Allorestricted cytotoxic T cells specific for human CD45 show potent antileukemic activity

Blood ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 1007-1014 ◽  
Author(s):  
Persis J. Amrolia ◽  
Steven D. Reid ◽  
Liquan Gao ◽  
Beate Schultheis ◽  
Gianpietro Dotti ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Myeloid Leukemia ◽  
Mononuclear Cells ◽  
Donor Cell ◽  
Healthy Controls ◽  
Specific Ctls ◽  
Haploidentical Transplantation ◽  
Cell Engraftment

Abstract Recent advances have made haploidentical transplantation for leukemia feasible, but the rigorous T-cell depletion used contributes to the high relapse rates observed. We have attempted to improve the graft-versus-leukemia (GVL) effect by generating allorestricted cytotoxic T lymphocytes (CTLs) directed against human CD45. Such CTLs should recognize patient hematopoietic cells including leukemia, enhancing donor cell engraftment and improving the GVL effect, but they should not recognize host nonhematopoietic tissues or donor cells from the graft. Using the T2 binding assay, 4 CD45-derived peptides were found to bind HLA-A2 molecules. These peptides were used to generate cytotoxic T-cell lines from HLA-A2− donors by sequential stimulation with peptide-pulsed HLA-A2+ stimulators, and the lines obtained were screened for peptide-specific cytotoxicity. Using one of these peptides (P1218), it was possible to generate peptide-specific, allorestricted CTLs in 3 of 7 responders. P1218-specific CTL lines show potent cytotoxicity against hematopoietic cell lines coexpressing HLA-A2 and CD45 but not CD45 loss variants. Studies with stable transfectants of 293 cells demonstrated recognition by P1218-specific CTLs of endogenously expressed CD45. Likewise P1218-specific CTLs recognized peripheral blood mononuclear cells (PBMCs) from HLA-A2+ patients with chronic myeloid leukemia (CML) and leukemic blasts in HLA-A2+ patients with acute myeloid leukemia (AML), but they were unable to lyse HLA-A2+ fibroblasts or HLA-A2− normal PBMCs. Coculture of CD34+ PBMCs and bone marrow mononuclear cells (BMMCs) with P1218-specific CTL significantly inhibited colony-forming unit–granulocyte macrophage (CFU-GM) formation in HLA-A2+healthy controls and CML patients but resulted in no significant inhibition in HLA-A2− healthy controls. These studies demonstrate that P1218-specific cytotoxic T lymphocytes (CTLs) have potent activity against leukemic progenitors and suggest that adoptive immunotherapy with allorestricted CTLs directed against CD45 epitopes may be useful in restoring the GVL effect after HLA-A2–mismatched haploidentical transplantation. Further, because P1218-specific CTLs also recognize healthy HLA-A2+ progenitors, such CTLs could also contribute to host myeloablation and enhance donor cell engraftment.

Target Epitopes of Human T Lymphotropic Virus 1 Recognized by Class I MHC-Restricted Cytotoxic T Lymphocytes in HAM/TSP Patients and Infected Patients with Autoimmune Disorders.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2660-2660
Author(s):  
Tomohiro Kozako ◽  
Masaki Akimoto ◽  
Yohann White ◽  
Shingo Toji ◽  
Kakushi Matsushita ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Spastic Paraparesis ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Class I Mhc ◽  
Collagen Diseases ◽  
Specific Ctls ◽  
Significant Difference

Abstract Abstract 2660 Poster Board II-636 Human T-cell leukemia virus-1 (HTLV-1) causes adult T cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) after long-term infection. A variety of collagen diseases such as Sjögren's syndrome and systemic lupus erythematosus has been reported in HTLV-1-infected individuals, although the precise relationship between these disorders and HTLV-1 infection remains unknown. We have previously reported the decreased frequency and function of HTLV-1 Tax-specific cytotoxic T lymphocytes (CTLs) in ATL patients due to insufficient cytolytic effector molecules compared with asymptomatic carriers (ACs) (ASH annual meeting 2007). However, there is no report on the repertoire of HTLV-1-specific CTLs in HAM/TSP patients or carriers with collagen diseases (CCs), both of which are characterized by abnormal immune states. In order to characterize HTLV-1-specific CTLs in ACs, HAM/TSP patients and CCs, we examined the frequency and diversity of HTLV-1-specific CTLs using 16 distinct HTLV-1 Tax and Env HLA-A*0201 and HLA-A*2402 tetramers. There was no statistically significant difference in the proportion of patients having HTLV-1 Tax specific CTLs among ACs, HAM/TSP and CC patients, but a greater proportion of patients with HAM/TSP or collagen disease had HTLV-1 Env-specific CTLs compared with ACs (Env175-183, Env239-246, Env442-450 for HLA-A*0201 and Env11-19, Env21-29, Env153-161 for HLA-A*2402) (Table 1). Within CD8+ lymphocyte subsets, the percentage of cells binding either Tax11-19/HLA-A*0201 or Tax 301-309/HLA-A*2402 tetramer (Figure 1), was significantly higher for HAM/TSP patients compared to ACs, being lowest among CCs. Additionally, the number of epitope repertoires found on HTLV-1 Env-specific CD8+ cells in ACs was considerably lower than for HAM/TSP patients and CCs (ACs, 1/6; HAM/TSP, 4/6; CCs, 6/6). This study demonstrates the relatively greater importance of CTLs recognizing HTLV-1 envelope tetramers compared to HTLV-1 Tax tetramers, thereby suggesting that the diversity, frequency and repertoire of HTLV-1-specific CTL clones, especially Env-CTLs, may be related to the hyperimmune response in HAM/TSP and carriers with collagen diseases. Disclosures: No relevant conflicts of interest to declare.

Ex vivo induction of multiple myeloma–specific cytotoxic T lymphocytes

Blood ◽  
2003 ◽  
Vol 102 (4) ◽  
pp. 1435-1442 ◽  
Author(s):  
Toshiaki Hayashi ◽  
Teru Hideshima ◽  
Masaharu Akiyama ◽  
Noopur Raje ◽  
Paul Richardson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
T Cell ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Stimulation Index ◽  
T Cell Proliferation ◽  
Autologous Tumor

Abstract Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by immunosuppression. In this study, we identified factors in patients' bone marrow (BM) sera inhibiting autologous anti-MM immunity and developed an ex vivo strategy for inducing MM-specific cytotoxic T lymphocytes (CTLs). We found that sera from BM of MM patients inhibited induction of dendritic cells (DCs), evidenced by both phenotype and only weak stimulation of T-cell proliferation. Anti–vascular endothelial growth factor (anti-VEGF) and/or anti–interleukin 6 (anti–IL-6) antibodies neutralized this inhibitory effect, confirming that VEGF and IL-6, at least in part, mediate immunosuppression in MM patients. To induce MM-specific CTLs ex vivo, immature DCs were generated by culture of adherent mononuclear cells in medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 5 days and then cocultured with apoptotic MM bodies in the presence of tumor necrosis factor α (TNF-α) for 3 days to induce their maturation. Autologous BM or peripheral blood mononuclear cells were stimulated weekly with these DCs, and cytotoxicity was examined against the MM cells used to pulse DCs. DCs cultured with apoptotic bodies stimulated significantly greater T-cell proliferation (stimulation index [SI] = 23.2 at a T-DC ratio of 360:1) than T cells stimulated by MM cells only (SI = 5.6), DCs only (SI = 9.3), or MM lysate–pulsed DCs (SI = 13.5). These CTLs from MM patients demonstrated specific cytotoxicity (24.7% at the effector-target [E/T] ratio of 40:1) against autologous primary MM cells. These studies therefore show that CTLs from MM patients can recognize and lyse autologous tumor cells and provide the framework for novel immunotherapy to improve patient outcome in MM.

scholarly journals Detection of JC Virus-Specific Cytotoxic T Lymphocytes in Healthy Individuals

2004 ◽  
Vol 78 (18) ◽  
pp. 10206-10210 ◽  
Author(s):  
R. A. Du Pasquier ◽  
J. E. Schmitz ◽  
J. Jean-Jacques ◽  
Y. Zheng ◽  
J. Gordon ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Mononuclear Cells ◽  
Demyelinating Disease ◽  
Jc Virus ◽  
T Cell Response ◽  
Healthy Individuals ◽  
Peripheral Blood Mononuclear ◽  
Specific Ctls ◽  
The Central Nervous System

ABSTRACT The polyomavirus JC (JCV) infects 85% of healthy individuals, and its reactivation in a limited number of immunosuppressed people causes progressive multifocal leukoencephalopathy (PML), a severe demyelinating disease of the central nervous system. We hypothesized that JCV-specific cytotoxic T lymphocytes (CTLs) might control JCV replication in healthy individuals, blocking the evolution of PML. Using 51Cr release and tetramer staining assays, we show that 8 of 11 HLA-A*0201+ healthy subjects (73%) harbor detectable JCV-specific CD8+ CTLs that recognize one or two epitopes of JCV VP1 protein, the HLA-A*0201-restricted VP1p36 and VP1p100 epitopes. We determined that the frequency of JCV VP1 epitope-specific CTLs varied from less than 1/100,000 to 1/2,494 peripheral blood mononuclear cells. More individuals had JCV VP1-specific than cytomegalovirus-specific CTLs (8 of 11 subjects [73%] versus 2 of 10 subjects [20%], respectively). These results show that a CD8+-T-cell response against JCV is commonly found in immunocompetent people and suggest that these cells might protect against the development of PML.

The kinase insert domain-containing receptor is an angiogenesis-associated antigen recognized by human cytotoxic T lymphocytes

Blood ◽  
2006 ◽  
Vol 107 (4) ◽  
pp. 1476-1483 ◽  
Author(s):  
Yuansheng Sun ◽  
Stefan Stevanović ◽  
Mingxia Song ◽  
Astrid Schwantes ◽  
C. James Kirkpatrick ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Antigenic Property ◽  
Cellular Immunotherapy ◽  
Target Cells ◽  
Peripheral Blood Mononuclear ◽  
Specific Ctls ◽  
Class 1

Antigen-specific cancer immunotherapy directed toward tumor-nourishing angiogenic blood vessels holds the promise of high efficacy, low toxicity, and ease of application. To evaluate whether the human angiogenic kinase insert domain-containing receptor (KDR) can serve as a target for cellular immunotherapy, 19 peptide sequences with HLA-A*0201 motifs were selected by computer-based algorithms. Five peptides (KDR82-90, KDR288-297, KDR766-774, KDR1093-1101, KDR1035-1044) stimulated specific cytotoxic T lymphocytes (CTLs) from peripheral-blood mononuclear cells (PBMCs) of 3 HLA-A*0201 donors. The decapeptide KDR288-297 was efficient in sensitizing target cells for recognition by a CTL clone at a concentration of 10 nM. More important, KDR288-297 - specific CTLs lysed target cells transfected with HLA-A2/KDR cDNAs and a range of HLA-matched KDR+ angiogenic endothelial cells (aECs) and also recognized CD34+ endothelial progenitor cells. The specificity of CTLs was further confirmed by tetramer assay and cold-target inhibition assay. In addition, ex vivo exposure of aECs to the inflammatory cytokines enhanced CTL reactivity, which is in keeping with up-regulated KDR and HLA class 1 expression. In Matrigel assays, recognition of aECs by specific CTLs triggered an antivascular effect. These findings provide the first proof of the antigenic property of KDR protein and may be useful for devising new immunotherapeutic approaches to human cancers.

Generation and Expansion of PRAME-Specific Cytotoxic T-Lymphocytes for Adoptive T-Cell Therapy of Hematological Malignancies.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2205-2205
Author(s):  
Concetta Quintarelli ◽  
Gianpietro Dotti ◽  
Fabrizio Pane ◽  
Cliona M. Rooney ◽  
Malcolm K. Brenner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Line ◽  
Chronic Myelogenous Leukemia ◽  
Cytotoxic T Lymphocytes ◽  
Peripheral Blood ◽  
Myelogenous Leukemia ◽  
Specific Ctls ◽  
Irrelevant Peptide

Abstract The cancer testis antigen PRAME is a potential target for T-cell based adoptive immunotherapy for many myeloid and lymphoid malignancies, since these cells frequently either overexpress the antigen constitutively, or following induction with demethylating agents. Although several HLA class I A*0201 PRAME-derived T cell epitopes have been previously identified, the ex-vivo generation of PRAME-specific cytotoxic T lymphocytes (CTLs) has proved to be a major challenge. We have now optimized a method that consistently and reproducibly generates peptide tumor-specific CTLs. CD8+ cells selected from peripheral blood mononuclear cells. HLA A*0201 donors were first primed with autologous CD40L-activated B blasts loaded with A2-restricted peptides in the presence of low doses of IL-2, IL-7 and IL-15. Following this initial priming, we re-stimulated the T cells with an artificial antigen presenting cell line (AAPC), consisting of the human chronic myelogenous leukemia cell line K562 genetically modified to stably express the HLA-A*0201 molecule and the CD80 co-stimulatory molecule (K562/A*0201/CD80). These AAPC were loaded with HLA-A2 restricted peptides and used to stimulate CTLs in the presence of IL-2. Table 1 shows that this method consistently generated CTLs recognizing multiple HLA-A2 restricted peptides derived from well-characterized tumor-associated antigens including hTERT and PR1. The frequency of expanded T-lymphocytes was evaluated by IFNγ Elispot assay. Table 1. Irrelevant peptide Mart1-ELA Mage3-KVA PR1-VLQ WT1-RMF hTERT-RLV hTERT-ILA Tyr-YMD Number of IFNγ Spot Forming Cells (SFC)/10^5 cells (4 donors) 63±12 895±116 676±37 325±71 560±54 650±70 1019±240 897±127 8±3 810±30 488±19 15±10 48±18 13±7 101±12 503±20 7±2 1379±105 54±11 140±10 631±19 744±33 465±8 1103±97 3±2 1430±52 2±0 3±1 3±1 20±18 376±11 632±121 The specificity of the response was confirmed by tetramer analysis and cytotoxic activity with 51Cr release assay on peptide-loaded PHA blasts. We then evaluated whether this approach could be used to generate and expand PRAME-specific CTLs. In 5 HLA A*0201 healthy donors, after priming with B-blasts and 4 stimulations with K562/A*0201/CD80 cells loaded with the PRAME-derived peptide ALY, we obtained 22±7 fold T-cell expansion. T cells were PRAME specific, as the frequency of IFNγ+ T cells after exposure to the ALY peptide was significantly higher compared to exposure to irrelevant peptide (511±260 IFNg SFC/105 cells vs. 30±10 IFNg SFC/105 cells) in 4 of the 5 donors tested. In addition, CTLs significantly lysed autologous-PHA blasts loaded with ALY (84±12% at 20:1 E:T ratio) while lysis of blasts loaded with the irrelevant peptide was <10%. We then tested whether PRAME-CTLs recognized primary tumor cells, using CD33+ blast cells selected from the peripheral blood of 2 HLA A*0201 patients with PRAME expressing Chronic Myelogenous Leukemia (as assessed by Real Time PCR). The frequency of IFNγ+ T cells was 320±31 SFC/105 when ALY-CTLs were used as effector cells, but only 6±2 SFC/105 for CTLs expanded from the same donors using an irrelevant antigen. In conclusion, our data show that we can efficiently stimulate and expand PRAME-specific CTLs, and suggest that our approach could be developed for clinical application.

Fiber-modified adenoviruses generate subgroup cross-reactive, adenovirus-specific cytotoxic T lymphocytes for therapeutic applications

Blood ◽  
2004 ◽  
Vol 103 (3) ◽  
pp. 1011-1019 ◽  
Author(s):  
Ann M. Leen ◽  
Uluhan Sili ◽  
Barbara Savoldo ◽  
Alan M. Jewell ◽  
Pedro A. Piedra ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Target Cells ◽  
Cell Transplant ◽  
Peripheral Blood Mononuclear ◽  
Hematopoietic Stem ◽  
Considerable Morbidity

AbstractAdenovirus (Ad) infections are responsible for considerable morbidity and mortality, particularly in pediatric hematopoietic stem cell transplant (HSCT) recipients. To date there is no therapy. The present study was motivated by the potential for using adoptive immunotherapy as either prophylaxis or treatment for Ad infections and associated diseases. The authors have developed a protocol to reactivate Ad-specific memory T cells from peripheral blood mononuclear cells (PBMCs) using a clinical-grade adenoviral vector. Such lines contain a specific CD4 and CD8 T-cell component and are capable of recognizing and lysing target cells infected with wild-type Ad serotypes from different Ad groups. Furthermore, the frequency of Ad-specific precursors can be determined in PBMCs ex vivo and used as a means to assess changes in Ad-specific T-cell memory responses after infusion. This is the first report of a simple and reproducible method to activate and expand Ad-specific cytotoxic T lymphocytes (CTLs), which should be protective against the range of different Ad subtypes that affect transplant recipients. (Blood. 2004;103:1011-1019)

scholarly journals Generation of Pure Highly Functional Human Anti-Tumor Specific Cytotoxic T Lymphocytes With Stem Cell-Like Memory Features for Melanoma Immunotherapy

2021 ◽  
Vol 12 ◽  
Author(s):  
Mohamad Hamieh ◽  
Jean-François Chatillon ◽  
Estelle Dupel ◽  
Florence Bayeux ◽  
Emilie Fauquembergue ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Adoptive Immunotherapy ◽  
Mouse Fibroblast ◽  
Rapid Expansion ◽  
Effector Memory ◽  
Memory T Cell ◽  
Specific Ctls

Adoptive immunotherapy based on the transfer of anti-tumor cytotoxic T lymphocytes (CTLs) is a promising strategy to cure cancers. However, rapid expansion of numerous highly functional CTLs with long-lived features remains a challenge. Here, we constructed NIH/3T3 mouse fibroblast-based artificial antigen presenting cells (AAPCs) and precisely evaluated their ability to circumvent this difficulty. These AAPCs stably express the essential molecules involved in CTL activation in the HLA-A*0201 context and an immunogenic HLA-A*0201 restricted analogue peptide derived from MART-1, an auto-antigen overexpressed in melanoma. Using these AAPCs and pentamer-based magnetic bead-sorting, we defined, in a preclinical setting, the optimal conditions to expand pure MART-1-specific CTLs. Numerous highly purified MART-1-specific CTLs were rapidly obtained from healthy donors and melanoma patients. Both TCR repertoire and CDR3 sequence analyses revealed that MART-1-specific CTL responses were similar to those reported in the literature and obtained with autologous or allogeneic presenting cells. These MART-1-specific CTLs were highly cytotoxic against HLA-A*0201+ MART-1+ tumor cells. Moreover, they harbored a suitable phenotype for immunotherapy, with effector memory, central memory and, most importantly, stem cell-like memory T cell features. Notably, the cells harboring stem cell-like memory phenotype features were capable of self-renewal and of differentiation into potent effector anti-tumor T cells. These “off-the-shelf” AAPCs represent a unique tool to rapidly and easily expand large numbers of long-lived highly functional pure specific CTLs with stem cell-like memory T cell properties, for the development of efficient adoptive immunotherapy strategies against cancers.

scholarly journals Simultaneous Induction of Multiple Antigen-Specific Cytotoxic T Lymphocytes in Nonhuman Primates by Immunization with a Mixture of Four Plasmodium falciparum DNA Plasmids

1998 ◽  
Vol 66 (9) ◽  
pp. 4193-4202 ◽  
Author(s):  
Ruobing Wang ◽  
Denise L. Doolan ◽  
Yupin Charoenvit ◽  
Richard C. Hedstrom ◽  
Malcolm J. Gardner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Mononuclear Cells ◽  
T Cell Responses ◽  
Peripheral Blood Mononuclear ◽  
Effector Cells ◽  
Cell Responses

ABSTRACT CD8+ T cells have been implicated as critical effector cells in protective immunity against malaria parasites developing within hepatocytes. A vaccine that protects against malaria by inducing CD8+ T cells will probably have to include multiple epitopes on the same protein or different proteins, because of parasite polymorphism and genetic restriction of T-cell responses. To determine if CD8+ T-cell responses against multiple P. falciparum proteins can be induced in primates by immunization with plasmid DNA, rhesus monkeys were immunized intramuscularly with a mixture of DNA plasmids encoding four P. falciparumproteins or with individual plasmids. All six monkeys immunized with PfCSP DNA, seven of nine immunized with PfSSP2 DNA, and five of six immunized with PfExp-1 or PfLSA-1 DNA had detectable antigen-specific cytotoxic T lymphocytes (CTL) after in vitro restimulation of peripheral blood mononuclear cells. CTL activity was genetically restricted and dependent on CD8+ T cells. By providing the first evidence for primates that immunization with a mixture of DNA plasmids induces CD8+ T-cell responses against all the components of the mixture, these studies provide the foundation for multigene immunization of humans.

scholarly journals Identification of Two New HLA-A*1101-Restricted Tax Epitopes Recognized by Cytotoxic T Lymphocytes in an Adult T-Cell Leukemia Patient after Hematopoietic Stem Cell Transplantation

2005 ◽  
Vol 79 (15) ◽  
pp. 10088-10092 ◽  
Author(s):  
Nanae Harashima ◽  
Ryuji Tanosaki ◽  
Yukiko Shimizu ◽  
Kiyoshi Kurihara ◽  
Takao Masuda ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
T Lymphocytes ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cytotoxic T Lymphocytes ◽  
Cell Leukemia ◽  
Hematopoietic Stem ◽  
Adult T Cell Leukemia

ABSTRACT We previously reported that Tax-specific CD8+ cytotoxic T lymphocytes (CTLs), directed to single epitopes restricted by HLA-A2 or A24, expanded in vitro and in vivo in peripheral blood mononuclear cells (PBMC) from some adult T-cell leukemia (ATL) patients after but not before allogeneic hematopoietic stem cell transplantation (HSCT). Here, we demonstrated similar Tax-specific CTL expansion in PBMC from another post-HSCT ATL patient without HLA-A2 or A24, whose CTLs equally recognized two newly identified epitopes, Tax88-96 and Tax272-280, restricted by HLA-A11, suggesting that these immunodominant Tax epitopes are present in the ATL patient in vivo.

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