topical IMQ treatment and anti-PD-1 antibody may be used for primary skin lesions or skin metastases of various cancers, the efficacy of each monotherapy for these lesions is insufficient. Using a murine tumor model and human samples, we aimed to elucidate the detailed mechanisms of the IMQ-induced antitumor effect and analyzed the antitumor effect of combination therapy of topical IMQ plus anti-PD-1 antibody. Topical IMQ significantly suppressed the tumor growth of MC38 in wildtype mice. IMQ upregulated interferon γ (IFN-γ) expression in CD8+ T cells in both the lymph nodes and the tumor, and the antitumor effect was abolished in both Rag1-deficient mice and IFN-γ-deficient mice, indicating that IFN-γ produced by CD8+ T cells play a crucial role in the IMQ-induced antitumor effect. IMQ also upregulated PD-1 expression in T cells as well as PD-L1/PD-L2 expression in myeloid cells, suggesting that IMQ induces not only T-cell activation but also T-cell exhaustion by enhanced PD-1 inhibitory signaling. Combination therapy of topical IMQ plus anti-PD-1 antibody exerted a significantly potent antitumor effect when compared with each single therapy, indicating that the combination therapy is a promising therapy for the skin lesions of various cancers.

scholarly journals Generation of Human CD8 T Regulatory Cells by CD40 Ligand–activated Plasmacytoid Dendritic Cells

2002 ◽  
Vol 195 (6) ◽  
pp. 695-704 ◽  
Author(s):  
Michel Gilliet ◽  
Yong-Jun Liu
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Transforming Growth Factor ◽  
Cd8 T Cell ◽  
Cd40 Ligand ◽  
Plasmacytoid Dendritic Cells ◽  
Target Cells ◽  
Ifn Γ

Although CD8 T cell–mediated immunosuppression has been a well-known phenomenon during the last three decades, the nature of primary CD8 T suppressor cells and the mechanism underlying their generation remain enigmatic. We demonstrated that naive CD8 T cells primed with allogeneic CD40 ligand–activated plasmacytoid dendritic cells (DC)2 differentiated into CD8 T cells that displayed poor secondary proliferative and cytolytic responses. By contrast, naive CD8 T cells primed with allogeneic CD40 ligand–activated monocyte-derived DCs (DC1) differentiated into CD8 T cells, which proliferated to secondary stimulation and killed allogeneic target cells. Unlike DC1-primed CD8 T cells that produced large amounts of interferon (IFN)-γ upon restimulation, DC2-primed CD8 T cells produced significant amounts of interleukin (IL)-10, low IFN-γ, and no IL-4, IL-5, nor transforming growth factor (TGF)-β. The addition of anti–IL-10–neutralizing monoclonal antibodies during DC2 and CD8 T cell coculture, completely blocked the generation of IL-10–producing anergic CD8 T cells. IL-10–producing CD8 T cells strongly inhibit the allospecific proliferation of naive CD8 T cells to monocytes, and mature and immature DCs. This inhibition was mediated by IL-10, but not by TGF-β. IL-10–producing CD8 T cells could inhibit the bystander proliferation of naive CD8 T cells, provided that they were restimulated nearby to produce IL-10. IL-10–producing CD8 T cells could not inhibit the proliferation of DC1-preactivated effector T cells. This study demonstrates that IL-10–producing CD8 T cells are regulatory T cells, which provides a cellular basis for the phenomenon of CD8 T cell–mediated immunosuppression and suggests a role for plasmacytoid DC2 in immunological tolerance.

scholarly journals Chronic Hepatitis C Virus Infection Impairs M1 Macrophage Differentiation and Contributes to CD8+ T-Cell Dysfunction

Cells ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 374 ◽  
Author(s):  
Faria Ahmed ◽  
Andrea Ibrahim ◽  
Curtis L. Cooper ◽  
Ashok Kumar ◽  
Angela M. Crawley
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Hcv Infection ◽  
M1 Macrophage ◽  
T Cell Dysfunction ◽  
Chronic Hcv Infection ◽  
Ifn Γ ◽  
Chronic Hcv

Chronic hepatitis C virus (HCV) infection causes generalized CD8+ T cell impairment, not limited to HCV-specific CD8+ T-cells. Liver-infiltrating monocyte-derived macrophages (MDMs) contribute to the local micro-environment and can interact with and influence cells routinely trafficking through the liver, including CD8+ T-cells. MDMs can be polarized into M1 (classically activated) and M2a, M2b, and M2c (alternatively activated) phenotypes that perform pro- and anti-inflammatory functions, respectively. The impact of chronic HCV infection on MDM subset functions is not known. Our results show that M1 cells generated from chronic HCV patients acquire M2 characteristics, such as increased CD86 expression and IL-10 secretion, compared to uninfected controls. In contrast, M2 subsets from HCV-infected individuals acquired M1-like features by secreting more IL-12 and IFN-γ. The severity of liver disease was also associated with altered macrophage subset differentiation. In co-cultures with autologous CD8+ T-cells from controls, M1 macrophages alone significantly increased CD8+ T cell IFN-γ expression in a cytokine-independent and cell-contact-dependent manner. However, M1 macrophages from HCV-infected individuals significantly decreased IFN-γ expression in CD8+ T-cells. Therefore, altered M1 macrophage differentiation in chronic HCV infection may contribute to observed CD8+ T-cell dysfunction. Understanding the immunological perturbations in chronic HCV infection will lead to the identification of therapeutic targets to restore immune function in HCV+ individuals, and aid in the mitigation of associated negative clinical outcomes.

scholarly journals The Level of Viral Antigen Presented by Hepatocytes Influences CD8 T-Cell Function

2007 ◽  
Vol 81 (6) ◽  
pp. 2940-2949 ◽  
Author(s):  
Adam J. Gehring ◽  
Dianxing Sun ◽  
Patrick T. F. Kennedy ◽  
Esther Nolte-'t Hoen ◽  
Seng Gee Lim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Viral Antigen ◽  
Cell Function ◽  
Cd8 T Cell ◽  
T Cell Function ◽  
Tnf Α ◽  
Antiviral Function ◽  
Ifn Γ

ABSTRACT CD8 T cells exert their antiviral function through cytokines and lysis of infected cells. Because hepatocytes are susceptible to noncytolytic mechanisms of viral clearance, CD8 T-cell antiviral efficiency against hepatotropic viruses has been linked to their capacity to produce gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). On the other hand, intrahepatic cytokine production triggers the recruitment of mononuclear cells, which sustain acute and chronic liver damage. Using virus-specific CD8 T cells and human hepatocytes, we analyzed the modulation of virus-specific CD8 T-cell function after recognition peptide-pulsed or virally infected hepatocytes. We observed that hepatocyte antigen presentation was generally inefficient, and the quantity of viral antigen strongly influenced CD8 T-cell antiviral function. High levels of hepatitis B virus production induced robust IFN-γ and TNF-α production in virus-specific CD8 T cells, while limiting amounts of viral antigen, both in hepatocyte-like cells and naturally infected human hepatocytes, preferentially stimulated CD8 T-cell degranulation. Our data document a mechanism where virus-specific CD8 T-cell function is influenced by the quantity of virus produced within hepatocytes.

scholarly journals IL-1 enhances expansion, effector function, tissue localization, and memory response of antigen-specific CD8 T cells

2013 ◽  
Vol 210 (3) ◽  
pp. 491-502 ◽  
Author(s):  
Shlomo Z. Ben-Sasson ◽  
Alison Hogg ◽  
Jane Hu-Li ◽  
Paul Wingfield ◽  
Xi Chen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Granzyme B ◽  
Interleukin 1 ◽  
Cd8 T Cell ◽  
Cell Receptor ◽  
In Vivo Models ◽  
Ifn Γ

Here, we show that interleukin-1 (IL-1) enhances antigen-driven CD8 T cell responses. When administered to recipients of OT-I T cell receptor transgenic CD8 T cells specific for an ovalbumin (OVA) peptide, IL-1 results in an increase in the numbers of wild-type but not IL1R1−/− OT-I cells, particularly in spleen, liver, and lung, upon immunization with OVA and lipopolysaccharide. IL-1 administration also results in an enhancement in the frequency of antigen-specific cells that are granzyme B+, have cytotoxic activity, and/ or produce interferon γ (IFN-γ). Cells primed in the presence of IL-1 display enhanced expression of granzyme B and increased capacity to produce IFN-γ when rechallenged 2 mo after priming. In three in vivo models, IL-1 enhances the protective value of weak immunogens. Thus, IL-1 has a marked enhancing effect on antigen-specific CD8 T cell expansion, differentiation, migration to the periphery, and memory.

Ex-Vivo Characterization of Polyclonal Memory CD8+ T-Cell Responses to PRAME-Specific Peptides in Patients with Acute Leukemia and Chronic Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2910-2910
Author(s):  
Katayoun Rezvani ◽  
Agnes S. M. Yong ◽  
Abdul Tawab ◽  
Behnam Jafarpour ◽  
Rhoda Eniafe ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
High Avidity ◽  
Cell Responses ◽  
Ifn Γ

Abstract PRAME (Preferentially expressed antigen of melanoma) is aberrantly expressed in hematological malignancies and may be a useful target for immunotherapy in leukemia. We studied CD8+ T-cell responses to four HLA-A*0201-restricted PRAME-derived epitopes (PRA100, PRA142, PRA300, PRA425) in HLA-A*0201-positive patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and healthy donors, using PRA300/HLA-A*0201 tetramer staining, intracellular cytokine (IC) assay and ex-vivo and cultured ELISPOT analysis. CD8+ T-cells recognizing PRAME peptides were detected directly ex-vivo in 4/10 ALL, 6/10 AML, 3/10 CML patients and 3/10 donors. The frequency of PRAME-specific CD8+ T-cells was greater in patients with AML, CML and ALL than in healthy controls. All peptides were immunogenic in patients, whilst PRA300 was the only immunogenic peptide in donors. High PRAME expression in patient peripheral blood mononuclear cells was associated with responses to two or more PRAME epitopes (4/7 vs. 0/23 in individuals with low PRAME expression, P = 0.001), suggesting a PRAME-driven T-cell response. In 2 patients studied PRA300/HLA-A*0201+ CD8+T-cells were found to be a mixture of effector and central memory phenotypes. To determine the functional avidity of the PRAME T-cell response, the response of CD8+ T-cells to stimulation with 2 concentrations of peptide was measured by IC-IFN-γ staining. High-avidity CD8+ T-cells were defined as those capable of producing IFN-γ in response to the lower concentration of peptide (0.1μM), while low-avidity CD8+ T-cells were those that only produced IFN-γ in response to the higher concentration of peptide (10 μM). Both high and low-avidity CD8+ T-cell responses could be detected for all peptides tested (median 1.05, 0.90, 0.52, 0.40 high/lowavidity ratios for PRA100, PRA142, PRA300 and PRA425 respectively). In patients with high PRAME expression (&gt;0.001 PRAME/ABL) low-avidity CD8+ T-cell responses to PRAME peptides were more prominent than high-avidity responses, suggesting selective deletion of high-avidity T-cells. In contrast, in some patients with levels &lt;0.001 PRAME/ABL, we could detect the presence of high-avidity CD8+ T-cell responses to PRAME. PRAME-specific CD8+ T-cells were further characterized by IC staining for IL-2, IL-4 and IL-10 production and CD107a mobilization (as a marker of cytotoxicity). Following stimulation with the relevant PRAME peptide, there was no significant production of IL-2, IL-4 or IL-10, suggesting a Tc1 effector response but no significant CD107a mobilization was detected despite significant CD107a mobilization in the same patient in response to CMVpp65495. This finding suggests that patients with leukemia have a selective functional impairment of PRAME-specific CD8+ T-cells, consistent with PRAME-specific T cell exhaustion. However, PRAME-specific T-cells were readily expanded in the presence of cytokines in short-term cultures in-vitro to produce IFN-γ, suggesting that it may be possible to improve the functional capacity of PRAME-specific T-cells for therapeutic purposes. These results provide evidence for spontaneous T-cell reactivity against multiple epitopes of PRAME in ALL, AML and CML and support the usefulness of PRAME as a target for immunotherapy in leukemia. The predominance of low-avidity PRAME-specific CD8+ T-cells suggests that achievement of a state of minimal residual disease may be required prior to peptide vaccination to augment T-cell immune surveillance.

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