Aberrant Expression of LMO2, TAL1 (SCL) and T- RALDH2 in the T-Cell Clone of a Patient with T-ALL like Syndrome after Gene Therapy for X1-SCID.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1114-1114
Author(s):  
Gerlinde Layh-Schmitt ◽  
Antonia Rosenstiel ◽  
Chuck Klanke ◽  
Kathleen Anderson ◽  
Salim Hacein-Bey-Abina ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cell Proliferation ◽  
T Cells ◽  
Retinoic Acid ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Clone ◽  
Truncated Form ◽  
T Cell Clone

Abstract In a gene therapy trial with patients suffering from X-linked Severe Combined Immunodeficiency (X1-SCID) led by Fischer and Cavazzana-Calvo (Hacein-Bey-Abina et al., Science, 2003) full restoration of the immune function was observed after retrovirally mediated gamma c transfer. However, three years after treatment, two out of eleven patients developed a T cell lymphoproliferative disorder, which was associated with LMO2 activation as a result of integration of the retroviral vector into the LMO2 locus. LMO2 is required for normal hematopoiesis and is usually only expressed in erythroid cells and immature T-cells as a component of a multifactorial transcription regulation complex consisting of TAL1, LMO2 (mediating protein-protein interaction), GATA1/2, Ldb-1, and E2A. A number of studies with transgenic mice and observations in T-ALL patients suggest that in addition to aberrant LMO2 expression, secondary events, such as mutations in an oncogene like SCL (TAL1) or in tumor suppressor genes are responsible for the onset of malignancy. The goal of this project is to unravel on molecular level specific events which might occur after retroviral mediated gamma c transfer and to determine possible secondary independent events which finally lead to uncontrolled clonal T-cell proliferation. Experiments were initiated with the T-cell clone of one of the patients (patient 5) who developed leukemia-like symptoms after gene therapy. Gene transcription profiling using whole genome gene chips (Affymetrix) revealed, that in addition to LMO2, TAL1 as well as RALDH2 (retinaldehyde dehydrogenase) were among genes which were aberrantly expressed in the patient 5 T-cell clone, whereas in normal T-cell controls none of the three genes were transcribed. By immunoblot analyses and RT-PCR we were able to confirm over expression of LMO2 and TAL1 in the patient lymphoblasts as well as in Jurkat (T-ALL cell line) and K562 (erythroleukemia cell line) cells. In the patient T-cell clone and in the Jurkat cells, RALDH2 was found to be expressed as an N-terminal truncated form on both the mRNA and protein level, suggesting that the molecular mechanism leading to the T-ALL-like lymphoproliferation in patient 5 resembles the findings in T-ALL cell lines described by Ono et al., (Molecular and Cellular Biol., 1998). Ono et al. identified N-terminal truncated (T) RALDH2 as a target gene for a protein complex consisting of LMO2, TAL1, E47 and GATA3 in T-ALL cell lines, in which GATA 3 mediates DNA binding of the complex. Retinoic acid is known to induce cell proliferation and to inhibit activation induced apoptosis of T-cells. We have cloned and overexpressed the T-RALDH variant by transfection and retrovirus transduction to test whether the truncated form of RALDH2 is still capable of converting retinal to retinoic acid and to establish siRNA procedures to examine whether depletion of T-RALDH2 in T-ALL cells changes cellular proliferation.

RALDH2 Is Regulated by an LMO/SCL(TAL1) Associated Protein Complex in T Cell Acute Lymphoblastic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1225-1225
Author(s):  
Gerlinde Layh-Schmitt ◽  
Wei Wang ◽  
Antonia Rosenstiel ◽  
Stefanie Koch ◽  
Chuck Klanke ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Protein Complex ◽  
Lymphoblastic Leukemia ◽  
Jurkat Cells ◽  
Truncated Form ◽  
Down Regulation ◽  
Aberrant Expression ◽  
T Cell Clone

Abstract Three and five years after an otherwise successful gene therapy trial for X1-SCID (gamma c deficiency) three out of 11 patients developed T cell leukemia due to insertional activation of the proto-oncogene LMO2 after retroviral mediated gamma c transfer into autologous CD34+ cells. In one of the patient’s T-cells SCL(TAL1) was expressed in addition to LMO2 because of a SIL-SCL fusion, where the deletion of the SIL gene brings SCL(TAL1) under the control of the constitutively active SIL promoter. LMO2 and SCL(TAL1) are components of a multifactorial transcription regulation complex consisting of SCLTAL1, LMO2, GATA1/2, Ldb-1, and E2A which is essential for normal hematopoiesis; however, LMO2 and SCL(TAL1) are normally down regulated after the DN2 stage of T cell maturation and expression of both proteins after DN2 leads to a block of T cell differentiation which precedes the onset of malignancy as shown in transgeneic mouse models. Experiments with T cell lymphoblasts of the patient, who developed LMO2/SCL(TAL1) associated T-ALL after gene therapy were initiated, in order to determine genes which are deregulated by aberrant expression of LMO2/SCL(TAL1) in mature T-cells leading to the onset of T-ALL. We established a xenotransplant model in NOD/SCID mice with the patient’s leukemic T-cells, which will serve as an in vivo model to examine the mechanisms underlying LMO2/SCL(TAL1) associated T-ALL. By immuno precipitation using LMO2 specific antibodies and nuclear extracts of the patient’s leukemic T-cells, we were able to isolate a protein complex containing LMO2, SCL(TAL) and E47 which resembles the LMO1 associated protein complex described by Ono et al. for the T-ALL cell line Jurkat. Gene transcription profiling using whole genome gene chips (Affymetrix) revealed, that in addition to LMO2 and TAL1, RALDH2 (retinaldehyde dehydrogenase) were among genes which were aberrantly expressed in the patient 5 T-cell clone, whereas in normal T-cell controls none of the three genes are transcribed. Immunoblot analyses and RT-PCR revealed an N-terminal truncation of RALDH2 (T-RALDH2) in the patient’s T-ALL cells. A cellular based activity test using protein extracts of the patient’s T-cells, K562 cells (positive control) and HL-60 cells (negative control) revealed, that the truncated form of RALDH2 is enzymatically active and converts retinaldehyde into retinoic acid which was described to induce cell proliferation and to inhibit activation induced apoptosis of T-cells. To test the role and regulation of RALDH2 in T-ALL we designed RALDH2 and LMO1 specific siRNA for down regulation of RALHD2 and LMO1 in a T-ALL cell culture model (Jurkat cells). We found that down regulation of LMO1 led to a decrease of RALDH2 expression whereas SCL(TAL1) and house keeping genes were not affected. This result confirms the finding of Ono et al. who showed that LMO and SCL(TAL1) activate a truncated form of RALDH2. Both, down regulation of LMO1 or RALDH2 resulted in a decrease of cell viability between 24 and 48 hours post siRNA transfection. We conclude that RALDH2 is a target gene of an LMO1(2)/SCL(TAL1) associated transcription regulating complex in T-ALL and might play a crucial role in the onset of TALL by interfering with proliferation and apoptotic processes.

Possible Protective Role for Vδ1+ γδT Lymphocytes in Epstein-Barr Virus Infection after Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3225-3225
Author(s):  
Masumi Fujishima ◽  
Makoto Hirokawa ◽  
Naohito Fujishima ◽  
Junsuke Yamashita ◽  
Hirobumi Saitoh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytotoxic Activity ◽  
Cell Line ◽  
Cell Clone ◽  
Cell Subset ◽  
Allogeneic Hsct ◽  
T Cell Clone ◽  
Term Survivor

Abstract Background: The posttransplant reconstitution of the αβ TCR repertoire correlates with the control of viral infections. We have previously demonstrated that using a limited number of patients cohort some had a skew of the Vδ1+ TCRγδ+ T cell repertoire after allogeneic HSCT, and that clonal predominance of Vδ1+ TCRγδ+ T cells was already present in blood of some donors and those clones were transferred to the recipients. The proliferation of Vδ1+ γδ T cells has been described in various infections including HIV, CMV and malaria. Although the functions of human Vδ1+ γδ T cells in vivo remain obscure, aforementioned results have raised the hypothesis that Vδ1+ T cells may have the repertoire against microorganisms widely infected in humans. Objective: We sought to explore the biological role for this T cell subset by investigating the reconstitution of the Vδ1+ γδT cell repertoire after human allogeneic HSCT, and the cytotoxic activity against EBV-infected cells of this T cell subset isolated from the long-term survivor. Methods: The size distribution and sequences of CDR3 of TCR δ-chains were determined by using an automated DNA sequencer. Immunophenotypes of the cells were analyzed by flow cytometry. PBMCs were stimulated with autologous EBV-LCL in the presence of IL-2 followed by positive selection with a MACS TCRγ/δ MicroBead kit. Positively selected γδ T lymphocytes were cultured with irradiated autologous EBV-LCL and allogeneic PBMCs as feeder cells, PHA (1 μg/ml) and IL-2 (10 U/ml) for 7 days. Vδ1 T cells were enriched by depletion of αβ T cells, Vδ2+ and Vδ3+ T cells with mAbs and an anti-FITC MicroBead kit and LD columns. Cytotoxicity was measured by standard 4-hr 51Cr release assay. Results: We observed skewed TCR repertoires of the Vδ1+ γδT cells in 27 out of 44 patients receiving allogeneic HSCT. The -WGI- amino acid motif was observed in CDR3 of clonally expanded Vδ1+ T cells in half of the patients. A skew was also detected in certain healthy donors, and the Vδ1+ T cell clone derived from the donor mature T cell pool was persisting in the recipient’s blood even ten years after transplant. In addition, this T cell clone expanded in vitro against the stimulation with autologous EBV-LCL (Fig. 1), and the Vδ1+ T cell line expanded in vitro from the same patient showed cytotoxicity against autologous EBV-LCL (Fig. 2). EBV-infected cells could also induce oligoclonal expansion of Vδ1+ T cells in healthy individuals as well. Conclusion: The skewing of Vδ1+ γδT cell repertoires may be the result of the responses to latently infectious antigens, and human Vδ1+ T cells may have a role in the protection against EBV infection. Figure 1. Expansion of the Vdelta 1+ T cell clone persistently present in the long-term survivor of allo-HSCT in response to autologous EBV-LCL Figure 1. Expansion of the Vdelta 1+ T cell clone persistently present in the long-term survivor of allo-HSCT in response to autologous EBV-LCL Figure 2. Cytotoxic activity of the Vdelta 1+ T cell line against autologous EBV-LCL established form the long-term survivor of allo-HSCT. Figure 2. Cytotoxic activity of the Vdelta 1+ T cell line against autologous EBV-LCL established form the long-term survivor of allo-HSCT.

Transfer of Human T-Cell Receptors (TCR) Containing Murine Chimeric Constant Beta-Gamma-Chain Sequences Reduces the Risk of Mixed Heterodimers and Shows Enhanced in Vitro-Accumulation of TCR-Tranduced Effector Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3583-3583
Author(s):  
Angela Krackhardt ◽  
Xiaoling Liang ◽  
Ingrid G Schuster ◽  
Luise U Weigand ◽  
Elfriede Eppinger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Therapeutic Approach ◽  
Cell Clone ◽  
Target Cells ◽  
Malignant Diseases ◽  
Effector Cells ◽  
T Cell Clone

Abstract Abstract 3583 Poster Board III-520 Introduction Adoptive transfer of T-cell receptor (TCR)-transduced T cells may represent an attractive and promising novel approach to specifically treat malignant diseases and has been previously successfully applied in the clinic. This approach promises the availability of sufficient numbers of effector cells with defined specificity for any tumor-associated antigen as also TCR from T cells with specificity for tumor-associated self antigens usually deleted in the autologous host may be isolated from an allorestricted or xenorestricted environment. We have previously identified the HLA-A2-allorestricted T-cell clone (SK22) with specificity for a peptide derived from Formin-like protein 1 (FMNL1) restrictedly expressed in hematopoietic tissue and overexpressed in diverse leukemias and other malignant tissue. SK22 demonstrated specific cytotoxicity against FMNL1-overexpressing cells as EBV-transformed B cells, lymphoma cell lines and native malignant cells derived from patients with chronic lymphocytic leukemia whereas healthy tissue was mainly spared. The TCR of this T cell clone may therefore represent a suitable tool for the treatment of diverse malignant diseases using TCR-transduced T cells. However, there are different concerns which need to be addressed to further improve this therapeutic approach. First, the formation of heterodimers between endogenous TCR chains and transduced TCR chains derived from receptors with low interchain affinity may abrogate specific TCR function and harbours a particular risk for unknown specificities. Although a number of TCR chain modifications has been previously applied to solve this problem further improvements are necessary. Second, longterm survival of TCR-transduced T cells has been demonstrated to be critical for the effectivity of this approach and novel approaches are needed. Methods and Results We have isolated the TCR-chain genes of the FMNL1-specific T cell clone SK22 and cloned them into the retroviral vector pMP71. Transduction of unmodified TCR-chain genes of SK22 in CD8α-transfected TCR-deficient Jurkat76 cells resulted in multimer-positive cells indicating that correct TCR-chain genes have been isolated. However, peripheral blood mononuclear cells (PBMC) transduced with these native TCR chains did neither show TCR expression nor specific T-cell function suggesting that TCR SK22 represents a weak TCR with low interchain affinity. Expression and function of this TCR could be significantly improved by current optimization strategies as codon-optimization and murinization of constant chains. Effector cells transduced with these optimized TCR chain genes showed reactivity against transformed cells of different origin whereas non-transformed HLA-A2 positive target cells as lung fibroblasts, embryonic cardiomyocytes, CD4- and CD8-positive T cells as well as activated PBMC were not recognized. However, substantial mispairing persisted despite of murinization of constant chain sequences. Using human TCR chain genes containing murinized chimeric constant βγ-chains previously reported to exert improved signaling in murine T cells and cell lines, we created a hybrid TCR with high functional efficiency after transfer in human effector cells. Moreover, usage of murinized chimeric constant βγ-chains of SK22 clearly reduced the formation of heterodimers in human PBMC. In addition, we observed enhanced in vitro-accumulation of CD8- and CD4-positive cells expressing the transgenic receptor when optimized murinized chimeric constant βγ-chains in comparison to optimized murinized constant β-chains without γ-chain sequences were used. These results could be confirmed after transfer of two alternative TCR with specificities for HER2/neu and GP100 containing murinized chimeric constant βγ-chains. Conclusion These data show that transfer of the optimized TCR SK22 may be an attractive therapeutic tool for the treatment of malignancies of hematologic and other origin. Moreover, the transfer of TCR chain genes containing optimized murinized chimeric constant βγ-chains may have a significant impact on the improvement of safety and efficiency of this therapeutic approach. Disclosures: No relevant conflicts of interest to declare.

CD8 T cells are not required for islet destruction induced by a CD4+ islet-specific T-cell clone

Diabetes ◽  
1992 ◽  
Vol 41 (12) ◽  
pp. 1603-1608 ◽  
Author(s):  
B. J. Bradley ◽  
K. Haskins ◽  
F. G. La Rosa ◽  
K. J. Lafferty
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Clone ◽  
T Cell Clone

scholarly journals HL-T, a new cell line derived from HL-60 promyelocytic leukemia cell cultures expressing terminal transferase and secreting suppressor activity

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1151-1160 ◽  
Author(s):  
E Paietta ◽  
RJ Stockert ◽  
T Calvelli ◽  
P Papenhausen ◽  
SV Seremetis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Cultures ◽  
Germ Line ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Chromosome 9 ◽  
Terminal Transferase

A cell line with immature blast cell morphology was isolated from HL-60 promyelocytic leukemia cell cultures and designated HL-T. This new cell type is biphenotypic, expressing terminal transferase (TdT) together with myelomonocytoid immunologic features. TdT enzymatic activity, undetectable in HL-60, was determined to be 140 to 180 units/10(8) HL-T cells by the dGTP-assay, approximately 20% of the activity found in lymphoblastoid cell lines. HL-T predominantly synthesize the known 58- kDa TdT-protein plus a minor 54/56-kDa doublet. The 58-kDa steady state form is nonglycosylated and is phosphorylated. Precursor antigens S3.13 and MY-10, absent on HL-60, are expressed by HL-T; however, the cells are negative for HLA-Dr. Southern blot analysis by hybridization with immunoglobulin heavy chain (JH) and T cell-receptor chain gene (T beta) probes shows JH to be in the germ-line configuration in both cell lines and the T beta gene to be in germ-line in HL-60 but to be rearranged in HL-T. Truncation of the gene encoding the granulocyte-macrophage-colony- stimulating factor (GM-CSF), as found in HL-60, is not observed in HL- T. HL-T are resistant to differentiation-induction by retinoic acid and 1,25-dihydroxyvitamin D3. Cytogenetically HL-T share with HL-60 a deletion of the short arm of chromosome 9 at breakpoint p13, an aberration frequently found in patients with T cell leukemia. In addition, HL-T display t(8;9)(p11;p24) and trisomy 20. Tetraploidy is observed in 80% of HL-T metaphases with aberrations identical to those in the diploid karyotype. Like HL-60, the new line shows some surface- antigenic-T cell characteristics. Despite an antigenic pattern most consistent with that of helper-inducer T cells (T4+, D44+/-, 4B4+, 2H4- , TQ1+/-), HL-T cells and their conditioned culture medium suppress antigen, mitogen, and mixed-leukocyte-culture-mediated lymphocyte proliferation.

scholarly journals A Novel Approach for Treatment of T-Cell Malignancies: Targeting T-Cell Receptor Vβ Families

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 28-29
Author(s):  
Jie Wang ◽  
Katarzyna Urbanska ◽  
Prannda Sharma ◽  
Mathilde Poussin ◽  
Reza Nejati ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Receptor ◽  
Brentuximab Vedotin ◽  
T Cell Lymphomas ◽  
T Cell Malignancies

Background: Peripheral T-cell lymphomas (PTCL) encompass a highly heterogeneous group of T-cell malignancies and are generally associated with a poor prognosis. Combination chemotherapy results in consistently poorer outcomes for T-cell lymphomas compared with B-cell lymphomas.1 There is an urgent clinical need to develop novel approaches to treatment of PTCL. While CD19- and CD20-directed immunotherapies have been successful in the treatment of B-cell malignancies, T-cell malignancies lack suitable immunotherapeutic targets. Brentuximab Vedotin, a CD30 antibody-drug conjugate, is not applicable to PTCL subtypes which do not express CD30.2 Broadly targeting pan-T cell markers is predicted to result in extensive T-cell depletion and clinically significant immune deficiency; therefore, a more tumor-specific antigen that primarily targets the malignant T-cell clone is needed. We reasoned that since malignant T cells are clonal and express the same T-cell receptor (TCR) in a given patient, and since the TCR β chain in human α/β TCRs can be grouped into 24 functional Vβ families targetable by monoclonal antibodies, immunotherapeutic targeting of TCR Vβ families would be an attractive strategy for the treatment of T-cell malignancies. Methods: We developed a flexible approach for targeting TCR Vβ families by engineering T cells to express a CD64 chimeric immune receptor (CD64-CIR), comprising a CD3ζ T cell signaling endodomain, CD28 costimulatory domain, and the high-affinity Fc gamma receptor I, CD64. T cells expressing CD64-CIR are predicted to be directed to tumor cells by Vβ-specific monoclonal antibodies that target tumor cell TCR, leading to T cell activation and induction of tumor cell death by T cell-mediated cytotoxicity. Results: This concept was first evaluated in vitro using cell lines. SupT1 T-cell lymphoblasts, which do not express a native functioning TCR, were stably transduced to express a Vβ12+ MART-1 specific TCR, resulting in a Vβ12 TCR expressing target T cell line.3 Vβ family specific cytolysis was confirmed by chromium release assays using co-culture of CD64 CIR transduced T cells with the engineered SupT1-Vβ12 cell line in the presence of Vβ12 monoclonal antibody. Percent specific lysis was calculated as (experimental - spontaneous lysis / maximal - spontaneous lysis) x 100. Controls using no antibody, Vβ8 antibody, and untransduced T cells did not show significant cytolysis (figure A). Next, the Jurkat T cell leukemic cell line, which expresses a native Vβ8 TCR, was used as targets in co-culture. Again, Vβ family target specific cytolysis was achieved in the presence of CD64 CIR T cells and Vβ8, but not Vβ12 control antibody. Having demonstrated Vβ family specific cytolysis in vitro using target T cell lines, we next evaluated TCR Vβ family targeting in vivo. Immunodeficient mice were injected with SupT1-Vβ12 or Jurkat T cells with the appropriate targeting Vβ antibody, and either CD64 CIR T cells or control untransduced T cells. The cell lines were transfected with firefly luciferase and tumor growth was measured by bioluminescence. The CD64 CIR T cells, but not untransduced T cells, in conjunction with the appropriate Vβ antibody, successfully controlled tumor growth (figure B). Our results provide proof-of-concept that TCR Vβ family specific T cell-mediated cytolysis is feasible, and informs the development of novel immunotherapies that target TCR Vβ families in T-cell malignancies. Unlike approaches that target pan-T cell antigens, this approach is not expected to cause substantial immune deficiency and could lead to a significant advance in the treatment of T-cell malignancies including PTCL. References 1. Coiffier B, Brousse N, Peuchmaur M, et al. Peripheral T-cell lymphomas have a worse prognosis than B-cell lymphomas: a prospective study of 361 immunophenotyped patients treated with the LNH-84 regimen. The GELA (Groupe d'Etude des Lymphomes Agressives). Ann Oncol Off J Eur Soc Med Oncol. 1990;1(1):45-50. 2. Horwitz SM, Advani RH, Bartlett NL, et al. Objective responses in relapsed T-cell lymphomas with single agent brentuximab vedotin. Blood. 2014;123(20):3095-3100. 3. Hughes MS, Yu YYL, Dudley ME, et al. Transfer of a TCR Gene Derived from a Patient with a Marked Antitumor Response Conveys Highly Active T-Cell Effector Functions. Hum Gene Ther. 2005;16(4):457-472. Figure Disclosures Schuster: Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria.

scholarly journals T Cell Activity Correlates with Oligomeric Peptide-Major Histocompatibility Complex Binding on T Cell Surface

2001 ◽  
Vol 276 (50) ◽  
pp. 47320-47328 ◽  
Author(s):  
Jennifer Buslepp ◽  
Rui Zhao ◽  
Debora Donnini ◽  
Douglas Loftus ◽  
Mohamed Saad ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Cell Clone ◽  
Cell Activity ◽  
Class I ◽  
Antigenic Peptides ◽  
Major Histocompatibility ◽  
Class I Mhc ◽  
T Cell Clone

Recognition of virally infected cells by CD8+T cells requires differentiation between self and nonself peptide-class I major histocompatibility complexes (pMHC). Recognition of foreign pMHC by host T cells is a major factor in the rejection of transplanted organs from the same species (allotransplant) or different species (xenotransplant). AHIII12.2 is a murine T cell clone that recognizes the xenogeneic (human) class I MHC HLA-A2.1 molecule (A2) and the syngeneic murine class I MHC H-2 Dbmolecule (Db). Recognition of both A2 and Dbare peptide-dependent, and the sequences of the peptides recognized have been determined. Alterations in the antigenic peptides bound to A2 cause large changes in AHIII12.2 T cell responsiveness. Crystal structures of three representative peptides (agonist, null, and antagonist) bound to A2 partially explain the changes in AHIII12.2 responsiveness. Using class I pMHC octamers, a strong correlation is seen between T cell activity and the affinity of pMHC complexes for the T cell receptor. However, contrary to previous studies, we see similar half-lives for the pMHC multimers bound to the AHIII12.2 cell surface.

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