scholarly journals Tolerance checkpoint bypass permits emergence of pathogenic T cells to neuromyelitis optica autoantigen aquaporin-4

2016 ◽  
Vol 113 (51) ◽  
pp. 14781-14786 ◽  
Author(s):  
Sharon A. Sagan ◽  
Ryan C. Winger ◽  
Andrés Cruz-Herranz ◽  
Patricia A. Nelson ◽  
Sarah Hagberg ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Neuromyelitis Optica ◽  
Severe Disease ◽  
Clinical Signs ◽  
Cell Receptor ◽  
Aquaporin 4 ◽  
T Cell Epitopes ◽  
Donor T Cells ◽  
Polarized Cells

Aquaporin-4 (AQP4)-specific T cells are expanded in neuromyelitis optica (NMO) patients and exhibit Th17 polarization. However, their pathogenic role in CNS autoimmune inflammatory disease is unclear. Although multiple AQP4 T-cell epitopes have been identified in WT C57BL/6 mice, we observed that neither immunization with those determinants nor transfer of donor T cells targeting them caused CNS autoimmune disease in recipient mice. In contrast, robust proliferation was observed following immunization of AQP4-deficient (AQP4−/−) mice with AQP4 peptide (p) 135–153 or p201–220, peptides predicted to contain I-Ab–restricted T-cell epitopes but not identified in WT mice. In comparison with WT mice, AQP4−/−mice used unique T-cell receptor repertoires for recognition of these two AQP4 epitopes. Donor T cells specific for either determinant from AQP4−/−, but not WT, mice induced paralysis in recipient WT and B-cell–deficient mice. AQP4-specific Th17-polarized cells induced more severe disease than Th1-polarized cells. Clinical signs were associated with opticospinal infiltrates of T cells and monocytes. Fluorescent-labeled donor T cells were detected in CNS lesions. Visual system involvement was evident by changes in optical coherence tomography. Fine mapping of AQP4 p201–220 and p135–153 epitopes identified peptides within p201–220 but not p135–153, which induced clinical disease in 40% of WT mice by direct immunization. Our results provide a foundation to evaluate how AQP4-specific T cells contribute to AQP4-targeted CNS autoimmunity (ATCA) and suggest that pathogenic AQP4-specific T-cell responses are normally restrained by central tolerance, which may be relevant to understanding development of AQP4-reactive T cells in NMO.

scholarly journals Characterization of Changes in the T-Cell Receptor Repertoire in Patients with Acute Myeloid Leukemia with Durable Remission Following Allogeneic Stem Cell Transplant

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5186-5186
Author(s):  
Ronald M. Paranal ◽  
Hagop M. Kantarjian ◽  
Alexandre Reuben ◽  
Celine Kerros ◽  
Priya Koppikar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Cell Receptor ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Donor T Cells ◽  
Durable Remission

Introduction: Allogeneic hematopoietic stem-cell transplantation (HSCT) is curative for many patients with advanced hematologic cancers, including adverse-risk acute myeloid leukemia (AML). This is principally through the induction of a graft-versus-leukemia (GVL) immune effect, mediated by donor T-cells. The incredible diversity and specificity of T-cells is due to rearrangement between V, D, and J regions and the random insertion/deletion of nucleotides, taking place in the hypervariable complementarity determining region 3 (CD3) of the T-cell receptor (TCR). Massively parallel sequencing of CDR3 allows for a detailed understanding of the T-cell repertoire, an area relatively unexplored in AML. Therefore, we sought out to characterize the T-cell repertoire in AML before and after HSCT, specifically for those with a durable remission. Methods: We identified 45 bone marrow biopsy samples, paired pre- and post-HSCT, from 14 patients with AML in remission for > 2 years as of last follow-up. We next performed immunosequencing of the TCRβ repertoire (Adaptive Biotechnologies). DNA was amplified in a bias-controlled multiplex PCR, resulting in amplification of rearranged VDJ segments, followed by high-throughput sequencing. Resultant sequences were collapsed and filtered in order to identify and quantitate the absolute abundance of each unique TCRβ CDR3 region. We next employed various metrics to characterize changes in the TCR repertoire: (1) clonality (range: 0-1; values closer to 1 indicate a more oligoclonal repertoire), it accounts for both the number of unique clonotypes and the extent to which a few clonotypes dominate the repertoire; (2) richness with a higher number indicating a more diverse repertoire with more unique rearrangements); (3) overlap (range: 0-1; with 1 being an identical T-cell repertoire). All calculations were done using the ImmunoSeq Analyzer software. Results: The median age of patients included in this cohort was 58 years (range: 31-69). Six patient (43%) had a matched related donor, and 8 (57%) had a matched unrelated donor. Baseline characteristics are summarized in Figure 1A. Six samples were excluded from further analysis due to quality. TCR richness did not differ comparing pre- and post-HSCT, with a median number pre-HSCT of 3566 unique sequences (range: 1282-22509) vs 3720 (range: 1540-12879) post-HSCT (P = 0.7). In order to assess whether there was expansion of certain T-cell clones following HSCT, we employed several metrics and all were indicative of an increase in clonality (Figure 2B). Productive clonality, a measure of reactivity, was significantly higher in post-transplant samples (0.09 vs 0.02, P = 0.003). This is a measure that would predict expansion of sequences likely to produce functional TCRs. The Maximum Productive Frequency Index was higher post-HSCT indicating that the increase in clonality was driven by the top clone (most prevalent per sample). Similarly for the Simpson's Dominance index, another marker of clonality which was higher post-HSCT (0.01 vs 0.0009, P = 0.04). In order to determine whether this clonal expansion was driven by TCR clones shared among patients, we compared the degree of overlap in unique sequences among pre and post-HSCT samples. We found there was very little overlap between samples in the pre and the post-transplant setting and no change in the Morisita and Jaccard Overlap Indices. Conclusions: In conclusion, we show in this analysis an increase in clonality of T-cells following HSCT in patients with AML. This is likely related to the GVL effect after recognition of leukemia antigens by donor T cells and subsequent expansion of these T-cells. These expanded T-cell clonotypes were unlikely to be shared by patients in this cohort, likely reflecting the variety of antigens leading to the GVL effect. This could have direct implications on TCR-mediated immune-therapies given the likely need for a personalized, patient-specific design for these therapies. Figure 1 Disclosures Kantarjian: BMS: Research Funding; Novartis: Research Funding; AbbVie: Honoraria, Research Funding; Jazz Pharma: Research Funding; Astex: Research Funding; Immunogen: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Takeda: Honoraria; Amgen: Honoraria, Research Funding; Cyclacel: Research Funding; Ariad: Research Funding; Pfizer: Honoraria, Research Funding. Short:Takeda Oncology: Consultancy, Research Funding; AstraZeneca: Consultancy; Amgen: Honoraria. Cortes:Takeda: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Sun Pharma: Research Funding; BiolineRx: Consultancy; Novartis: Consultancy, Honoraria, Research Funding; Astellas Pharma: Consultancy, Honoraria, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Immunogen: Consultancy, Honoraria, Research Funding; Biopath Holdings: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Forma Therapeutics: Consultancy, Honoraria, Research Funding. Jabbour:Cyclacel LTD: Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding. Molldrem:M. D. Anderson & Astellas Pharma: Other: Royalties.

Immunological and clinical consequences of treating a patient with natalizumab

2011 ◽  
Vol 18 (3) ◽  
pp. 335-344 ◽  
Author(s):  
Nicholas Schwab ◽  
Karin G Höhn ◽  
Tilman Schneider-Hohendorf ◽  
Imke Metz ◽  
Max-Philipp Stenner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Jc Virus ◽  
Clinical Signs ◽  
Brain Biopsy ◽  
Cell Receptor ◽  
Term Therapy ◽  
Cell Repertoire ◽  
Csf Analysis ◽  
Clinical Consequences

Background: Long-term therapy with natalizumab increases the risk of progressive multifocal leukoencephalopathy (PML). Objectives: We present a patient study through therapy, the diagnosis of PML (after 29 infusions), plasma exchange (PE) and development of immune reconstitution inflammatory syndrome (IRIS). Methods: Routine diagnostics, magnetic resonance imaging (MRI), immunological status (flow cytometry, T-cell migration assays and T-cell repertoire analysis), and brain biopsy with immunohistological analysis. Results: CD49d decreased after 12 months of treatment. At PML diagnosis, CD49d expression and migratory capacity of T cells was low and peripheral T-cell receptor (TCR) complexity showed severe perturbations. The distribution of peripheral monocytes changed from CCR5+ to CCR7+. After PE some changes reverted: CD49d increased and overshot earliest levels, migratory capacities of T cells recovered and peripheral TCR complexity increased. With no clinical, routine laboratory or cerebrospinal fluid (CSF) changes, MRI 2 months after PE demonstrated progressive lesion development. Brain histopathology confirmed the presence of infiltrates indicative of IRIS without clinical signs, immunologically accompanied by CCR7/CCR5 recovery of peripheral monocytes. Conclusion: Natalizumab-associated immunological changes accompanying PML were reversible after PE; IRIS can occur very late, remain asymptomatic and be elusive to CSF analysis. Our study may provide insights into the changes under treatment with natalizumab associated with JC virus control.

scholarly journals Analysis of T-cell repopulation after allogeneic bone marrow transplantation: significant differences between recipients of T-cell depleted and unmanipulated grafts

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3984-3992 ◽  
Author(s):  
E Roux ◽  
C Helg ◽  
F Dumont-Girard ◽  
B Chapuis ◽  
M Jeannet ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Allogeneic Bone Marrow Transplantation ◽  
Cell Receptor ◽  
Allogeneic Bone ◽  
Receptor Repertoire ◽  
Donor T Cells ◽  
Circulating T Cells ◽  
Cell Repopulation

We have studied the repopulation of the T-cell compartment in 27 patients transplanted with bone marrow from an (HLA)-identical sibling. Significant differences were found between recipients of unmanipulated and T-cell depleted grafts. Analysis of the T cells by a method based on amplification of minisatellite DNA regions showed that without depletion > 99.9% of the clones responding to a mitogenic stimulus after transplantation were of donor origin. In contrast, when the graft had been depleted with Campath-1M plus complement, a significant part of the T cells cloned during the first weeks after transplantation comprised of recipient T cells that had survived the preconditioning. This mixed population of low numbers donor and recipient T cells (19 +/- 31/mm3 at day 14) expanded rapidly (predominantly CD8+ T cells) during the first 2 months, without a significant change of the ratio of recipient/donor T cells. In 11 of 17 evaluable patients a late wave ( > 9 months) of donor T cells occurred. As a consequence, T-cell chimerism changed in favor of donor T cells and the CD4/CD8 ratio that had been reversed ( < 0.5) after the first expansion, normalized (1.5 +/- 0.51). Analysis of the T-cell receptor repertoire showed that in recipients of a T-cell depleted graft, the recipient as well as the donor T cells that repopulated the peripheral T-cell pool during the first month, were the progeny of a limited number of precursors. Because without depletion, when larger numbers of donor T cells had been cotransfused with the marrow, the repertoire was much more diverse, these data show that immediately after transplantation, the peripheral pool is repopulated primarily through expansion of circulating T cells.

scholarly journals Donor γδ T Lymphocytes Promote Allogeneic Engraftment Across the Major Histocompatibility Barrier in Mice

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 1100-1109 ◽  
Author(s):  
William R. Drobyski ◽  
David Majewski
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Γδ T Cells ◽  
Cell Receptor ◽  
Marrow Graft ◽  
Hematopoietic Reconstitution ◽  
Donor T Cells ◽  
Αβ T Cells

Abstract T cells that express the αβ T-cell receptor are thought to be the T-cell population primarily responsible for facilitating alloengraftment. The role of γδ+ T cells that comprise only a minority of mature T cells in promoting allogeneic engraftment, however, has not been extensively studied. The purpose of this study was to determine whether γδ T cells were capable of facilitating alloengraftment in murine recipients of major histocompatibility complex-mismatched marrow grafts. We developed a model where engraftment of C57BL/6 × 129/F2 (H-2b) marrow in sublethally irradiated (800 cGy) recipients (AKR/J, H-2k) is dependent on the presence of mature donor T cells in the marrow graft. In this model, donor T-cell engraftment was significantly augmented by as few as 1 × 105 αβ T cells. The role of γδ T cells was then investigated using transgenic donors (C57BL/6 × 129 background) in which a portion of the T-cell receptor–β chain gene was deleted by gene targeting so that these mice lack αβ T cells. Addition of 10 × 106 naive γδ T cells to T-cell depleted marrow grafts was required to significantly increase alloengraftment, although donor T cells averaged <50% of total splenic T cells. To determine whether higher doses of γδ T cells would improve donor engraftment and eradicate residual host T cells, γδ T cells were ex vivo expanded with a γδ T-cell–specific monoclonal antibody and interleukin-2 and then transplanted into irradiated recipients. Transplantation of ≥ 160 × 106 activated γδ T cells was necessary to consistently and significantly augment donor cell chimerism and enhance hematopoietic reconstitution when compared to control mice, but host T cells persisted in these chimeras. Addition of 2.5 × 104 mature αβ T cells, which alone were incapable of facilitating engraftment, to T-cell depleted marrow grafts containing 160 × 106 activated γδ T cells resulted in long-term (<100 day) complete donor engraftment, indicating that limiting numbers of αβ T cells were required in the marrow graft for the eradication of residual host T cells. Using serial weight curves and B-cell reconstitution as end points, clinically significant graft-versus-host disease was not observed in these chimeras under these experimental conditions. These data show that, whereas less potent than αβ T cells, γδ T cells are able to promote engraftment and enhance hematopoietic reconstitution in allogeneic marrow transplant recipients.

scholarly journals Antigen-specific T-cell receptor signatures of cytomegalovirus infection

2018 ◽  
Author(s):  
Alina Huth ◽  
Xiaoling Liang ◽  
Stefan Krebs ◽  
Helmut Blum ◽  
Andreas Moosmann
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Response ◽  
Ex Vivo ◽  
Cell Receptor ◽  
T Cell Response ◽  
The Body ◽  
T Cell Epitopes ◽  
Virus Reactivation

AbstractCytomegalovirus (CMV) is a prevalent human pathogen. The virus cannot be eliminated from the body, but is kept in check by CMV-specific T cells. Patients with an insufficient T-cell response, such as transplant recipients, are at high risk of developing CMV disease. However, the CMV-specific T-cell repertoire is complex, and is not yet clear which T cells protect best against virus reactivation and disease. Here we present a highly resolved characterization of CMV-specific CD8+ T cells based on enrichment by specific peptide stimulation and mRNA sequencing of their T-cell receptor β chains (TCRβ). Our analysis included recently identified T-cell epitopes restricted through HLA-C, whose presentation is resistant to viral immunomodulation, and well-studied HLA-B-restricted epitopes. In 8 healthy virus carriers, we identified a total of 1052 CMV-specific TCRβ chains. HLA-C-restricted, CMV-specific TCRβ clonotypes theex vivoT-cell response, and contributed the highest-frequency clonotype of the entire repertoire in 2 of 8 donors. We analyzed sharing and similarity of CMV-specific TCRβ sequences and identified 63 public or related sequences belonging to 17 public TCRβ families. In our cohort and in an independent cohort of 352 donors, the cumulative frequency of these public TCRβ family members was a highly discriminatory indicator of carrying both CMV infection and the relevant HLA type. Based on these findings, we propose CMV-specific TCRβ signatures as a biomarker for an antiviral T-cell response to identify patients in need of treatment and to guide future development of immunotherapy.

scholarly journals Vβ14+ T Cells Mediate the Vaccine-Enhanced Disease Induced by Immunization with Respiratory Syncytial Virus (RSV) G Glycoprotein but Not with Formalin-Inactivated RSV

2004 ◽  
Vol 78 (16) ◽  
pp. 8753-8760 ◽  
Author(s):  
Teresa R. Johnson ◽  
Steven M. Varga ◽  
Thomas J. Braciale ◽  
Barney S. Graham
Keyword(s):  
T Cells ◽  
Respiratory Syncytial Virus ◽  
T Cell ◽  
Cytokine Production ◽  
Severe Disease ◽  
Cell Receptor ◽  
Pulmonary Eosinophilia ◽  
Cell Repertoire ◽  
Cytokine Levels ◽  
Syncytial Virus

ABSTRACT Mice immunized with respiratory syncytial virus (RSV) G glycoprotein or with formalin-inactivated RSV (FI-RSV) exhibit severe disease following RSV challenge. This results in type 2 cytokine production and pulmonary eosinophilia, both hallmarks of vaccine-enhanced disease. RSV G-induced T-cell responses were shown to be restricted to CD4+ T cells expressing Vβ14 in the T-cell receptor (TCR), and the deletion of these T cells resulted in less severe disease. We therefore examined the role of Vβ14+ T cells in FI-RSV-induced disease. BALB/c mice were immunized with vaccinia virus expressing secreted RSV G (vvGs) or with FI-RSV. At the time of challenge with live RSV, mice were injected with antibody to the Vβ14 component of the TCR. vvGs-immunized mice treated with anti-Vβ14 had reduced cytokine levels in the lung. Eosinophil recruitment to the lung was also significantly reduced. In contrast, depletion of Vβ14+ T cells in FI-RSV-immunized mice had little impact on cytokine production or pulmonary eosinophilia. An analysis of TCR Vβ chain usage confirmed a bias toward Vβ14 expression on CD4+ T cells from vvGs-immunized mice, whereas the CD4+ T cells in FI-RSV-immunized mice expressed a diverse array of Vβ chains. These data show that although FI-RSV and vvGs induce responses resulting in similar immunopathology, the T-cell repertoire mediating the response is different for each immunogen and suggest that the immune responses elicited by RSV G are not the basis for FI-RSV vaccine-enhanced disease.

scholarly journals Blockade of osteopontin reduces alloreactive CD8+ T cell–mediated graft-versus-host disease

Blood ◽  
2011 ◽  
Vol 117 (5) ◽  
pp. 1723-1733 ◽  
Author(s):  
Fang Zhao ◽  
Yi Zhang ◽  
Hao Wang ◽  
Min Jin ◽  
Shan He ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Cell Receptor ◽  
Hematopoietic Stem ◽  
Host Disease ◽  
Graft Versus Host ◽  
Donor T Cells

Abstract Graft-versus-host disease (GVHD), a life-threatening complication after allogeneic hematopoietic stem cell transplantation, is caused by alloreactive donor T cells that trigger host tissue damage. The inflammatory environment inside recipients is critical for GVHD pathogenesis, but the underpinning mechanisms remain elusive. Using mouse model of human GVHD, we demonstrate osteopontin (OPN), a potent proinflammatory cytokine, plays an important role in regulating activation, migration, and survival of alloreactive T cells during GVHD. OPN was significantly elevated after irradiation and persisted throughout the course of GVHD. Blockade of OPN attenuated GVHD with reduced accumulation of donor T cells in recipient organs. Amelioration was the result of migration and survival suppression caused by anti-OPN treatment on donor-derived T cells for 2 reasons. First, OPN promoted the migration and infiltration of naive and alloreactive CD8+ T cells into host organs. Second, it also facilitated activation and viability of donor-derived CD8+ T cells via synergizing with T-cell receptor/CD3 signaling. Finally, anti-OPN treatment retained graft-versus-leukemia effect of alloreactive CD8+ T cells. This study demonstrates, to our knowledge for the first time, the critical effect of OPN in the initiation and persistence of CD8+ T cell-mediated GVHD and validates OPN as a potential target in GVHD prevention.

scholarly journals Rapid selection and identification of functional CD8+ T cell epitopes from large peptide-coding libraries

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Govinda Sharma ◽  
Craig M. Rive ◽  
Robert A. Holt
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Epitope ◽  
Cell Receptor ◽  
T Cell Response ◽  
Target Cells ◽  
T Cell Epitopes ◽  
Peptide Epitopes ◽  
Histocompatibility Complex

Abstract Cytotoxic CD8+ T cells recognize and eliminate infected or malignant cells that present peptide epitopes derived from intracellularly processed antigens on their surface. However, comprehensive profiling of specific major histocompatibility complex (MHC)-bound peptide epitopes that are naturally processed and capable of eliciting a functional T cell response has been challenging. Here, we report a method for deep and unbiased T cell epitope profiling, using in vitro co-culture of CD8+ T cells together with target cells transduced with high-complexity, epitope-encoding minigene libraries. Target cells that are subject to cytotoxic attack from T cells in co-culture are isolated prior to apoptosis by fluorescence-activated cell sorting, and characterized by sequencing the encoded minigenes. We then validate this highly parallelized method using known murine T cell receptor/peptide-MHC pairs and diverse minigene-encoded epitope libraries. Our data thus suggest that this epitope profiling method allows unambiguous and sensitive identification of naturally processed and MHC-presented peptide epitopes.

Vigorous Priming of Minor Histocompatibility Antigen Specific T Cells by Frequent High Dose Peptide Vaccination May Lead to Prolonged T Cell Receptor Downregulation, Impaired T Cell Functionality, and Promotion of Tumor Escape.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1809-1809 ◽  
Author(s):  
Inge Jedema ◽  
Linda van Dreunen ◽  
Marleen M. van Loenen ◽  
Mirjam Heemskerk ◽  
Roel Willemze ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cell Receptor ◽  
Stem Cell Population ◽  
Target Cells ◽  
Tumor Escape ◽  
Cd34 Cells ◽  
Donor T Cells

Abstract Allogeneic stem cell transplantation can be successfully applied in the treatment of hematological malignancies and relies on the graft versus leukemia (GVL) effect mediated by donor T cells directed against minor histocompatibility antigens (mHag) selectively expressed on malignant hematopoietic cells of the patient. However, due to insufficient in-vivo priming of donor T cells the GVL response may not be adequately initiated or amplified. Vaccination strategies using immunogenic peptides derived from hematopoeisis specific mHag like HA-1 may form a strategy to initiate or boost the in-vivo GVL response. However, it has been reported that repetitive vaccination with HLA-class I binding 9-mer peptides can lead to the induction of T cell anergy. We hypothesized that repetitive strong priming of the mHag specific T cells may also lead to prolonged downregulation of the T cell receptor (TCR) resulting in inability of the T cells to subsequently attack tumor cells expressing the mHag, allowing tumor escape despite the presence of potentially effective T cells. We tested this hypothesis in an in-vitro model using CFSE-labeled HA-1+ CD34+ chronic myeloid leukemia (CML) cells as target/stimulator cells, and HA-1 specific T cells as effector cells. In previous studies we have demonstrated the resistance of a small population of quiescent CML stem cells to all high avidity T cells, allowing the subsequent outgrowth of malignant progeny from this population. To mimick a peptide vaccination strategy, we loaded CD34+ CML cells from an HA-1+ patient with various concentrations (E-12-E-6M) of the 9-mer HA-1 peptide, and investigated the direct and residual functional cytotoxic capacity of an HA-1 specific CD8+ T cell clone. In accordance with our previous results, we observed complete deletion of all proliferating CML precursor cells after 24–48 hours of exposure to the CTLs, whereas a small subpopulation of quiescent CD34+ cells was resistant to T cell attack. The exogenous peptide loading resulted in more rapid lysis and also attack of part of the quiescent stem cell population. However, in the next days malignant progeny was formed from the quiescent stem cell population in the conditions of high peptide stimulation despite the continuous presence of the T cells, suggesting impaired residual cytotoxic function of these T cells. Therefore, we analyzed the level of TCR downregulation after exposure to the HA-1 positive CML CD34+ cells in the absence or presence of E-12-E-6 M HA-1 peptide loaded to the target cells. We observed strong dose-dependent TCR downregulation as measured by specific tetramer staining (20%–78% decrease in fluorescence intensity after 24 hours of exposure to targets loaded with 0-E-6M HA-1 peptide). At high peptide concentrations it took 6–9 days before proper functional TCR expression could be again demonstrated. In conclusion, we here demonstrate that high affinity T cells show a prolonged TCR downregulation after vigorous stimulation by peptide loaded target cells. In this period the T cells showed a dramatic loss of function and allowed the outgrowth of a leukemic subpopulation expressing the HA-1 antigen. Milder vaccination strategies using longer peptides requiring uptake and processing by the target cells may lead to expression of more physiological levels of the mHag and less vigorous priming of the mHag specific T cells, thereby preserving their functional capacity and responsiveness.

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