Physiological TCR Modulation after Antigen Specific Triggering of Introduced TCRs under Control of a Retroviral Promotor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5537-5537
Author(s):  
Marleen M. van Loenen ◽  
Renate S. Hagedoorn ◽  
Esther H.M. van Egmond ◽  
Roel Willemze ◽  
J.H.Frederik Falkenburg ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Surface Expression ◽  
Cytolytic Activity ◽  
Cell Surface Expression ◽  
Chromium Release Assay ◽  
Chromium Release ◽  
Release Assay ◽  
Specific Stimulation ◽  
Stimulation Of

Abstract TCR transfer to engineer tumor specific T cells may be an alternative strategy for adoptive immunotherapy. We have previously shown that TCR-transduced T cells are capable of recognizing targets both via their endogenous TCR and the introduced TCR. Since the introduced TCR is regulated by a different promotor, we investigated whether triggering and modulation of the introduced TCR occur in a physiological manner compared to the endogenous TCR. Because introduction of a TCRα-chain and TCRβ-chain will lead to formation of chimeric TCR-complexes with the endogenous TCRα- and β-chain, we used models in which both specificities of the TCRs were known and tetramers were available to distinguish each TCR. CMV PP65 specific T cells were transduced with a retroviral construct encoding the hematopoietic minor histocompatibility antigen HA-2 specific TCR (HA-2-TCR). TCR-transduced T cells were antigen specifically triggered using EBV-LCLs that presented endogenously processed PP65- or HA-2-antigen. At various time points after stimulation cell surface expression of the TCRαβ-complexes and the BV-chain of the endogenous TCR was studied with monoclonal antibodies (mAbs). No mAb directed against the BV-chain of the introduced TCR was available. Tetramers specific for the endogenous or introduced TCR were used to distinguish the TCRs from chimeric TCRαβ-complexes. We observed that after stimulation of either the endogenous or the introduced TCR the total amount of TCRs decreased to 40% and 70% of unstimulated cells, respectively. When the endogenous TCR was triggered, HA2- and PP65-tetramer stainings as well as the endogenous BV chain were diminished after 1 day. Stimulation of the introduced TCR decreased the introduced TCR expression after 1 day, and the endogenous TCR expression measured by tetramers and BV specific mAb was marginally decreased. Two days after stimulation of the introduced TCR and 3 days after stimulation of the endogenous TCR, the total amount of TCRαβ-complexes was restored. Staining with specific BV-mAb and tetramers demonstrated that the endogenous TCR expression, both after triggering of the endogenous as well as the introduced TCR, was still decreased at day 3. These data indicate that TCRαβ-complexes on the surface at days 2 and 3 mainly consisted of the introduced HA-2-TCR. TCR expression of non-transduced T cells was still decreased at day 3 after specific stimulation. Functional analysis of the T cells in a chromium release assay after 1 day of TCR triggering demonstrated that the T cells exerted reduced cytolytic activity that correlated with the downmodulation of TCR expression of either the introduced and endogenous TCR. The lytic activity of the T cells was restored at day 2 or 3 and correlated with the tetramer stainings. In conclusion, we observed physiological downmodulation of TCRs which were regulated by a retroviral promotor after antigen specific triggering. However, the introduced TCR was more quickly re-expressed at the cell surface, probably due to the increased retroviral promotor activity. The downmodulation upon specific triggering of both introduced and endogenous TCRs implies that cell mechanisms other than promotor activity are also involved in regulation of TCR cell surface expression.

Physiological Non-Responsiveness and Absence of Activation Induced Cell Death of T Cells Rapidly Re-Expressing Retrovirally Introduced TCRs after T Cell Activation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2301-2301
Author(s):  
Marleen van Loenen ◽  
Renate Hagedoorn ◽  
Esther van Egmond ◽  
Roelof Willemze ◽  
J.H. Frederik Falkenburg ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Cell Surface ◽  
Refractory Period ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Functional Study ◽  
Kinetics Of ◽  
Stimulation Of

Abstract TCR transfer to engineer tumor specific T cells may be an alternative strategy for adoptive immunotherapy. We previously have shown that TCR-transduced T cells are capable of recognizing targets both via the endogenous and the introduced TCR. Stimulation of the TCR induces internalization of TCRs leading to a refractory period with a high activation threshold. Since the introduced TCR is regulated by a viral promotor which is constantly activated, we investigated whether modulation of the introduced TCRs after antigen specific triggering occurred in a physiological manner compared to the endogenous TCR. CMV specific T cells were retrovirally transduced with the hematopoietic minor histocompatibility antigen HA-2 specific TCR. TCR transduced T cells were antigen specifically triggered via either the introduced HA-2 (HA-2 TCR) or the endogenous CMV specific TCR (CMV TCR). At various time points after stimulation cell surface expression of the TCRαß-complexes and the BV-chain of the CMV TCR and HA-2 TCR was studied with monoclonal antibodies. Tetramers specific for the CMV TCR or HA-2 TCR were used to distinguish the TCRs from chimeric TCRαß-complexes. Stimulation via the CMV TCR or the HA-2 TCR resulted in similar internalization of approximately 50% of the TCRs. Preferentially, but not solely, the triggered TCRs were internalized. In contrast to the kinetics of internalization, the kinetics of TCR re-expression after stimulation differed considerably between the endogenous CMV and the introduced HA-2 TCR. The introduced HA-2 TCR was already re-expressed at the cell surface 24h after stimulation, while 70% of the endogenous CMV TCR still was internalized 72h after stimulation. This rapid synthesis of HA-2 TCRs could lead to enhanced competition for cell surface expression. Indeed, when TCR cell surface expression of the HA-2 TCR restored, cell surface expression of the CMV TCR decreased even further. When T cells were analyzed 4h after stimulation for cytolytic reactivity, both T cells stimulated via the HA-2 and the CMV TCR were non-responsive, correlating with low TCR expression. Although the HA-2 TCR was re-expressed at the cell surface 48h after stimulation, still no cytolytic activity via either the HA-2 or the CMV TCR was found. At this timepoint the level of expression of adhesion molecules and the amount of intracellular granzyme B after an initial decline was comparable to non-stimulated T cells. However, cell surface expression of the CD8 coreceptor was still diminished, resulting in low T cell avidity. To analyze whether stimulation via rapidly re-expressed HA-2 TCRs would lead to antigen induced cell death (AICD), T cells were stimulated via the HA-2 TCR after an initial stimulation and analyzed at different timepoints. In agreement with their non-responsiveness in the functional study, no increased AICD after specific stimulation via the HA-2 TCR was observed. In conclusion, we observed physiological internalization of TCRs which were regulated by a retroviral promotor after antigen specific triggering, but the introduced TCR was more rapidly re-expressed at the cell surface. Despite different TCR make up early after stimulation, in these T cells physiological non-responsiveness and no increased AICD was found after stimulation of the re-expressed introduced TCR, illustrating that cell mechanisms other than TCR cell surface expression like CD8 downregulation are also involved in providing a protective refractory period.

Cell Surface Expression of CD154 Inhibits Alloantibody Responses: A Mechanism for the Prevention of Autoimmune Responses against Activated T Cells?

1999 ◽  
Vol 195 (2) ◽  
pp. 157-161 ◽  
Author(s):  
Adele Louise McCormick ◽  
Leopoldo Santos-Argumedo ◽  
Mark Stephen Thomas ◽  
Andrew William Heath
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Activated T Cells

Leukemia-Reactive Virus-Specific T Cells Generated by T Cell Receptor (TCR) Transfer Preserve Their Dual Specificity upon Repetitive Stimulation of the Endogenous TCR.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 136-136
Author(s):  
M.M. van Loenen ◽  
R.S. Hagedoorn ◽  
M. Hoogeboom ◽  
M.G.D. Kester ◽  
Roelof Willemze ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Repetitive Stimulation ◽  
Cell Surface Expression ◽  
Viral Antigens ◽  
Low Expression ◽  
Stimulation Of

Abstract TCR-transfer to engineer tumor-specific T cells may be a strategy for adoptive immunotherapy. For complete eradication of leukemic cells and to achieve long-term protection, potent effector T cell function and long-term T cell persistence are necessary. Therefore, we propose to use virus specific T cells for TCR transfer since such engineered dual specific T cells can be triggered via their endogenous TCR by latent presence of viral antigens, improving their long-term persistence. We have previously shown that virus specific T cells can be redirected towards anti-leukemic reactivity by transfer of the hematopoietic minor histocompatibility antigen HA-2 specific TCR (HA-2-TCR). The TCR-transferred virus specific T cells showed differences in TCR cell surface make up, which was stable for months after repetitive non-specific TCR triggering. The T cells expressed either both TCRs intermediately at the cell surface, or the endogenous TCR was highly expressed with a low expression of the introduced TCR, or the introduced TCR was highly expressed with a low expression of the endogenous TCR. It may be anticipated that frequent encounter with viral antigens in vivo leads to selective outgrowth of TCR-transferred dual specific T cells with high expression of the endogenous viral specific TCR but low expression of the introduced tumor specific TCR, resulting in reduced anti-leukemic reactivity. To address this issue, we generated CMVA2-specific T cells transduced with the HA-2-TCR. This resulted in dual specific cells with different TCR cell surface make up. The dual specific T cells were repetitively stimulated specifically either via their endogenous virus specific TCR or via the introduced HA-2 specific TCR. In time, the cell surface expression of the endogenous and introduced TCRs as measured with CMVA2 and HA-2A2 tetramers diverged. Repetitive stimulation of the endogenous TCR skewed the dual specific T cells towards a cell population that predominantly expressed the endogenous TCR. In contrast, repetitive stimulation of the introduced TCR skewed the cells towards T cells that predominantly expressed the introduced TCR. However, this divergence in tetramer stainings was shown to quickly revert after a single stimulation via the other TCR. To study whether this divergence was the result of a difference in TCR cell surface distribution or of selective outgrowth of different T cells, T cells were sorted that predominantly expressed either the endogenous or the introduced TCR. These cells were subsequently stimulated on the endogenous or introduced TCR, and compared regarding TCR cell surface expression and functional activity. Directly after sorting dual specific T cells preferentially expressing the endogenous TCR were still reactive against HA-2+ target cells, although the reactivity was reduced compared to cells preferentially expressing the introduced TCR. However, when restimulated on the introduced HA-2-TCR, the dual specific T cells expanded antigen specifically, and reverted within several days into cells with high expression of the introduced TCR that exerted potent HA-2 specific anti-leukemic effector functions. In conclusion, we demonstrate that these dual specific T cells are likely to persist in vivo due to repetitive encounter with viral antigens with preservation of anti-leukemic effector function. Moreover, in vivo exposure to the tumor associated antigen will further enhance the relevant specificity.

LMP2-Specific TCR Gene Therapy for Hodgkin Lymphoma - Design of a Dominant TCR Construct for Immunotherapy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2762-2762
Author(s):  
Daniel P. Hart ◽  
Sharyn Thomas ◽  
Shao-an Xue ◽  
Emma C. Morris ◽  
Hans J. Stauss
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Hodgkin Lymphoma ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Beta Chain ◽  
Antigen Specificity ◽  
Human T Cells ◽  
Beta Chain Genes ◽  
Tcr Gene Therapy

Abstract Hodgkin lymphoma (HL) is the second most commonly diagnosed cancer in the 15–29 year old population. EBV-positive Hodgkin lymphoma typically demonstrates latency II antigen expression, characterised by loss of most EBV antigens except for the latent membrane protein (LMP) 1 and 2 and the EBNA-1 protein. LMP2 is expressed in Reed Sternberg cells and may serve as a target for antigen-specific immunotherapy. However, LMP2 is poorly immunogenic and it is often difficult to generate autologous LMP2-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy. T cell receptor (TCR) gene transfer using retroviral vectors containing the TCR alpha and beta chain genes can reproducibly redirect the antigen specificity of a given population of T cells. Such an approach has been used here to generate LMP2-specific CTL independent of the immuno-competence of the patient. The goal of this study was to generate a retroviral TCR construct suitable for rapid and efficient production of LMP2-specific CTL. Retrovirally introduced TCRs compete with endogenous TCRs for a limited pool of CD3 molecules required for assembly of the TCR complex. Competition for CD3 molecules may limit surface expression of the introduced TCR resulting in a transduced T cell with poor functional avidity. In an attempt to generate a ‘highly competitive’ LMP2-TCR the following modifications were made to the retroviral vector construct: nucleotide sequences were codon optimised for efficient translation in human cells; the TCR chain constant regions were altered to contain murine sequences to enhance CD3 binding; and the TCR alpha and beta chain genes were linked by a self-cleaving 2A sequence from the porcine teschovirus to enhance equimolar expression of both TCR chains. The unmodified HLA-A2-restricted LMP2-specific TCR was poorly expressed in primary human T cells, suggesting that it competed inefficiently with endogenous TCR chains for cell surface expression. Very few CD8+Vβ13+ T cells were detectable after LMP2-TCR transduction (up to 2.5% of viable CD3+ T cells, as detected by FACs analysis using monoclonal anti-Vβ13 antibodies), which included 1.9% CD8+ T cells expressing endogenous Vβ13+ TCRs as quantified in mock-transduced control cells. Poor expression of the wild type LMP2-TCR was consistently observed in independent transduction experiments. However, transduction with the modified LMP2-TCR construct resulted in cell surface expression of the TCR in 55–65% viable CD3+ T cells. HLA-A2/LMP2 pentamer binding was demonstrated in 36–39% CD8+ CTL cells immediately post transduction. The transduced cells showed peptide-specific IFNγ and IL2 production and killed target cells displaying the LMP2 peptide. Of major importance, expression of the introduced LMP2-TCR completely suppressed the cell surface expression of almost the entire repertoire of endogenous TCR combinations, including ‘mis-paired’ TCRs in the transduced primary human T cells. ‘Mis-paired’ TCRs contain an introduced alpha chain paired with an endogenous beta chain and vice versa. The antigen specificity of such mispaired TCRs generated after transduction is unknown and could lead to unwanted side effects. The design of vectors containing modified TCR sequences, which produce ‘dominant’ TCRs may improve the efficacy of TCR gene therapy and reduce the risk of potential auto-reactivity of endogenous and ‘mis-paired’ TCR combinations. We have shown that LMP2-specific T cells can be readily generated by TCR gene transfer with minimal risk of autoreactivity.

Identification of a T cell lineage-specific RNA/DNA helicase and its cell surface expression during intrathymic education of αβ and γδ T cells

1997 ◽  
Vol 27 (12) ◽  
pp. 3269-3282 ◽  
Author(s):  
Arkadiusz Miazek ◽  
Manfred Brockhaus ◽  
Hanno Langen ◽  
Andrea Braun ◽  
Pawel Kisielow
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Cell Lineage ◽  
Γδ T Cells ◽  
Surface Expression ◽  
Dna Helicase ◽  
Cell Surface Expression

Retinoids induce Fas(CD95) ligand cell surface expression via RARγ andnur77in T cells

2004 ◽  
Vol 34 (3) ◽  
pp. 827-836 ◽  
Author(s):  
Beáta Tóth ◽  
Katalin Ludányi ◽  
Ildikó Kiss ◽  
Uwe Reichert ◽  
Serge Michel ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Cd95 Ligand

scholarly journals Immunotherapeutic Targeting of TSLPR with Chimeric Antigen Receptor T Cells in AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2789-2789
Author(s):  
Lindsey F Call ◽  
Sommer Castro ◽  
Thao T. Tang ◽  
Cynthia Nourigat-Mckay ◽  
LaKeisha Perkins ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Surface ◽  
Peripheral Blood ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Car T Cells ◽  
Car T

Abstract Adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) has achieved impressive outcomes in the treatment of refractory/relapsed B-ALL, providing potentially curative treatment options for these patients. The use of CAR T in AML, however, is still in its infancy with limitations due to the innate heterogeneity associated with AML and the lack of AML-specific targets for therapeutic development. The CRLF2 gene encodes for thymic stromal lymphopoietin receptor (TSLPR) and has previously been shown to be highly upregulated in a subset of children and adults with B-ALL. Targeting TSLPR with CAR T cells demonstrates potent anti-leukemia activity against TSLPR-positive B-ALL (PMID 26041741). Through Target Pediatric AML (TpAML), we profiled the transcriptome of nearly 3000 children and young adults with AML and identified CRLF2 (TSLPR) to be highly expressed in a subset of AML, including the majority of AML harboring KM2TA (aka MLL) fusions. TSLPR cell surface expression was validated in primary patient samples using flow cytometry, which showed uniform expression of TSLPR on AML blasts. Given that TSLPR is expressed in AML with confirmed cell surface expression, we developed TSLPR-directed CAR T for preclinical evaluation in AML. We generated a TSLPR-directed CAR using the single-chain variable fragment (scFv) derived from an anti-TSLPR binder (clone 3G1, MD Anderson), IgG4 spacer and 41-BB/CD3zeta signaling domains. The in vitro cytotoxicity of TSLPR CAR T cells was evaluated against the REH-1 cell line and primary AML specimens. TSLPR CAR T cells demonstrated anti-leukemia activity against REH-1 as well as against primary AML specimens. To evaluate the in vivo efficacy of TSLPR CAR T cells, we developed a patient-derived xenograft (PDX) model using bone marrow cells from a TSLPR-positive patient. These cells provided a robust model system to evaluate the in vivo activity of TSLPR CAR T cells, as they produced an aggressive leukemia in humanized NSG-SGM3 mice. The PDX generated from these cells died within 2 months of transplant with significant leukemia infiltration into the bone marrow, liver, and spleen. In the in vivo study, the leukemia burden was assessed by flow cytometric analysis of AML cells in the peripheral blood and bone marrow aspirates following treatment with unmodified control or TSLPR CAR T cells given at 10x10 6 T cells per mouse. After CAR T treatment, we detected a significant decrease in leukemia infiltration into the peripheral blood and bone marrow in the CAR T-treated mice compared to mice that received unmodified T cells. In this study, we report that similar to B-ALL, CRLF2 (TSLPR) is overexpressed in a subset of AML, providing a strategy to eliminate AML cells with CAR T cell therapy. We validated the cell surface expression of TSLPR and showed that the expression is uniform across AML specimens. We further demonstrate that CAR T cells targeting TSLPR were effective in eliminating AML cells in vitro and in vivo. Given that TSLPR is highly expressed in the KMT2A-rearranged AML, a subtype that is associated with poor outcomes, TSLPR-directed CAR T cells represent a promising immunotherapy for this high-risk AML subset. Disclosures Pardo: Hematologics, Inc.: Current Employment.

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