Shedding Light on Drug-Induced Liver Injury: Activation of T Cells From Drug Naive Human Donors With Tolvaptan and a Hydroxybutyric Acid Metabolite

2020 ◽  
Author(s):  
Sean Hammond ◽  
Andrew Gibson ◽  
Kanoot Jaruthamsophon ◽  
Sharin Roth ◽  
Merrie Mosedale ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Liver Injury ◽  
Antigen Processing ◽  
Hydroxybutyric Acid ◽  
T Cell Clones ◽  
Adaptive Immune ◽  
Cell Clones ◽  
T Cell Clone ◽  
Immune Attack

Abstract Exposure to tolvaptan is associated with a significant risk of liver injury in a small fraction of patients with autosomal dominant polycystic kidney disease. The observed delayed onset of liver injury of between 3 and 18 months after commencing tolvaptan treatment, along with rapid recurrence of symptoms following re-challenge is indicative of an adaptive immune attack. This study set out to assess the intrinsic immunogenicity of tolvaptan and pathways of drug-specific T-cell activation using in vitro cell culture platforms. Tolvaptan (n = 7), as well as oxybutyric (DM-4103, n = 1) and hydroxybutyric acid (DM-4107, n = 18) metabolite-specific T-cell clones were generated from tolvaptan naive healthy donor peripheral blood mononuclear cells. Tolvaptan and DM-4103 T-cell clones could also be activated with DM-4107, whereas T-cell clones originally primed with DM-4107 were highly specific to this compound. A signature cytokine profile (IFN-γ, IL-13, granzyme B, and perforin) for almost all T-cell clones was identified. Mechanistically, compound-specific T-cell clone activation was dependent on the presence of soluble drug and could occur within 4 h of drug exposure, ruling out a classical hapten mechanism. However, antigen processing dependence drug presentation was indicated in many T-cell clones. Collectively these data show that tolvaptan-associated liver injury may be attributable to an adaptive immune attack upon the liver, with tolvaptan- and metabolite-specific T cells identified as candidate effector cells in such etiology.

scholarly journals Emergence of CD52-, phosphatidylinositolglycan-anchor-deficient T lymphocytes after in vivo application of Campath-1H for refractory B- cell non-Hodgkin lymphoma

Blood ◽  
1995 ◽  
Vol 86 (4) ◽  
pp. 1487-1492 ◽  
Author(s):  
B Hertenstein ◽  
B Wagner ◽  
D Bunjes ◽  
C Duncker ◽  
A Raghavachar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Molecular Characteristics ◽  
T Cell Clones ◽  
Non Hodgkin Lymphoma ◽  
Cell Clones ◽  
Immune Attack

CD52 is a phosphatidylinositolglycan (PIG)-anchored glycoprotein (PIG- AP) expressed on normal T and B lymphocytes, monocytes, and the majority of B-cell non-Hodgkin lymphomas. We observed the emergence of CD52- T cells in 3 patients after intravenous treatment with the humanized anti-CD52 monoclonal antibody Campath-1H for refractory B- cell lymphoma and could identify the underlaying mechanism. In addition to the absence of CD52, the PIG-AP CD48 and CD59 were not detectable on the CD52- T cells in 2 patients. PIG-AP-deficient T-cell clones from both patients were established. Analysis of the mRNA of the PIG-A gene showed an abnormal size in the T-cell clones from 1 of these patients, suggesting that a mutation in the PIG-A gene was the cause of the expression defect of PIG-AP. An escape from an immune attack directed against PIG-AP+ hematopoiesis has been hypothesized as the cause of the occurrence of PIG-AP-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia. Our results support the hypothesis that an attack against the PIG-AP CD52 might lead to the expansion of a PIG-anchor-deficient cell population with the phenotypic and molecular characteristics of PNH cells.

Diversity of HLA Class I and Class II Restricted Minor Histocompatibility Antigens in Graft-Versus-Leukemia Reactivity.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4084-4084
Author(s):  
Marieke Griffioen ◽  
M. Willy Honders ◽  
Anita N. Stumpf ◽  
Edith D. van der Meijden ◽  
Cornelis A.M. van Bergen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hematopoietic Cells ◽  
Cd4 T Cell ◽  
Cell Recognition ◽  
T Cell Clones ◽  
T Cell Recognition ◽  
Cell Clones ◽  
T Cell Clone ◽  
Ifn Γ

Abstract Abstract 4084 Poster Board III-1019 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) encoded by single nucleotide polymorphisms (SNPs) on malignant cells of the recipient. Donor T-cells may also induce Graft-versus-Host Disease (GvHD) when directed against mHags with broad expression on non-malignant tissues. The aim of this study was to investigate the specificity and diversity of mHags recognized by T-cells in Graft-versus-Leukemia (GvL) reactivity. Activated (HLA-DR+) CD8+ and CD4+ T-cell clones were isolated from a patient successfully treated with DLI for relapsed chronic myeloid leukemia (CML) more than one year after HLA-matched alloSCT. GvL reactivity in this patient was accompanied with mild GvHD of the skin. Isolated T-cell clones were shown to recognize 13 different mHags. CD8+ T-cell clones were specific for HA-1 and HA-2 in HLA-A*0201, one unknown mHag in B*0801 and 4 unknown mHags in B*4001. CD4+ T-cell clones were specific for one unknown mHag in HLA-DQ and 5 unknown mHags in DR. By screening plasmid (class I) and bacteria (class II) cDNA libraries, we identified a mHag in HLA-DQ encoded by the PI4K2B gene (Griffioen et al., PNAS 2008), 4 mHags in HLA-DR encoded by the PTK2B, MR-1, LY75 and MTHFD1 genes (Stumpf et al., Blood 2009) and a mHag in B*4001 encoded by the TRIP10 gene. For the 3 T cell clones recognizing unknown mHags in B*4001, we performed Whole Genome Assocation scanning (WGAs). A panel of 60 EBV-LCL was retrovirally-transduced with B*4001 and tested for T-cell recognition. In parallel, genomic DNA was isolated and more than one million single nucleotide polymorphisms (SNPs) were determined by the Illumina beadchip array. Statistical analysis revealed significant association between T-cell recognition of EBV-LCL and the presence of coding SNPs in the SON DNA-binding protein and SWAP-70 genes. To get more insight into the role and potential use of the mHags in GvL reactivity and GvHD, all T-cell clones were analyzed in detail for reactivity against hematopoietic and non-hematopoietic cells. Hematopoietic cells included peripheral blood cells (monocytes, B-cells and T-cells), professional antigen presenting cells (APC) and leukemic cells (CML, ALL and AML). All CD8+ T-cell clones recognized (subsets of) peripheral blood cells as well as CML cells, except for the T-cell clone for TRIP10. Recognition of (subsets of) peripheral blood cells was also observed for all CD4+ T-cell clones, but CML cells were differentially recognized. CML cells were strongly recognized by the T-cell clones for MTHFD1 and the unknown mHag in HLA-DR, whereas no or low reactivity was observed for all other CD4+ T-cell clones. All CD8+ and CD4+ T-cell clones strongly recognized professional APC, including monocyte-derived dendritic cells and in vitro differentiated CML cells with APC phenotype. All T-cell clones were also capable of recognizing AML and ALL, except for the T-cell clone for TRIP10, which showed restricted recognition of AML-M4 and -M5 of monocytic origin. As non-hematopoietic cells, patient-derived fibroblasts were cultured with and without IFN-γ and tested for T-cell recognition. In the absence of IFN-γ, all T-cell clones failed to recognize fibroblasts, except for the T-cell clone for the unknown mHag in B*0801. After treatment with IFN-γ, additional reactivity was observed for the T-cell clones for SON DNA-binding protein and the unknown mHag in B*4001. Our data showed the specificity and diversity of mHags recognized by T-cells induced in a patient successfully treated with DLI for relapsed CML. The T-cell response was directed against 13 different mHags, of which 10 mHags in HLA class I and class II have now been identified by different techniques. Detailed analysis of T-cell recognition of hematopoietic and non-hematopoietic cells provides evidence that the mHags played different roles in the onset and execution of GvL and GvHD. Moreover, only one of the 10 identified mHags was expressed on fibroblasts after treatment with IFN-γ, indicating the characterization of mHags with potential relevance for T-cell based immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Hairy Cell Leukemia-Specific Recognition by Multiple Autologous HLA-DQ or DP-Restricted T-Cell Clones

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 251-259
Author(s):  
Lisette van de Corput ◽  
Hanneke C. Kluin-Nelemans ◽  
Michel G.D. Kester ◽  
Roel Willemze ◽  
J.H. Frederik Falkenburg
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hairy Cell Leukemia ◽  
Lymphoblastic Leukemia ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
T Cell Clones ◽  
Cell Clones ◽  
Hla Dq ◽  
T Cell Clone

We studied in patients with hairy cell leukemia (HCL) whether autoreactive T cells could be isolated with specific reactivity to the HCL cells. HCL cells were activated via triggering of CD40 on the cell membrane and used as stimulator cells to generate autologous T-cell clones. Two types of CD4+BV2+ T-cell clones with different CDR3 rearrangements and one type of CD4+BV8S3+ T-cell clone were generated from the spleen or blood. These clones specifically recognized the autologous HCL cells, without reactivity to autologous peripheral blood mononuclear cells (PBMC), phytohemagglutinin blasts, or Epstein-Barr virus–transformed B cells in a primed lymphocyte test. Blocking and panel studies using HCL cells from 11 other patients showed that recognition of the HCL cells by the BV2+ T cells was restricted by HLA-DQA1*03/DQB1*0301, and the BV8S3+ T cells were restricted by DPB1*04. The T-cell clones did not recognize DPB1*04+ or DQ3+ PBMC from healthy donors or DP/DQ matched malignant cells from patients with other hematologic malignancies, except for one patient with acute lymphoblastic leukemia. These HCL-specific T-cell clones may be used for the detection of an HCL-specific tumor antigen.

scholarly journals Anti-IE1 CD4+ T-cell clones kill peptide-pulsed, but not human cytomegalovirus-infected, target cells

2007 ◽  
Vol 88 (9) ◽  
pp. 2441-2449 ◽  
Author(s):  
Sandra Delmas ◽  
Pierre Brousset ◽  
Danièle Clément ◽  
Emmanuelle Le Roy ◽  
Jean-Luc Davignon
Keyword(s):  
T Cells ◽  
T Cell ◽  
Human Cytomegalovirus ◽  
Antigen Processing ◽  
Hcmv Infection ◽  
Target Cells ◽  
T Cell Clones ◽  
Early Protein ◽  
Cell Clones ◽  
Precise Role

Cellular immunity plays a major role in the control of human cytomegalovirus (HCMV) infection. CD4+ T lymphocytes have been shown to contribute to this function but their precise role is a matter of debate. Although CD4+ T cells have been shown to kill target cells through the perforin/granzyme pathway, whether HCMV-specific CD4+ T cells are capable of killing HCMV-infected targets has not yet been documented. In the present paper, we have taken advantage of well established cellular reagents to address this issue. Human CD4+ T-cell clones specific for the major immediate-early protein IE1 were shown to perform perforin-based cytotoxicity against peptide-pulsed targets. However, when tested on infected anitgen presenting cell targets, cytotoxicity was not detectable, although gamma interferon (IFN-γ) production was significant. Furthermore, cytotoxicity against peptide-pulsed targets was inhibited by HCMV infection, whereas IFN-γ production was not modified, suggesting that antigen processing was not altered. Remarkably, degranulation of CD4+ T cells in the presence of infected targets was significant. Together, our data suggest that impaired cytotoxicity is not due to failure to recognize infected targets but rather to a mechanism specifically related to cytotoxicity.

scholarly journals Association of Donor T Cell Repertoire in Host Lymphoid and Target Organs and Gvhd Development

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4525-4525
Author(s):  
Yongxia Wu ◽  
Jianing Fu ◽  
Anusara Daenthanasanmak ◽  
Hung D Nguyen ◽  
Mohammed Hanief Sofi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Clone ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
T Cell Clones ◽  
Cell Clones ◽  
Target Organs ◽  
T Cell Clone ◽  
Donor T Cells

Abstract The diversity and composition of T cell receptor (TCR) repertoire, which is the result of V, D and J gene recombination in TCR gene locus, has been found to impact immune responses in autoimmune and infectious diseases. The correlation of T-cell repertoire with the pathogenesis and outcome of graft-versus-host disease (GVHD) remain undefined. Here, by utilizing high-throughput sequencing of the gene encoding the TCRβ-chain, we comprehensively analyzed the profile of T-cell repertoire in host lymphoid and GVHD target organs after bone marrow transplantation (BMT). To understand whether T-cell repertoire is affected by different strength of alloantigen stimulation, we transferred same donor T cells derived from C57BL/6 (B6) mice into irradiated BALB/c (MHC-fully mismatched), B6D2F1 (MHC-haploidentical), BALB.b (MHC-matched ) and B6 recipients (syngeneic). Fourteen days later, T cells were isolated from recipient peripheral blood, spleen, peripheral lymphoid nodes (pLN), mesenteric lymphoid nodes (mLN), liver, lung, gut and skin for TCR sequencing. Clonality of donor T cells, which is inversely associated with TCR diversity, was significantly increased in either syngeneic or allogeneic recipients when compared with naïve donor T-cells, consistent with the concept that TCR diversity is reduced after T-cell activation and expansion. Increased TCR clonality was observed in lymphoid organs of allogeneic compared with syngeneic recipients, confirming that donor T cells were further activated in allogeneic recipients. However, decreased TCR clonality was observed in GVHD target organs of allogeneic compared with syngeneic recipients, suggesting that only limited donor T-cell clones were able to migrate in target organs in syngeneic compared to allogeneic recipients. The frequency of top clones in total productive rearrangements was increased in GVHD target organs especially liver of allogenic than syngeneic receipts. Interestingly, the frequency of top clones was positively associated with MHC disparity between donor and host, ranging from low to high in syngeneic, MHC-matched, haploidentical, and fully-mismatched recipients, respectively. To understand the extent to which TCR rearrangement is shared among different organs after BMT, we analyzed the overlap of TCR clones across different organs in the same recipients. T-cell clones were highly overlapping across organs, especially among GVHD target organs, in the same recipients after allogeneic BMT, although much lower overlapping in recipients after syngeneic BMT. The results suggest that alloantigen stimulation selectively activate certain T-cell clones and enrich antigen specific clones. On the other hand, much fewer shared clones were found among different recipients within the same group, regardless of MHC-disparity between donor and host. These results suggest that specific T-cell clones activated and expanded by alloantigens stimulation were highly different in individual recipients even with the same MHC-disparity between donor and host. Interestingly, the levels of clone overlapping were different in distinct organs among individual recipients. The level of T-cell clone overlapping was found high in liver of individual recipients regardless of the strength of alloantigen stimulation. The level of T cell clone overlapping was relatively high in pLNs and skin of the recipients after haploidentical BMT; whereas the level of T cell clone overlapping was relatively high in mLNs and gut of the recipients after MHC-matched BMT. These results suggest that skin may be a dominant target in haploidentical BMT and gut as a dominant target in MHC-matched BMT; whereas liver is a common target organ regardless. In conclusion, the current study establishes the association between MHC disparity, T-cell activation, and GVHD development in the level of donor T-cell repertoire. While TCR repertoire of donor T cells in peripheral blood or lymph nodes likely is representative in any individual recipient/patient, it is nearly impossible to identify T-cell clones that are pathogenic and shared among groups of recipients/patients even with the same MHC-disparity between donor and host. Disclosures No relevant conflicts of interest to declare.

Hairy Cell Leukemia-Specific Recognition by Multiple Autologous HLA-DQ or DP-Restricted T-Cell Clones

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 251-259 ◽  
Author(s):  
Lisette van de Corput ◽  
Hanneke C. Kluin-Nelemans ◽  
Michel G.D. Kester ◽  
Roel Willemze ◽  
J.H. Frederik Falkenburg
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hairy Cell Leukemia ◽  
Lymphoblastic Leukemia ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
T Cell Clones ◽  
Cell Clones ◽  
Hla Dq ◽  
T Cell Clone

Abstract We studied in patients with hairy cell leukemia (HCL) whether autoreactive T cells could be isolated with specific reactivity to the HCL cells. HCL cells were activated via triggering of CD40 on the cell membrane and used as stimulator cells to generate autologous T-cell clones. Two types of CD4+BV2+ T-cell clones with different CDR3 rearrangements and one type of CD4+BV8S3+ T-cell clone were generated from the spleen or blood. These clones specifically recognized the autologous HCL cells, without reactivity to autologous peripheral blood mononuclear cells (PBMC), phytohemagglutinin blasts, or Epstein-Barr virus–transformed B cells in a primed lymphocyte test. Blocking and panel studies using HCL cells from 11 other patients showed that recognition of the HCL cells by the BV2+ T cells was restricted by HLA-DQA1*03/DQB1*0301, and the BV8S3+ T cells were restricted by DPB1*04. The T-cell clones did not recognize DPB1*04+ or DQ3+ PBMC from healthy donors or DP/DQ matched malignant cells from patients with other hematologic malignancies, except for one patient with acute lymphoblastic leukemia. These HCL-specific T-cell clones may be used for the detection of an HCL-specific tumor antigen.

Demonstration of Frequent Occurrence of Clonal T Cells in the Peripheral Blood But Not in the Skin of Patients With Small Plaque Parapsoriasis

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1409-1417 ◽  
Author(s):  
J. Marcus Muche ◽  
Ansgar Lukowsky ◽  
Jürgen Heim ◽  
Markus Friedrich ◽  
Heike Audring ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Cell Clone ◽  
Small Plaque ◽  
Blood Samples ◽  
T Cell Clones ◽  
Cell Clones ◽  
T Cell Clone

Clinical, immunohistological, and molecular biological data suggest the chronic dermatosis small plaque parapsoriasis (SPP) to be a precursor of mycosis fungoides (MF). However, most data are contradictory and confusing due to inexact definition of SPP. Recently, clonal T cells were detected in skin and blood samples of early MF. Because demonstration of identical T-cell clones in skin and blood of SPP patients would indicate a close relationship of SPP to MF, we investigated the clonality of skin and blood specimens from 14 well-defined SPP patients. By a polymerase chain reaction (PCR) amplifying T-cell receptor γ rearrangements and subsequent high-resolution electrophoresis, clonal T cells were detected in 9 of 14 initial and 32 of 49 follow-up blood samples, but in 0 of 14 initial skin specimens. Even a clone-specific PCR showing the persistence of the initial blood T-cell clone in 20 of 20 follow-up samples, failed to detect the T-cell clone in the skin. In 2 patients, the clonal T cells were shown to be CD4+. For the first time, the majority of SPP patients was shown to carry a T-cell clone in the peripheral blood. Although a relation between circulating clonal T cells and SPP cannot directly be proven by the applied techniques, our results indicate blood T-cell clonality to be a characteristic feature of SPP and CTCL because analysis of multiple controls and clinical workup of our SPP patients excluded other factors simulating or causing a clonal T-cell proliferation. A sufficient cutaneous antitumor response but also an extracutaneous origin of the T-cell clones might explain the failure to detect skin infiltrating clonal T cells.

scholarly journals Human CD4+ T-Cell Clone Expansion Leads to the Expression of the Cysteine Peptidase Inhibitor Cystatin F

2021 ◽  
Vol 22 (16) ◽  
pp. 8408
Author(s):  
Milica Perišić Nanut ◽  
Graham Pawelec ◽  
Janko Kos
Keyword(s):  
T Cells ◽  
T Cell ◽  
Natural Killer Cells ◽  
Natural Killer ◽  
Cd4 T Cell ◽  
Killer Cells ◽  
T Cell Clones ◽  
Cysteine Cathepsins ◽  
Cell Clones ◽  
T Cell Clone

The existence of CD4+ cytotoxic T cells (CTLs) at relatively high levels under different pathological conditions in vivo suggests their role in protective and/or pathogenic immune functions. CD4+ CTLs utilize the fundamental cytotoxic effector mechanisms also utilized by CD8+ CTLs and natural killer cells. During long-term cultivation, CD4+ T cells were also shown to acquire cytotoxic functions. In this study, CD4+ human T-cell clones derived from activated peripheral blood lymphocytes of healthy young adults were examined for the expression of cytotoxic machinery components. Cystatin F is a protein inhibitor of cysteine cathepsins, synthesized by CD8+ CTLs and natural killer cells. Cystatin F affects the cytotoxic efficacy of these cells by inhibiting the major progranzyme convertases cathepsins C and H as well as cathepsin L, which is involved in perforin activation. Here, we show that human CD4+ T-cell clones express the cysteine cathepsins that are involved in the activation of granzymes and perforin. CD4+ T-cell clones contained both the inactive, dimeric form as well as the active, monomeric form of cystatin F. As in CD8+ CTLs, cysteine cathepsins C and H were the major targets of cystatin F in CD4+ T-cell clones. Furthermore, CD4+ T-cell clones expressed the active forms of perforin and granzymes A and B. The levels of the cystatin F decreased with time in culture concomitantly with an increase in the activities of granzymes A and B. Therefore, our results suggest that cystatin F plays a role in regulating CD4+ T cell cytotoxicity. Since cystatin F can be secreted and taken up by bystander cells, our results suggest that CD4+ CTLs may also be involved in regulating immune responses through cystatin F secretion.

scholarly journals The Composition of a Primary T Cell Response Is Largely Determined by the Timing of Recruitment of Individual T Cell Clones

1999 ◽  
Vol 189 (10) ◽  
pp. 1591-1600 ◽  
Author(s):  
Philippe Bousso ◽  
Jean-Pierre Levraud ◽  
Philippe Kourilsky ◽  
Jean-Pierre Abastado
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Response ◽  
Cell Clone ◽  
Early Time ◽  
T Cell Response ◽  
Specific Response ◽  
T Cell Clones ◽  
Cell Clones ◽  
T Cell Clone

Primary T cell responses rely on the recruitment and proliferation of antigen-specific T cell precursors. The extent of expansion of each individual T cell clone may depend on (a) its frequency before immunization, (b) its proliferative capacity, and (c) the time at which it first encounters its cognate antigen. In this report, we have analyzed the relative contribution of each of these parameters to the shaping of immune repertoires in the T cell response specific for the epitope 170-179 derived from HLA-Cw3 and presented by Kd. By means of hemisplenectomy, we compared immune and naive repertoires in the same animal and found that the frequency of all expanded T cell clones was extremely low before immunization. In particular, the most expanded clones did not derive from high-frequency precursors. In addition, recruited T cells were found to proliferate at the same rate, irrespective of their T cell antigen receptor sequence. Finally, we showed that only T cells that encounter the antigen at early time points account for a significant part of the specific response. Therefore, the contribution of a T cell clone to the immune response is mostly determined by the time of its entry into the immune repertoire, i.e., the time of first cell division after antigen encounter.

High Avidity PRAME Specific T Cells Derived From In Vivo HLA Mismatched Transplantation Setting Potentially Useful for Immunotherapeutic Strategies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4087-4087 ◽  
Author(s):  
Avital L. Amir ◽  
Dirk M. van der Steen ◽  
Renate S. Hagedoorn ◽  
Marieke Griffioen ◽  
J.H. Frederik Falkenburg ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Epithelial Cells ◽  
Cell Lines ◽  
High Avidity ◽  
T Cell Clones ◽  
Cell Clones ◽  
T Cell Clone ◽  
Self Antigens

Abstract Abstract 4087 Poster Board III-1022 Adoptive T cell therapy is an attractive strategy to provide cancer patients with antigen specific T cells. For this approach T cells with specificity for non-polymorphic tumor-associated self-antigens that are shared between various tumors are promising candidates. However, the isolation of high avidity T cells specific for nonpolymorphic tumor associated self-antigens is difficult, because of self-tolerance. During thymic selection T cells that exhibit high avidity for self-antigens presented by self-HLA are deleted by positive and negative selection. This explains why most T cells directed against non-polymorphic tumor associated self-antigens characterized to date exhibit low to intermediate avidity. After HLA mismatched stem cell transplantation (SCT) however, T cells educated in the donor have not encountered the allogeneic HLA molecules from the patient during thymic selection. Consequently, these T cells can exhibit high avidity for tumor associated antigens presented by allogeneic patient HLA molecules. In this study we aimed to identify T cells directed against non-polymorphic tumor associated antigens using an in vivo HLA mismatched transplantation setting. Alloreactive T cell clones were isolated and expanded from a patient that experienced graft versus leukemia as well as acute graft versus host disease following HLA-A2 mismatched SCT and donor lymphocyte infusion for the treatment of AML. All isolated T-cell clones were allo-HLA-A2 reactive, and by loading of T2 cells with HPLC fractionations of peptides eluted from HLA-A2 we were able to demonstrate that all alloreactive clones recognized one single fraction, indicative for peptide specific recognition. By two additional peptide HPLC fractionation rounds and mass spectrometry we were able to characterize the peptides of 8 different allo-HLA-A2 reactive T cell clones. One of the T cell clones, was of particular interest since this T cell clone recognized the peptide SLLQHLIGL derived from the preferentially expressed antigen on melanomas (PRAME). Recognition by the clone of HLA-A2 positive COS cells transfected with PRAME, and tetramer staining of the clone, confirmed the specificity against the PRAME derived peptide. Peptide titration demonstrated that the PRAME specific T cell clone exhibited high affinity for the SLL peptide. Since PRAME is overexpressed in a large fraction of tumors, we analyzed whether the PRAME specific clone could recognize HLA-A2 expressing tumor cell lines. The results demonstrated that all 8 tested melanoma cell lines, 2 of 3 RCC cell lines, 1 of 2 mamma carcinoma cell lines and 1 of 2 lung carcinoma cell lines were recognized by the clone. In addition, 5 out of 10 primary acute myeloid leukemia cells, and 2 out of 3 acute lymphoblastic leukemia cell lines were recognized. Since it has been described that also certain normal tissues express low levels of PRAME, the clone was tested against numerous HLA-A2 positive non-malignant cells. No reactivity against fibroblasts, keratinocytes, bronchus epithelial cells, hepatocytes, billiair duct epithelial cells, colon epithelial cells, mesenchymal stem cells, (activated) B-cells, (activated) T cells, monocytes and CD34+ cells was observed. However, the clone demonstrated high reactivity against monocyte derived DC's and a low but significant reactivity against primary tubular epithelial cells. By quantitative PCR we demonstrated that the level of recognition of the different cell types is correlated with the expression levels of PRAME. The results demonstrate that the high avidity PRAME specific T cells clone, derived from an in vivo allo-HLA-A2 immune response, is solely PRAME specific and exerts high reactivity against numerous tumors and limited of target toxicity. Based on these results we conclude that the high affinity TCR from this high avidity PRAME specific T cell may be an effective tool for adoptive T cell therapy using TCR gene modified T cells for the treatment of cancer patients. Disclosures: No relevant conflicts of interest to declare.

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