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 ◽  
...  

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.

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 ◽  
...  

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.


1997 ◽  
Vol 27 (12) ◽  
pp. 3269-3282 ◽  
Author(s):  
Arkadiusz Miazek ◽  
Manfred Brockhaus ◽  
Hanno Langen ◽  
Andrea Braun ◽  
Pawel Kisielow

1985 ◽  
Vol 161 (6) ◽  
pp. 1575-1580 ◽  
Author(s):  
T R Malek ◽  
J D Ashwell

Stimulation of a class II-restricted, antigen-specific T cell clone with interleukin 2 (IL-2) resulted in substantial increases in both cell surface IL-2 receptor (IL-2-R) and cytoplasmic IL-2-R messenger RNA (mRNA), whereas no increase was observed for cell-surface expression of Thy-1 and L3T4 antigens, and only a modest increase in Thy-1 mRNA was observed. These experiments demonstrate that, after initial acquisition of the IL-2-R, IL-2 as well as antigen is able to directly upregulate both the level of IL-2-R mRNA and cell surface IL-2-R molecules.


Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3085-3092 ◽  
Author(s):  
Manisha D. Nath ◽  
Francis W. Ruscetti ◽  
Cari Petrow-Sadowski ◽  
Kathryn S. Jones

AbstractLittle is known about the requirements for human T-cell leukemia virus type I (HTLV-I) entry, including the identity of the cellular receptor(s). Recently, we have generated an HTLV-I surface glycoprotein (SU) immunoadhesin, HTSU-IgG, which binds specifically to cell-surface protein(s) critical for HTLV-I–mediated entry in cell lines. Here, expression of the HTLV-I SU binding protein on primary cells of the immune system was examined. The immunoadhesin specifically bound to adult T cells, B cells, NK cells, and macrophages. Cell stimulation dramatically increased the amount of binding, with the highest levels of binding on CD4+ and CD8+ T cells. Naive (CD45RAhigh, CD62Lhigh) CD4+ T cells derived from cord blood cells, in contrast to other primary cells and all cell lines examined, bound no detectable HTLV-I SU. However, following stimulation, the level of HTSU-IgG binding was rapidly induced (fewer than 6 hours), reaching the level of binding seen on adult CD4+ T cells by 72 hours. In contrast to HTLV-I virions, the soluble HTSU-IgG did not effect T-cell activation or proliferation. When incubated with human peripheral blood mononuclear cells in a mixed leukocyte reaction, HTSU-IgG inhibited proliferation at less than 1 ng/mL. These results indicate that cell-surface expression of the HTLV SU binding protein is up-regulated during in vitro activation and suggest a role for the HTLV-I SU binding proteins in the immunobiology of CD4+ T cells.


Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4019-4019
Author(s):  
Haneen Shalabi ◽  
Haiying Qin ◽  
Kelsey Wanhainen ◽  
Jillian Smith ◽  
Rimas Orentas ◽  
...  

Abstract Background: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an uncommon childhood leukemia that has been associated with very poor clinical outcomes in some studies. ETP-ALL cells arrest at a more immature differentiation stage than other T-lymphoblasts, and are hypothesized to retain multi-lineage differentiation potential, which may contribute to chemoresistance with standard lymphoid-directed therapy. Based on the recent clinical success of chimeric antigen receptor (CAR)-modified T-cells in children with B-ALL, we sought to identify potential surface protein targets on ETP lymphoblasts using differential gene expression analysis combined with a bioinformatic algorithm to predict surface expression. Methods: Cell-surface targets on ETP-ALL were predicted by identifying overexpressed transcripts based on gene expression and a bioinformatic algorithm to predict surface expression. Using several gene expression platforms and reference databases, (Oncogenomics website-Pediatric Oncology Branch, NCI, Gene Expression Omnibus, Gene Ontology, Human Protein References Database) ETP-ALL samples were compared to peripheral blood mononuclear cell (PBMC) controls on an individual transcript basis. A list of the top 25 transcripts was generated based on cell surface proteins, and the resultant list ordered by the degree of difference from PBMC controls. We next used human leukemia cells from six established ETP-ALL patient-derived xenograft (PDX) models using flow cytometry to evaluate for cell surface expression of proteins encoded by the overexpressed transcripts. Additionally, since CD7 and CD33 expression on ETP-ALL patient samples is universal with minimal normal tissue distribution, we developed two new second-generation anti-CD7 or anti-CD33 CAR constructs using a 41-BB/CD3ζ backbone. Results: Multiple gene transcripts encoding cell surface proteins potentially amenable to CAR T-cell targeting were overexpressed in ETP-ALL cells in comparison to PBMC controls. Many of these proteins are involved in cell signaling, cell adhesion, and metastasis, and thus potentially important for leukemic cell survival. TSPAN7 (also known as TALLA-1) was the strongest differentially expressed transcript. Despite identification of several transcripts, we did not detect increased surface expression of multiple antigens that were identified as top 25 transcripts, including TALLA-1, MCAM, EPHB6, or TSLPR. Interestingly, TALLA-1 was expressed on the more mature T-cell ALL lines, JURKAT and HPB-AU, suggesting that the surface expression of TALLA protein may be developmentally regulated. Although a new target could not be identified, given the universal expression of CD7 and CD33 on ETP-ALL, we proceeded with development of CARs targeting these antigens. CD33 CAR T-cells had excellent in vitro activity in human AML cell line MOLM-14 with minimal anti-leukemia activity in six tested ETP-ALL PDX models, perhaps due to their lower CD33 expression. We next tested T-cells transduced with a bicistronic CD7-redirected CAR with a truncated EGFR (EGFRt) to facilitate measurement of transduction efficiency and to provide a CAR deletion method. Despite high EGFRt surface expression in transduced T-cells, these CD7 CAR T-cells did not demonstrate in vitro activity against ETP-ALL or mature T-ALL samples despite high CD7 surface expression on all leukemia cell lines. We postulated that abnormal CAR distribution within the T-cell itself could be a potential factor in the observed lack of CD7 CAR T-cell activity. Using fluorescent-labeling to assess CAR surface membrane distribution, we detected high intracellular expression of the CD7 CAR, and noted that it did not traffic to the cell surface. Conclusions: We applied multimodal techniques to evaluate for cell surface expression on ETP-ALL that could serve as a target for immunotherapy. Although novel targets could not be identified, we were able to design an active anti-CD33 CAR. Further studies are in progress to evaluate what degree of antigen expression is needed to be amenable to targeted therapy. Additionally, ongoing studies are assessing whether optimization of CAR design can enhance cell surface trafficking and thereby potentially improve the anti-leukemia efficacy of CD7 CAR T-cells. Disclosures Orentas: Lentigen Technology, Inc.: Employment. Maude:Novartis: Consultancy. Teachey:Novartis: Research Funding.


Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1753-1753
Author(s):  
Mirjam H.M. Heemskerk ◽  
Manja Hoogeboom ◽  
Renate Hagedoorn ◽  
Michel G.D. Kester ◽  
Roel Willemze ◽  
...  

Abstract The genetic engineering of T lymphocytes is an attractive strategy to specifically redirect T cell immunity towards viral infections and malignancies. Transfer of virus- or tumor-specific TCRs has demonstrated to endow T cells with redirected antigen specificity. We demonstrated redirected anti-leukemic reactivity of CMV specific T cells using gene transfer of minor histocompatibility antigen HA-2 specific TCRs. The HA-2-TCR-modified T cells exerted high cytolytic activity against HA-2 expressing target cells, including leukemic cells, and not against target cells negative for the HA-2 mHag. After cloning of the TCR-transferred T cells, we demonstrated that the HA-2-TCR cell surface expression, measured by HA-2-tetramer staining, was variable on the transduced T cell clones, and that the cytolytic capacity of the T cells correlated with the level of HA-2-TCR expression. Since we could demonstrate that this variation in HA-2-TCR expression was not due to differences in transgene expression, we investigated whether the endogenous TCRs influenced the expression of the introduced TCR. CMV-A2 specific T cells were isolated from peripheral blood and transduced with the HA-2-TCR. In control transduced CMV specific T cells we observed 5 different high affinity CMV specific TCRs. CMV specific T cells transduced with the HA-2-TCR that expressed predominantly the HA-2-TCR, expressed only one of these types of CMV-TCR, and in CMV specific T cells with low HA-2-TCR expression two different types of CMV-TCRs were found. These data indicated that the level of expression of the introduced TCR is strongly influenced by the endogenous TCR. To investigate whether this was due to differences in promotor activity of the endogenous and retrovirally introduced TCR, the three CMV-TCRs were characterized and transferred into unselected peripheral T cells. T cells transferred with the weak competitior CMV-TCR that was strongly downregulated in CMV specific T cells by introduction of the HA-2-TCR, showed low CMV specific cytotoxicity and no tetramer staining. In contrast, T cells transferred with the strong competitor CMV-TCR that was modestly downregulated in CMV specific T cells by introduction of the HA-2-TCR, revealed strong CMV specific cytotoxic activity and tetramer staining. These data demonstrate that the introduced and endogenous TCRs compete for cell surface expression, and that this competition is dependent on characteristics of the different TCRs and independent of whether the TCR is retrovirally introduced or naturally expressed. To investigate whether the cell surface expression of the different TCRs was determined by preferential pairing properties of the individual TCR chains, TCR α and β deficient Jurkat 76 cells were transduced with the three CMV-specific TCRs or with chimeric TCRs consisting of the TCR α chain of one TCR with the TCR β chain of another TCR. TCRαβ membrane expression revealed that TCRs with a strong competitor phenotype expressed higher levels of TCRαβ than the TCR that was a weak competitor. TCRαβ expression of Jurkat cells transduced with chimeric TCRs indicated that the expression level of the different TCRs was determined by the pairing properties of the individual TCR α and β chains and not by differences in protein expression. In conclusion these data demonstrated that introduced and endogenous TCRs compete for cell surface expression in favor of the TCR that has the highest intrinsic pairing properties.


Blood ◽  
2006 ◽  
Vol 109 (1) ◽  
pp. 235-243 ◽  
Author(s):  
Mirjam H. M. Heemskerk ◽  
Renate S. Hagedoorn ◽  
Menno A. W. G. van der Hoorn ◽  
Lars T. van der Veken ◽  
Manja Hoogeboom ◽  
...  

Abstract Genetic engineering of T lymphocytes is an attractive strategy to specifically redirect T-cell immunity toward viral infections and malignancies. We previously demonstrated redirected antileukemic reactivity of cytomegalovirus (CMV)–specific T cells by transfer of minor histocompatibility antigen HA-2–specific T-cell receptors (TCRs). HA-2–TCR-transferred CMV-specific T cells were potent effectors against HA-2–expressing leukemic cells, as well as CMV-expressing cells. Functional activity of these T cells correlated with TCR cell-surface expression. In the present study we analyzed which properties of transferred and endogenous TCRs are crucial for efficient cell-surface expression. We demonstrate that expression of the introduced TCR is not a random process but is determined by characteristics of both the introduced and the endogenously expressed TCR. The efficiency of TCR cell-surface expression is controlled by the intrinsic quality of the TCR complex. In addition, we demonstrate that chimeric TCRs can be formed and that efficiency of TCR expression is independent of whether TCRs are retrovirally introduced or naturally expressed. In conclusion, introduced, endogenous, and chimeric TCRs compete for cell-surface expression in favor of the TCR-CD3 complex with best-pairing properties.


2004 ◽  
Vol 200 (4) ◽  
pp. 493-505 ◽  
Author(s):  
Tomoo Ueno ◽  
Fumi Saito ◽  
Daniel H.D. Gray ◽  
Sachiyo Kuse ◽  
Kunio Hieshima ◽  
...  

Upon TCR-mediated positive selection, developing thymocytes relocate within the thymus from the cortex to the medulla for further differentiation and selection. However, it is unknown how this cortex–medulla migration of thymocytes is controlled and how it controls T cell development. Here we show that in mice deficient for CCR7 or its ligands mature single-positive thymocytes are arrested in the cortex and do not accumulate in the medulla. These mutant mice are defective in forming the medullary region of the thymus. Thymic export of T cells in these mice is compromised during the neonatal period but not in adulthood. Thymocytes in these mice show no defects in maturation, survival, and negative selection to ubiquitous antigens. TCR engagement of immature cortical thymocytes elevates the cell surface expression of CCR7. These results indicate that CCR7 signals are essential for the migration of positively selected thymocytes from the cortex to the medulla. CCR7-dependent cortex–medulla migration of thymocytes plays a crucial role in medulla formation and neonatal T cell export but is not essential for maturation, survival, negative selection, and adult export of thymocytes.


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 ◽  
...  

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.


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