Targeted deletion of T-cell clones using alpha-emitting suicide MHC tetramers

Blood ◽  
2004 ◽  
Vol 104 (8) ◽  
pp. 2397-2402 ◽  
Author(s):  
Rui Rong Yuan ◽  
Phillip Wong ◽  
Michael R. McDevitt ◽  
Ekaterina Doubrovina ◽  
Ingrid Leiner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immunosuppressive Agents ◽  
Cd8 T Cell ◽  
Model Systems ◽  
Clonal Deletion ◽  
T Cell Clones ◽  
Cell Clones ◽  
Mhc Tetramers

Abstract Immunosuppressive agents in current use are nonspecific. The capacity to delete specific CD8 T-cell clones of unique specificity could prove to be a powerful tool for dissecting the precise role of CD8+ T cells in human disease and could form the basis for a safe, highly selective therapy of autoimmune disorders. Major histocompatibility complex (MHC) tetramers (multimeric complexes capable of binding to specific CD8 T-cell clones) were conjugated to 225Ac (an alpha-emitting atomic nanogenerator, capable of single-hit killing from the cell surface) to create an agent for CD8 T-cell clonal deletion. The “suicide” tetramers specifically bound to, killed, and reduced the function of their cognate CD8 T cells (either human anti–Epstein-Barr virus (EBV) or mouse anti-Listeria in 2 model systems) while leaving the nonspecific control CD8 T-cell populations unharmed. Such an approach may allow a pathway to selective ablation of pathogenic T-cell clones ex vivo or in vivo without disturbing general immune function.

ROR1 Specific T Cell Clones from Healthy Individuals Show Common T Cell Receptor Motifs

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3364-3364
Author(s):  
Falk Heidenreich ◽  
Elke Ruecker-Braun ◽  
Juliane S. Stickel ◽  
Anne Eugster ◽  
Denise Kühn ◽  
...  
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
T Cell ◽  
Mhc Class I ◽  
T Cell Receptor ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Cell Receptor ◽  
T Cell Clones ◽  
Cell Clones

Abstract Background Immunotherapy for CLL with new antibodies or T-cells with modified TCR relies on attractive targets. ROR1 is such a promising target since it is highly overexpressed in CLL. Chimeric antigen receptor engineered T cells and antibodies directed against the extracellular part of ROR1 have already been developed and tested in vitro or in animal models, but still there is no MHC-class I presented peptide serving as target structure for CD8+ T cells (with or without a genetically modified T cell receptor) available. Aim The aim of this study was (1) to identify an immunogenic MHC-class I presented ROR1 peptide, (2) to generate respective ROR1 peptide specific CD8+ T cell clones, and (3) to analyze the nucleotide sequence of the CDR3 region of the expressed alpha and beta T cell receptor chain. Results In mass spectrometric-based analyses of the HLA-ligandome a HLA-B*07 presented ROR1 peptide was identified in primary CLL cells of two patients. Six T cell clones specific for this particular ROR1-peptide were generated from single CD8+ T cells from 2 healthy individuals with 3 T cell clones generated from each donor. Functionality and specificity of those T cell clones were tested in cytotoxicity assays. All 6 dextramer+ CD8+ T cell clones lysed peptide loaded and HLA-B*07+ transduced K562 cells (kindly provided by Lorenz Jahn, [Jahn et al., Blood, 2015 Feb 5;125(6):949-58]). Two selected clones (XD8 and XB6) were tested for their cytotoxic potential against 2 ROR1+ HLA-B*07+ tumor cell lines (with the ROR1 peptide identified by mass spectrometry for both of them) and against 2 primary CLL cell samples. Tested clones showed a significant lysis of the respective target cells. CDR3 regions of the alpha and beta T cell receptor chain were sequenced on a single cell level. The CDR3 alpha region from each of the 3 ROR1 specific T cell clones from donor A showed some similarities to T cell clones derived from donor B (Table 1). Conclusion For the first time a MHC-class I presented ROR1 peptide antigen is reported. ROR1 positive CLL cells can be targeted by specific HLA-B*07 restricted CTLs. Respective CD8+ T cell clones with anti-leukemic activity from 2 donors share some amino acid motifs of the CDR3 alpha and beta regions. In conclusion, this information provides the possibility of generating ROR1 specific CD8+ T cells with genetically modified T cell receptors for immunotherapy and for tracking those cells after administration with next generation sequencing in peripheral blood samples of patients. Furthermore, data suggest the existence of public TCR motifs in leukemia antigen specific CTLs, which needs to be proven in follow-up experiments with larger cohorts of donors and patients. Finally, the presented strategy to identify leukemia specific peptide antigens for CD8+ T cells might be an attractive method for similar projects. Table 1 Amino acid sequences of CDR3 alpha and beta regions of the TCR of ROR1 specific CD8+ T cell clones. When comparing two clones, matching amino acids are depicted in red. The aromatic amino acids phenylalanine (F) and tyrosine (Y) are shown in blue when situated at the same position. Gaps inserted during the sequence alignment process are indicated by a hyphen '-'. Table 1. Amino acid sequences of CDR3 alpha and beta regions of the TCR of ROR1 specific CD8+ T cell clones. When comparing two clones, matching amino acids are depicted in red. The aromatic amino acids phenylalanine (F) and tyrosine (Y) are shown in blue when situated at the same position. Gaps inserted during the sequence alignment process are indicated by a hyphen '-'. Disclosures Middeke: Sanofi: Honoraria. Schetelig:Sanofi: Honoraria.

scholarly journals Functional Characterization and Modulation of Cytokine Production by CD8+ T Cells from Human Immunodeficiency Virus-Infected Individuals

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3672-3681 ◽  
Author(s):  
Enrico Maggi ◽  
Roberto Manetti ◽  
Francesco Annunziato ◽  
Lorenzo Cosmi ◽  
Maria Grazia Giudizi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Clones ◽  
Cell Clones ◽  
Immunodeficiency Virus ◽  
Ifn Γ ◽  
Hiv Seropositive

CD8+ T-cell clones were generated from peripheral blood mononuclear cells (PBMC) of three human immunodeficiency virus (HIV)-seronegative individuals and six HIV-seropositive individuals and assessed for their cytokine secretion profile, cytolytic potential, and chemokine production. While the great majority of CD8+ T-cell clones generated from HIV-seronegative individuals produced interferon (IFN)-γ, but not interleukin-4 (IL-4), that is a type 1 cytotoxic (Tc1) profile, high numbers of CD8+ T-cell clones generated from HIV-seropositive individuals produced IL-4 in addition to IFN-γ or IL-4 alone, thus showing a type 0 cytotoxic (Tc0)- or a type 2 cytotoxic (Tc2) profile, respectively. Tc0/Tc2 cells displayed lower cytolytic activity than Tc1 cells, including a reduced ability to lyse autologous targets pulsed with HIV or HIV peptides. By contrast, the production of chemokines RANTES and macrophage inflammatory protein-1α was comparable in Tc1, Tc0, and Tc2 clones irrespective of whether they were derived from HIV-seronegative or HIV-seropositive individuals. When CD8+ T-cell clones were generated from PBMC cultures of HIV-seronegative individuals conditioned with IL-4 plus an anti–IL-12 antibody (Ab), a shift towards the Tc0/Tc2-like profile was observed. Conversely, the addition to PBMC cultures of IL-12 plus an anti – IL-4 Ab shifted the differentiation of CD8+ T cells from HIV-infected individuals towards the Tc1-like profile, whereas IL-12 or anti–IL-4 Ab alone had a lower Tc1-promoting effect. Irradiated PBMC from HIV-infected individuals, used as feeder cells, shifted the differentiation of CD8+ T cells from a healthy HIV-seronegative individual towards the Tc0/Tc2-like profile. On the other hand, a shift towards the Tc1-like profile was noted in CD8+ T-cell clones generated from the skin specimens of two HIV-seropositive patients with Kaposi's sarcoma, successfully treated with IFN-α, in comparison to CD8+ clones generated from the same skin areas before treatment. The IFN-α–induced Tc1 shift could be prevented by the incubation of skin-infiltrating CD8+ T cells with IL-4 before cloning. Taken together, these data indicate that both defective production of IL-12 and abnormal IL-4 production in bulk PBMC populations of HIV-infected individuals may contribute to the development of high numbers of CD8+ T-cell clones showing a Tc0/Tc2-like phenotype and reduced cytolytic potential against HIV itself. They also suggest that the cytokine profile of CD8+ T-cell clones can be modulated by cytokines (or anticytokine Ab) both in vitro and in vivo.

scholarly journals Regional and clonal T cell dynamics at single cell resolution in immune checkpoint blockade

2021 ◽  
Author(s):  
Joy A. Pai ◽  
Andrew Chow ◽  
Jennifer Sauter ◽  
Marissa Mattar ◽  
Hira Rizvi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Cd8 T Cell ◽  
Immune Checkpoint Blockade ◽  
Rna Seq ◽  
Cell Dynamics ◽  
T Cell Clones ◽  
Cell Clones

Paired T cell receptor and RNA single cell sequencing (scTCR/RNA-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scTCR/RNA-seq dataset of 162,062 single T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients who underwent resections for progressing lung cancers after immune checkpoint blockade (ICB). We found marked regional heterogeneity in tumor persistence that was associated with heterogeneity in CD4 and CD8 T cell phenotypes; regions with persistent cancer cells were enriched for follicular helper CD4 T cells (TFH), regulatory T cells (Treg), and exhausted CD8 T cells. Clonal analysis demonstrated that highly-expanded T cell clones were predominantly of the CD8 subtype, were ubiquitously present across all sampled regions, found in the peripheral circulation, and expressed gene signatures of 'large' and 'dual-expanded' clones that have been predictive of response to ICB. Longitudinal tracking of CD8 T cell clones in the peripheral blood revealed that the persistence of ubiquitous CD8 T cell clones, as well as phenotypically distinct clones with tumor-reactive features, correlated with systemic tumor control. Finally, tracking CD8 T cell clones across tissues revealed the presence of TCF-1+ precursor exhausted CD8 T cells in tumor draining LNs that were clonally linked to expanded exhausted CD8 T cells in tumors. Altogether, this comprehensive scTCR/RNA-seq dataset with regional, longitudinal, and clonal resolution provides fundamental insights into the tissue distribution, persistence, and differentiation trajectories of ICB-responsive T cells that underlie clinical responses to ICB.

scholarly journals Functional Characterization and Modulation of Cytokine Production by CD8+ T Cells from Human Immunodeficiency Virus-Infected Individuals

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3672-3681 ◽  
Author(s):  
Enrico Maggi ◽  
Roberto Manetti ◽  
Francesco Annunziato ◽  
Lorenzo Cosmi ◽  
Maria Grazia Giudizi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Clones ◽  
Cell Clones ◽  
Immunodeficiency Virus ◽  
Ifn Γ ◽  
Hiv Seropositive

Abstract CD8+ T-cell clones were generated from peripheral blood mononuclear cells (PBMC) of three human immunodeficiency virus (HIV)-seronegative individuals and six HIV-seropositive individuals and assessed for their cytokine secretion profile, cytolytic potential, and chemokine production. While the great majority of CD8+ T-cell clones generated from HIV-seronegative individuals produced interferon (IFN)-γ, but not interleukin-4 (IL-4), that is a type 1 cytotoxic (Tc1) profile, high numbers of CD8+ T-cell clones generated from HIV-seropositive individuals produced IL-4 in addition to IFN-γ or IL-4 alone, thus showing a type 0 cytotoxic (Tc0)- or a type 2 cytotoxic (Tc2) profile, respectively. Tc0/Tc2 cells displayed lower cytolytic activity than Tc1 cells, including a reduced ability to lyse autologous targets pulsed with HIV or HIV peptides. By contrast, the production of chemokines RANTES and macrophage inflammatory protein-1α was comparable in Tc1, Tc0, and Tc2 clones irrespective of whether they were derived from HIV-seronegative or HIV-seropositive individuals. When CD8+ T-cell clones were generated from PBMC cultures of HIV-seronegative individuals conditioned with IL-4 plus an anti–IL-12 antibody (Ab), a shift towards the Tc0/Tc2-like profile was observed. Conversely, the addition to PBMC cultures of IL-12 plus an anti – IL-4 Ab shifted the differentiation of CD8+ T cells from HIV-infected individuals towards the Tc1-like profile, whereas IL-12 or anti–IL-4 Ab alone had a lower Tc1-promoting effect. Irradiated PBMC from HIV-infected individuals, used as feeder cells, shifted the differentiation of CD8+ T cells from a healthy HIV-seronegative individual towards the Tc0/Tc2-like profile. On the other hand, a shift towards the Tc1-like profile was noted in CD8+ T-cell clones generated from the skin specimens of two HIV-seropositive patients with Kaposi's sarcoma, successfully treated with IFN-α, in comparison to CD8+ clones generated from the same skin areas before treatment. The IFN-α–induced Tc1 shift could be prevented by the incubation of skin-infiltrating CD8+ T cells with IL-4 before cloning. Taken together, these data indicate that both defective production of IL-12 and abnormal IL-4 production in bulk PBMC populations of HIV-infected individuals may contribute to the development of high numbers of CD8+ T-cell clones showing a Tc0/Tc2-like phenotype and reduced cytolytic potential against HIV itself. They also suggest that the cytokine profile of CD8+ T-cell clones can be modulated by cytokines (or anticytokine Ab) both in vitro and in vivo.

Transfer of Specific T Cell Clones with Graft-Versus-Leukemia (GVL) Activity from Donor to Recipient in Stem Cell Transplantation: Identification, Quantification and Characterization.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2958-2958 ◽  
Author(s):  
Katayoun Rezvani ◽  
David A. Price ◽  
Jason Brenchley ◽  
Yasemin Kilical ◽  
Emma Gostick ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Clones ◽  
Memory Phenotype ◽  
Cell Clones ◽  
Healthy Donors

Abstract The self-antigens PR1 and WT1 that are aberrantly expressed on malignant cells may be important target antigens for GVL effects from donor-derived anti-leukemia T cells. It is now clear that T cells recognizing these antigens circulate in transplant recipients and can be detected in small numbers in healthy individuals. To determine whether the same T cell clones in the donor are transferred to the recipient and induce GVL effects we sought for presence of leukemia-reactive T cell clones in healthy donors and their transfer into the patient after transplant and following DLI. We identified CD8+ T cell clones specific for PR1 and WT1 from 2 healthy donors. The HLA-A2/PR1-binding and HLA-A2/WT1-binding CD8+ T cells were purified by flow cytometric cell sorting and analyzed for their T cell receptor (TCR) usage by template switch anchored RT-PCR. This showed an oligoclonal population of WT1-specific CD8+ T cells and a polyclonal population of PR1-specific CD8+ T cells. In addition, using a fluorescent peptide/MHC class I multimeric complex incorporating mutations in the a3 domain that abrogate binding to the CD8 coreceptor, we selectively isolated WT1-specific CD8+ T cells of high functional avidity and demonstrated that high avidity T cells comprise a single clonotype. One patient with CML received an alloSCT from the donor in whom PR1-specific CD8+ T cell clones were detected. Using quantitative real-time PCR for IFN-g production and HLA-A2/PR1 tetrameric complexes, we showed the emergence of PR1-specific CD8+ T cells in the blood of the recipient 10 weeks after SCT and again 8 weeks post-DLI given to treat a molecular relapse of CML. HLA-A2/PR1 tetramer-positive CD8+ T cells were sorted by flow cytometry post-alloSCT and again following DLI. By comparing TCRb CDR3 sequences, we confirmed direct transfer and expansion of PR1-specific CD8+ T cell clones from the donor into the recipient and the reemergence of the same PR1-specific clones following DLI. The appearance of these HLA-A2/PR1 tetramer-positive CD8+ T cells was followed by complete molecular remission of CML by sensitive PCR for BCR/ABL The PR1-specific CD8+ T cells in the donor were of memory phenotype and expanded in the recipient after both alloSCT and DLI. During the early phase post-transfer in the recipient, the majority of PR1-specific CD8+ T cells had an effector memory phenotype (CD45RO+ CD57+). There was a shift towards a central memory phenotype (CD45 RO+ CD57−) during the course of the GVL effect. This is the first direct demonstration of the transfer of leukaemia-reactive specific T cell clones from a healthy donor to a patient with leukemia. Further, the identification and monitoring of T cell clones that mediate the GVL effect as described here can be undertaken before stem cell transplantation and could aid donor selection.

Anti-IL-10 Antibody Enhances the Proliferation of CD8+ T Cell Clones: Autoregulatory Role of Murine IL-10 in CD8+ T Cells

1994 ◽  
Vol 154 (1) ◽  
pp. 193-201 ◽  
Author(s):  
Ken-ichi Nishijima ◽  
Tatsuhiro Hisatsune ◽  
Yuji Minai ◽  
Masako Kohyama ◽  
Shuichi Kaminogawa
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Clones ◽  
Cell Clones

scholarly journals CD30 expression by CD8+ T cells producing type 2 helper cytokines. Evidence for large numbers of CD8+CD30+ T cell clones in human immunodeficiency virus infection.

1994 ◽  
Vol 180 (6) ◽  
pp. 2407-2411 ◽  
Author(s):  
R Manetti ◽  
F Annunziato ◽  
R Biagiotti ◽  
M G Giudizi ◽  
M P Piccinni ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Ifn Gamma ◽  
T Cell Clones ◽  
Cell Clones ◽  
Immunodeficiency Virus

A large panel of CD8+ T cell clones generated from peripheral blood lymphocytes (PBL) of healthy donors or human immunodeficiency virus (HIV)-infected individuals were assessed for both cytokine secretion profile and CD30 expression and release. The great majority of CD8+ T cell clones generated from healthy individuals showed the ability to produce interferon gamma (IFN-gamma), but not interleukin 4 (IL-4), and none of them either expressed membrane CD30 or released substantial amounts of soluble CD30 (sCD30) in their supernatant. In contrast, high numbers of CD8+ T cell clones generated from HIV-infected individuals, which produced IL-4 (and IL-5) in addition to IFN-gamma or IL-4 (and IL-5) alone, expressed membrane CD30 and released detectable amounts of sCD30 in their supernatants. Indeed, CD30 expression appeared to be positively correlated with the ability of CD8+ T cell clones to produce IL-4 and IL-5 and inversely correlated with their ability to produce IFN-gamma, whereas no correlation between CD30 expression and production of IL-10 was observed. These data suggest that CD30 is a marker for CD8+ T cells that have switched to the production of type 2 helper cytokines.

scholarly journals TCR-MHC Interaction Strength Defines Trafficking and Resident Memory Status of CD8 T cells in the Brain

2018 ◽  
Author(s):  
Anna Sanecka ◽  
Nagisa Yoshida ◽  
Elizabeth Motunrayo Kolawole ◽  
Harshil Patel ◽  
Brian D. Evavold ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Chronic Infection ◽  
Cd8 T Cell ◽  
T Cell Clones ◽  
Cell Clones ◽  
T Cell Clone ◽  
Wide Range ◽  
The Brain

AbstractT cell receptor-Major histocompatibility complex (TCR-MHC) affinities span a wide range in a polyclonal T cell response, yet it is undefined how affinity shapes long-term properties of CD8 T cells during chronic infection with persistent antigen. Here, we investigate how the affinity of the TCR-MHC interaction shapes the phenotype of memory CD8 T cells in the chronically Toxoplasma gondii-infected brain. We employed CD8 T cells from three lines of transnuclear (TN) mice that harbour in their endogenous loci different T cell receptors specific for the same Toxoplasma antigenic epitope ROP7. The three TN CD8 T cell clones span a wide range of affinities to MHCI-ROP7. These three CD8 T cell clones have a distinct and fixed hierarchy in terms of effector function in response to the antigen measured as proliferation capacity, trafficking, T cell maintenance and memory formation. In particular, the T cell clone of lowest affinity does not home to the brain. The two higher affinity T cell clones show differences in establishing resident memory populations (CD103+) in the brain with the higher affinity clone persisting longer in the host during chronic infection. Transcriptional profiling of naïve and activated ROP7-specific CD8 T cells revealed that Klf2 encoding a transcription factor that is known to be a negative marker for T cell trafficking is upregulated in the activated lowest affinity ROP7 clone. Our data thus suggest that TCR-MHC affinity dictates memory CD8 T cell fate at the site of infection.

Profound Shut-Down of Antigen Specific T-Cell Effector Functions by Dasatinib.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1348-1348
Author(s):  
Ralf Weichsel ◽  
Carolin Dix ◽  
Linda Wooldrige ◽  
Matthew Clement ◽  
Angharad Fenton-May ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Cd8 T Cells ◽  
Kinase Inhibitor ◽  
Cd8 T Cell ◽  
Inhibition Of Proliferation ◽  
T Cell Clones ◽  
Cell Functions ◽  
Cell Clones

Abstract Dasatinib (Sprycel®, Bristol-Myers Squibb) is a dual BCR-ABL/SRC kinase inhibitor. In 2006, it was approved for the treatment of imatinib-refractory/-resistant CML and Ph+ ALL. An increased infection rate has been described in patients undergoing dasatinib treatment; we postulated that this might be due to inhibition of the SRC kinases LCK and FYN, which play an important role in T cell signalling following antigen recognition. We evaluated dasatinib’s immunobiological effects on purified human CD3+ T cells from healthy blood donors using the promiscuous tyrosine kinase inhibitor staurosporine as a comparator; we also used antigen-specific T cell clones to dissect early T cell signaling events. All assays were performed at clinically relevant doses of dasatinib (1–100nM). In CFSE dilution assays, a dose-dependent inhibition of T cell proliferation was detected with dasatinib (IC50=11nM) and staurosporine (IC50=5nM). These effects of dasatinib, but not staurosporine, were reversible; thus, T cells that were pre-incubated with dasatinib for 24h and then washed, proliferated as well as untreated T cells in the absence of dasatinib. Furthermore, we observed significant inhibition of OKT3-induced up-regulation of the activation marker CD69 (dasatinib IC50=11nM, staurosporine IC50=5nM) and reduced IL-2 production (IC50=2nM, measured by ELISA, for both dasatinib and staurosporine) in purified T cells treated with dasatinib and staurosporine. CD4+ T cells were more sensitive than CD8+ T cells to the inhibitory effects of dasatinib on activation and proliferation (activation IC50=10nM for CD4+ and 15nM for CD8+ T cells; proliferation IC50=10nM for CD4+ and 13nM for CD8+ T cells) while no differences in sensitivity were observed for staurosporine (IC50=4–5 for activation and proliferation in both CD4+ and CD8+ T cells). Since CD8+ T cell-mediated immunity is essential for long-term control of persistent DNA viruses, we evaluated dasatinib’s impact on antigen-specific alpha/beta CD8+ T cell populations specific for CMV and EBV. Profound inhibition of proliferation, cytokine secretion and degranulation was observed in all cases; these observations potentially explain the increased frequency of viral infections in dasatinib-treated patients beyond the induction of myelosuppression. Of note, gamma/delta T cell functions were also inhibited by dasatinib. Dasatinib resulted in significant upregulation of TCR/CD8 at the T cell surface of EBV specific T cell clones indicating that the drug blocks TCR downregulation and recycling at the T cell surface and therefore exerts its effects by blockade of proximal signalling pathways. Overall, these findings provide a rationale to explore the potential of dasatinib as an immunosuppressant in the settings of transplantation and T cell driven autoimmune diseases.

Detailed Analysis of CD8+ T Cell Immunity and Identification of a Novel Minor Histocompatibility Antigen Contributing to Graft-Versus- Leukemia Reactivity.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3250-3250
Author(s):  
Marieke Griffioen ◽  
M. Willy Honders ◽  
Edith D. van der Meijden ◽  
Roelof Willemze ◽  
J.H. Frederik Falkenburg
Keyword(s):  
T Cells ◽  
T Cell ◽  
Detailed Analysis ◽  
Cd8 T Cells ◽  
Cell Clone ◽  
Hematopoietic Cells ◽  
Cd8 T Cell ◽  
T Cell Clones ◽  
Cell Clones ◽  
T Cell Clone

Abstract Donor lymphocyte infusion (DLI) can be an effective cellular immunotherapy for patients with hematological malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). The effect of DLI is mediated by donor derived T cells recognizing minor histocompatibility antigens (mHags) on malignant cells of the recipient. These donor originated T cells may also induce Graft-versus-Host Disease (GvHD) when directed against mHags with broad expression on non-malignant tissues of the patient. In this study, we performed a detailed analysis and characterization of mHags recognized by CD8+ T cells contributing to Graft-versus-Leukemia (GvL) reactivity in a patient treated with DLI for relapsed chronic myeloid leukemia (CML) more than one year after HLA-matched alloSCT. The GvL effect in this patient was accompanied with only mild GvHD of the skin. To investigate the specificity of the CD8+ T cell response induced in this patient, activated (HLA-DR+) CD8+ T cells were single cell sorted from a bone marrow sample obtained five weeks after DLI by flowcytometry. A number of isolated CD8+ T cell clones were shown to be specific for mHags, as determined by differential recognition of patient and donor EBV-transformed B cells (EBV-LCL) in IFN-g ELISA and 51Crrelease assays. By screening a panel of third party EBV-LCL sharing one or more HLA class I restriction molecules with the patient, CD8+ T cell clones directed against 7 different mHags were identified, including the known hematopoiesis restricted mHags HA-1 and HA-2, which are presented in HLA-A*0201. Of the 5 remaining specificities, 4 mHags were presented in HLA-B*4001 (B60) and one mHag in HLA-B*0801. To determine the tissue distribution patterns in more detail, we tested recognition of selected non-malignant hematopoietic cells (monocytes, B cells, T cells), malignant CD34+ CML precursor cells, and skin-derived fibroblasts. One HLA-B*4001-restricted T cell clone (clone ZRZ16) failed to recognize all primary hematopoietic cells and skin fibroblasts. The four remaining T cell clones were all capable of recognizing and lysing (specific subsets of) non-malignant hematopoietic cells and malignant CD34+ CML precursor cells. Fibroblast recognition could be demonstrated for two of these four T cell clones. Since clone ZRZ16 failed to recognize all hematopoietic and non-hematopoietic cells, except for EBV-LCL, we tested whether this clone selectively recognizes antigen presenting cells (APC), which are known to be required for efficient induction of immune responses in vivo. Clone ZRZ16 showed indeed strong recognition of monocyte-derived dendritic cells as well as in vitro differentiated CD34+ CML cells with APC phenotype. To identify the mHag recognized by the CD8+ T cell clone, we screened a cDNA expression library constructed from EBV-LCL from the patient. One single cDNA was isolated as the target for B*4001 restricted CD8+ T cell clone ZRZ16. The epitope recognized by this clone was derived from the 3’ untranslated region (UTR) of a cDNA encoding thyroid hormone receptor interactor 10 (TRIP10). The peptide epitope was translated in a reading frame different from the TRIP10 protein and comprises three single nucleotide polymorphisms, which were all different between patient and donor. Two of the three SNPs were shown to be important for recognition by clone ZRZ16. Despite ubiquitous tissue expression of the TRIP10 gene as determined by public microarray analysis, CD8+ T cells specific for the newly-identified LB-TRIP10-1EPC mHag selectively recognized APC and failed to recognize CD34+ CML precursor cells, suggesting a predominant role in the initiation, but not effector, phase of the anti-tumor response. In conclusion, our data show a detailed analysis of mHag specific CD8+ T cell immunity induced in a patient successfully treated with DLI for relapsed CML and provide evidence for differential involvement of HLA class I restricted mHags in the onset and execution of GvL reactivity.

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