scholarly journals Absence of H-2 restriction in primary and secondary mixed-lymphocyte reactions to strong M1s determinants.

1980 ◽  
Vol 151 (2) ◽  
pp. 407-417 ◽  
Author(s):  
K Molnar-Kimber ◽  
J Sprent
Keyword(s):  
T Cells ◽  
Positive Selection ◽  
Proliferative Response ◽  
Negative Selection ◽  
Selection Procedures ◽  
Per Se ◽  
Mixed Lymphocyte Reactions ◽  
Selection Of

Negative and positive selection procedures were used to establish whether the strong proliferative response of T cells to M1sa determinants is H-2 restricted. After negative selection of H-2 determinants in vivo, it was shown that T cells give high primary mixed lymphocyte reactions in vitro to M1sa determinants presented on H-2-incompatible stimulator cells. Other studies demonstrated that (a) negative selection of T cells to M1sa determinants on H-2-incompatible cells removed T cells with specificity for M1sa-bearing H-2-compatible cells, and (b) T cells primed in vitro or in vivo to M1sa determinants on H-2-compatible cells gave high secondary responses to M1sa determinants presented either on H-2-compatible or H-2-incompatible stimulator cells. From these data we conclude that T cells recognize M1sa determinants per se rather than an association of M1sa plus self or allo-H-2 determinants.

Artificial Thymic Organoids Permit Allelic Exclusion and Efficient Generation of Naïve TCR-Engineered T-Cells from Human Hematopoietic Stem Cells In Vitro

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4553-4553
Author(s):  
Christopher S Seet ◽  
Chongbin He ◽  
Michael Bethune ◽  
Suwen Li ◽  
Brent Chick ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Intellectual Property ◽  
Positive Selection ◽  
Allelic Exclusion ◽  
Hematopoietic Stem ◽  
Engineered T Cells ◽  
Selection Of

Abstract Engineered adoptive immunotherapies have shown unprecedented activity in the treatment of cancer and chronic viral infections. Current approaches rely on individualized ex vivo genetic modification of autologous T cells due to the risk of graft-versus-host disease from allogeneic T cells. These processes furthermore require activation and prolonged expansion of T cells, which may reduce in vivo efficacy and persistence. Direct in vitro differentiation of engineered T cells from hematopoietic stem and progenitor cells (HSPCs) may overcome these problems by permitting the suppression of endogenous TCR expression through allelic exclusion, and the de novo generation of naïve antigen-specific T cells. Existing methods of in vitro human T cell differentiation are subject to wide experimental variability and do not adequately support the positive selection of immature T cell precursors to mature T cells, and thus have not been suitable for clinical-scale production of engineered T cells. We report here the preclinical development of an artificial thymic organoid (ATO) system using off-the-shelf, serum-free components and a standardized stromal cell line that supports highly efficient in vitro differentiation and positive selection of native and TCR-engineered human T cells from cord blood (CB), bone marrow, and mobilized peripheral blood CD34+ HSPCs, and purified CD34+CD38- hematopoietic stem cells. ATOs closely recapitulated thymic T cell commitment and differentiation, resulting in greater than 80% CD7+CD5+ T-lineage cells and 50% CD4+CD8+ double positive (DP) T cell precursors by 4 weeks. By 6 weeks, 30-40% of ATO cells were CD3+TCRαβ+ T cells, of which 20-30% were mature CD8 single positive (SP) T cells. CD4SP cells were generated at a lower frequency and later in culture (2-14% of CD3+TCRαβ+ cells). ATO-derived T cells exhibited a naïve CD45RA+CD27+CCR7+CD62L+ phenotype, a diverse, thymic-like TCR repertoire, and robust TCR-dependent cytokine release and proliferation. Transduction of CB CD34+ HSPCs with an HLA-A*02:01-restricted αβ TCR specific for NY-ESO-1 resulted in a markedly increased cell output per ATO (>400-fold, relative to input HSPCs) and enhanced generation of naïve CD3+TCRαβ+CD8αβ+ conventional T cells, the majority of which were antigen-specific by tetramer staining. Positive selection of TCR-engineered naïve T cells could be further enhanced by expression of cognate HLA-A*02:01 in ATO stromal cells. ATO-derived TCR-engineered T cells exhibited a near complete lack of endogenous TCR Vβ expression, consistent with induction of allelic exclusion by the exogenous TCR during T cell development. ATO-derived engineered T cells underwent antigen-specific cytotoxic priming, polyfunctional cytokine release, and proliferation in response to artificial APCs; and exhibited antigen-specific killing of NY-ESO-1+ tumor cells in vitro and in vivo. ATOs thus present a highly efficient off-the-shelf platform for the generation of clinically relevant numbers of naïve and potentially non-alloreactive engineered T cells for adoptive immunotherapy. Clinical translation of the ATO system will be aided by its simplicity, scalability, use of serum-free components, and compatibility with irradiated stromal cells. In addition, genetic manipulation of stem or stromal cell components can be easily incorporated into the system to further enhance downstream T cell engraftment or function. Disclosures Seet: Kite Pharma: Patents & Royalties: Kite Pharma holds an exclusive license to certain intellectual property. Montel-Hagen:Kite Pharma: Patents & Royalties: Kite Pharma holds an exclusive license to certain intellectual property. Crooks:Kite Pharma: Patents & Royalties: Kite Pharma holds an exclusive license to certain intellectual property, Research Funding.

scholarly journals Interleukin 12 induces stable priming for interferon gamma (IFN-gamma) production during differentiation of human T helper (Th) cells and transient IFN-gamma production in established Th2 cell clones.

1994 ◽  
Vol 179 (4) ◽  
pp. 1273-1283 ◽  
Author(s):  
R Manetti ◽  
F Gerosa ◽  
M G Giudizi ◽  
R Biagiotti ◽  
P Parronchi ◽  
...  
Keyword(s):  
T Cells ◽  
Interferon Gamma ◽  
Specific Antigen ◽  
Th2 Cells ◽  
Interleukin 12 ◽  
T Helper ◽  
Ifn Gamma ◽  
Selection Of

Interleukin 12 (IL-12) facilitates the generation of a T helper type 1 (Th1) response, with high interferon gamma (IFN-gamma) production, while inhibiting the generation of IL-4-producing Th2 cells in polyclonal cultures of both human and murine T cells and in vivo in the mouse. In this study, we analyzed the effect of IL-12, present during cloning of human T cells, on the cytokine profile of the clones. The culture system used allows growth of clones from virtually every T cell, and thus excludes the possibility that selection of precommitted Th cell precursors plays a role in determining characteristics of the clones. IL-12 present during the cloning procedures endowed both CD4+ and CD8+ clones with the ability to produce IFN-gamma at levels severalfold higher than those observed in clones generated in the absence of IL-12. This priming was stable because the high levels of IFN-gamma production were maintained when the clones were cultured in the absence of IL-12 for 11 d. The CD4+ and some of the CD8+ clones produced variable amounts of IL-4. Unlike IFN-gamma, IL-4 production was not significantly different in clones generated in the presence or absence of IL-12. These data suggest that IL-12 primes the clone progenitors, inducing their differentiation to high IFN-gamma-producing clones. The suppression of IL-4-producing cells observed in polyclonally generated T cells in vivo and in vitro in the presence of IL-12 is not observed in this clonal model, suggesting that the suppression depends more on positive selection of non-IL-4-producing cells than on differentiation of individual clones. However, antigen-specific established Th2 clones that were unable to produce IFN-gamma with any other inducer did produce IFN-gamma at low but significant levels when stimulated with IL-12 in combination with specific antigen or insoluble anti-CD3 antibodies. This induction of IFN-gamma gene expression was transient, because culture of the established clones with IL-12 for up to 1 wk did not convert them into IFN-gamma producers when stimulated in the absence of IL-12. These results suggest that Th clones respond to IL-12 treatment either with a stable priming for IFN-gamma production or with only a transient low level expression of the IFN-gamma gene, depending on their stage of differentiation.

scholarly journals A Kinetic Threshold between Negative and Positive Selection Based on the Longevity of the T Cell Receptor–Ligand Complex

1999 ◽  
Vol 189 (10) ◽  
pp. 1531-1544 ◽  
Author(s):  
Calvin B. Williams ◽  
Deborah L. Engle ◽  
Gilbert J. Kersh ◽  
J. Michael White ◽  
Paul M. Allen
Keyword(s):  
T Cells ◽  
T Cell ◽  
Positive Selection ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Ligand Complex ◽  
Altered Peptide Ligands ◽  
Self Peptides ◽  
Selection Of

We have developed a unique in vivo system to determine the relationship between endogenous altered peptide ligands and the development of major histocompatibility complex class II– restricted T cells. Our studies use the 3.L2 T cell receptor (TCR) transgenic mouse, in which T cells are specific for Hb(64–76)/I-Ek and positively selected on I-Ek plus self-peptides. To this endogenous peptide repertoire, we have individually added one of six well-characterized 3.L2 ligands. This transgenic approach expands rather than constrains the repertoire of self-peptides. We find that a broad range of ligands produce negative selection of thymocytes in vivo. When compared with the in vitro TCR–ligand binding kinetics, we find that these negatively selecting ligands all have a half-life of 2 s or greater. Additionally, one of two ligands examined with no detectable binding to the 3.L2 TCR and no activity on mature 3.L2 T cells (Q72) enhances the positive selection of transgenic thymocytes in vivo. Together, these data establish a kinetic threshold between negative and positive selection based on the longevity of TCR–ligand complexes.

Mouse thymic epithelial cell lines expressing “Aire” and peripheral tissue-specific antigens reproduce in vitro negative selection of T cells

2011 ◽  
Vol 317 (14) ◽  
pp. 2019-2030 ◽  
Author(s):  
Yoshitaka Yamaguchi ◽  
Atsushi Takayanagi ◽  
Jiabing Chen ◽  
Kosuke Sakai ◽  
Jun Kudoh ◽  
...  
Keyword(s):  
T Cells ◽  
Epithelial Cell ◽  
Cell Lines ◽  
Negative Selection ◽  
Thymic Epithelial Cell ◽  
Tissue Specific ◽  
Epithelial Cell Lines ◽  
Specific Antigens ◽  
Selection Of

scholarly journals The influenza A virus nucleoprotein gene controls the induction of both subtype specific and cross-reactive cytotoxic T cells.

1984 ◽  
Vol 160 (2) ◽  
pp. 552-563 ◽  
Author(s):  
A R Townsend ◽  
J J Skehel
Keyword(s):  
T Cells ◽  
Influenza A ◽  
Cytotoxic T Cells ◽  
Target Cells ◽  
Spleen Cells ◽  
Influenza A Viruses ◽  
Group A ◽  
Selection Of

Using genetically typed recombinant influenza A viruses that differ only in their genes for nucleoprotein, we have demonstrated that repeated stimulation in vitro of C57BL/6 spleen cells primed in vivo with E61-13-H17 (H3N2) virus results in the selection of a population of cytotoxic T lymphocytes (CTL) whose recognition of infected target cells maps to the gene for nucleoprotein of the 1968 virus. Influenza A viruses isolated between 1934 and 1979 fall into two groups defined by their ability to sensitize target cells for lysis by these CTL: 1934-1943 form one group (A/PR/8/34 related) and 1946-1979 form the second group (A/HK/8/68 related). These findings complement and extend our previous results with an isolated CTL clone with specificity for the 1934 nucleoprotein (27, 28). It is also shown that the same spleen cells derived from mice primed with E61-13-H17 virus in vivo, but maintained in identical conditions by stimulation with X31 virus (which differs from the former only in the origin of its gene for NP) in vitro, results in the selection of CTL that cross-react on target cells infected with A/PR/8/1934 (H1N1) or A/Aichi/1968 (H3N2). These results show that the influenza A virus gene for NP can play a role in selecting CTL with different specificities and implicate the NP molecule as a candidate for a target structure recognized by both subtype-directed and cross-reactive influenza A-specific cytotoxic T cells.

scholarly journals In vivo and in vitro clonal deletion of double-positive thymocytes.

1992 ◽  
Vol 175 (5) ◽  
pp. 1307-1316 ◽  
Author(s):  
N J Vasquez ◽  
J Kaye ◽  
S M Hedrick
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytochrome C ◽  
Cyclosporin A ◽  
Negative Selection ◽  
Double Positive ◽  
Antigen Dose ◽  
Mhc Molecules

To study the processes of thymic development, we have established transgenic mice expressing and alpha/beta T cell antigen receptor (TCR) specific for cytochrome c associated with class II major histocompatibility complex (MHC) molecules. The transgenic TCR chains are expressed by most of the thymocytes in these mice, and these cells have been shown to efficiently mature in association with Ek- and Ab-encoded class II MHC molecules. This report describes a characterization of the negative selection of these transgenic thymocytes in vivo that is associated with the expression of As molecules. Negative selection by As molecules appears to result in the deletion of a late stage of CD4/CD8 double-positive thymocytes in that there is a virtual absence of transgenic TCR bearing CD4 single-positive thymocytes. This phenotype is accompanied by the appearance of CD4/CD8 double-negative thymocytes and peripheral T cells that are functionally antigen reactive. The process of negative selection has also been investigated using an in vitro culture system. Upon presentation of cytochrome c by Eb-expressing nonthymic antigen-presenting cells, there occurs an antigen dose-dependent deletion of the majority of CD4/CD8 double-positive thymocytes. In contrast, presentation of Staphylococcal enterotoxin A by Eb in vitro results in minimal deletion of double-positive thymocytes. In addition, we use this in vitro model to examine the effects of cyclosporin A on negative selection. In contrast to its effects on mature T cells, and the findings of others in vivo, cyclosporin A does not inhibit antigen-induced deletion of double-positive thymocytes. Finally, a comparison of the antigen dose responses for thymocyte deletion and for peripheral T cell activation indicates that double-positive thymocyte recognition is more sensitive than mature T cells to antigen recognition.

scholarly journals Normal Thymocyte Negative Selection in TRAIL-deficient Mice

2003 ◽  
Vol 198 (3) ◽  
pp. 491-496 ◽  
Author(s):  
Erika Cretney ◽  
Adam P. Uldrich ◽  
Stuart P. Berzins ◽  
Andreas Strasser ◽  
Dale I. Godfrey ◽  
...  
Keyword(s):  
Negative Selection ◽  
Fas Ligand ◽  
Critical Role ◽  
Death Receptor ◽  
Critical Factor ◽  
Immunological Tolerance ◽  
Trail Signaling ◽  
Selection Of

The molecular basis of thymocyte negative selection, which plays a critical role in establishing and maintaining immunological tolerance, is not yet resolved. In particular, the importance of the death receptor subgroup of the tumor necrosis factor (TNF)-family has been the subject of many investigations, with equivocal results. A recent report suggested that TRAIL was a critical factor in this process, a result that does not fit well with previous studies that excluded a role for the FADD-caspase 8 pathway, which is essential for TRAIL and Fas ligand (FasL) signaling, in negative selection. We have investigated intrathymic negative selection of TRAIL-deficient thymocytes, using four well-established models, including antibody-mediated TCR/CD3 ligation in vitro, stimulation with endogenous superantigen in vitro and in vivo, and treatment with exogenous superantigen in vitro. We were unable to demonstrate a role for TRAIL signaling in any of these models, suggesting that this pathway is not a critical factor for thymocyte negative selection.

Positive selection of T cells, an in vitro view

2010 ◽  
Vol 22 (5) ◽  
pp. 276-286 ◽  
Author(s):  
Dženetdina Dervović ◽  
Juan Carlos Zúñiga-Pflücker
Keyword(s):  
T Cells ◽  
Positive Selection ◽  
Selection Of

In Vivo T-Lymphocyte Tolerance in the Absence of Thymic Clonal Deletion Mediated by Hematopoietic Cells

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3856-3862 ◽  
Author(s):  
Joost P.M. van Meerwijk ◽  
H. Robson MacDonald
Keyword(s):  
T Cells ◽  
T Cell ◽  
Negative Selection ◽  
Apoptotic Cell ◽  
Hematopoietic Cells ◽  
Cell Receptor ◽  
Cell Repertoire ◽  
Clonal Deletion

Abstract Thymic negative selection renders the developing T-cell repertoire tolerant to self-major histocompatability complex (MHC)/peptide ligands. The major mechanism of induction of self-tolerance is thought to be thymic clonal deletion, ie, the induction of apoptotic cell death in thymocytes expressing a self-reactive T-cell receptor. Consistent with this hypothesis, in mice deficient in thymic clonal deletion mediated by cells of hematopoietic origin, a twofold to threefold increased generation of mature thymocytes has been observed. Here we describe the analysis of the specificity of T lymphocytes developing in the absence of clonal deletion mediated by hematopoietic cells. In vitro, targets expressing syngeneic MHC were readily lysed by activated CD8+ T cells from deletion-deficient mice. However, proliferative responses of T cells from these mice on activation with syngeneic antigen presenting cells were rather poor. In vivo, deletion-deficient T cells were incapable of induction of lethal graft-versus-host disease in syngeneic hosts. These data indicate that in the absence of thymic deletion mediated by hematopoietic cells functional T-cell tolerance can be induced by nonhematopoietic cells in the thymus. Moreover, our results emphasize the redundancy in thymic negative selection mechanisms.

scholarly journals Positive Selection of Cd4+ T Cells Is Induced in Vivo by Agonist and Inhibited by Antagonist Peptides

2001 ◽  
Vol 194 (4) ◽  
pp. 407-416 ◽  
Author(s):  
Piotr Kraj ◽  
Rafal Pacholczyk ◽  
Hanna Ignatowicz ◽  
Pawel Kisielow ◽  
Peter Jensen ◽  
...  
Keyword(s):  
T Cells ◽  
Positive Selection ◽  
Cd4 T Cells ◽  
In Vivo Model ◽  
Agonist Peptide ◽  
Histocompatibility Complex ◽  
Antagonist Peptides ◽  
Prevailing View ◽  
Selection Of

The nature of peptides that positively select T cells in the thymus remains poorly defined. Here we report an in vivo model to study the mechanisms of positive selection of CD4+ T cells. We have restored positive selection of TCR transgenic CD4+ thymocytes, arrested at the CD4+CD8+ stage, due to the lack of the endogenously selecting peptide(s), in mice deficient for H2-M and invariant chain. A single injection of soluble agonist peptide(s) initiated positive selection of CD4+ transgenic T cells that lasted for up to 14 days. Positively selected CD4+ T cells repopulated peripheral lymphoid organs and could respond to the antigenic peptide. Furthermore, coinjection of the antagonist peptide significantly inhibited agonist-driven positive selection. Hence, contrary to the prevailing view, positive selection of CD4+ thymocytes can be induced in vivo by agonist peptides and may be a result of accumulation of signals from TCR engaged by different peptides bound to major histocompatibility complex class II molecules. We have also identified a candidate natural agonist peptide that induces positive selection of CD4+ TCR transgenic thymocytes.

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