In Vitro Evidence That Cytokine Receptor Signals Are Required for Differentiation of Double Positive Thymocytes into Functionally Mature CD8+ T Cells

CD4+8+ double positive (DP) thymocytes differentiate into CD4+ and CD8+ mature T cells in response to TCR signals. However, TCR signals that are initiated in DP thymocytes are unlikely to persist throughout all subsequent differentiation steps, suggesting that other signals must sustain thymocyte differentiation after TCR signaling has ceased. Using an in vitro experimental system, we now demonstrate that cytokine receptor signals, such as those transduced by IL-7 receptors, are required for differentiation of signaled DP thymocytes into functionally mature CD8+ T cells as they: (a) up-regulate Bcl-2 expression to maintain thymocyte viability; (b) enhance CD4 gene silencing; (c) promote functional maturation;and (d) up-regulate surface expression of glucose transporter molecules, which improve nutrient uptake and increase metabolic activity. IL-7Rs appear to be unique among cytokine receptors in maintaining the viability of newly generated CD4−8+ thymocytes, whereas several different cytokine receptors can provide the trophic/differentiative signals for subsequent CD8+ thymocyte differentiation and maturation. Thus, cytokine receptors provide both survival and trophic/differentiative signals with varying degrees of redundancy that are required for differentiation of signaled DP thymocytes into functionally mature CD8+ T cells.

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The Checkpoint for Agonist Selection Precedes Conventional Selection in Human Thymus

Blood ◽

10.1182/blood.v128.22.860.860 ◽

2016 ◽

Vol 128 (22) ◽

pp. 860-860

Author(s):

Greet Verstichel ◽

David Vermijlen ◽

Liesbet Martens ◽

Glenn Goetgeluk ◽

Yvan Saeys ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Double Positive ◽

Human Thymus ◽

Tcr Signals

Abstract The thymus plays a central role in self-tolerance by preventing strongly self-reactive thymocytes from accumulating as naïve T cell receptor (TCR) αβ+ T cells in the periphery. The elimination of auto-reactive T cells from the naïve pool is in part mediated by deletion during conventional negative selection. Alternatively, self-reactive thymocytes can also be positively selected in response to strong TCR signals during agonist selection and functionally differentiate to innate TCRαβ + T cells such as the CD8αα+ double negative (DN) T cells. How thymocytes discriminate between these opposite outcomes remains unclear. We identified a novel agonist-selected PD-1+ CD8αα+ subset of mature CD8+ T cells in human thymus. Using the same markers a similar population was also identified in cord blood at about the same frequency as TCRγδ+ cells. This population expresses high levels of Helios, indicative of strong TCR engagement, and displays an effector phenotype associated with agonist selection. Indeed, PD-1+CD8αα+ T cells exhibit innate production of IFN-γ and an elevated T-bet to Eomes ratio typical of effector CD8 T cells. These cells are CD62L-, CXCR3+ and Hobit high suggesting that these cells leave the thymus and home to the tissues. Interestingly, in vitro CD3/TCR stimulation of sorted early post-β-selection thymocyte blasts uniquely gives rise to this innate subset, whereas small CD4+CD8+ double positive precursors fail to survive strong TCR signals. The generation of the innate subset seems to arise also in vivo from early post-β-selection thymocyte blasts as these two populations have an identical TCRα repertoire: ex vivo isolated PD-1+CD8αα+ thymocytes are skewed for early 3' TRAV and 5' TRAJ rearrangements compared to conventional CD8 T cells. A similar skewing was found in early post-β-selection thymocyte blasts. As TCRα rearrangements are terminated by TCR engagement of agonist selection, this is strong evidence for a precursor progeny relationship. Together, we conclude that human CD8αα+ T cells are preferentially selected by strong TCR engagement on a subset of progenitors that express a full TCRαβ early on, leading to the generation of a post-selection T cell population with innate functional capacity and a markedly distinct TCR repertoire. These findings uncover the heterogeneity among DP precursors in their potential to survive strong selection signals and suggests that the decision making in the thymus to divert immature thymocytes to the agonist selection pathway occurs early before conventional selection of DP cells. We propose that progression through the immature thymic developmental program influences the outcome of TCR engagement with early post-β-selection thymocytes triggered by strong TCR signals preferentially giving rise to innate CD8αα+ T cells in humans. Disclosures No relevant conflicts of interest to declare.

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Differential effects of γc cytokines on postselection differentiation of CD8 thymocytes

Blood ◽

10.1182/blood-2012-05-433508 ◽

2013 ◽

Vol 121 (1) ◽

pp. 107-117 ◽

Cited By ~ 21

Author(s):

Moutih Rafei ◽

Alexandre Rouette ◽

Sylvie Brochu ◽

Juan Ruiz Vanegas ◽

Claude Perreault

Keyword(s):

T Cells ◽

Cell Differentiation ◽

T Cell ◽

Positive Selection ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

T Cell Differentiation ◽

Thymic Epithelial Cells ◽

Double Positive

Abstract The primary consequence of positive selection is to render thymocytes responsive to cytokines and chemokines expressed in the thymic medulla. In the present study, our main objective was to discover which cytokines could support the differentiation of positively selected thymocytes. To this end, we have developed an in vitro model suitable for high-throughput analyses of positive selection and CD8 T-cell differentiation. The model involves coculture of TCRhiCD5intCD69− double-positive (DP) thymocytes with peptide-pulsed OP9 cells and γc-cytokines. We report that IL-4, IL-7, and IL-21 have nonredundant effects on positively selected DP thymocytes. IL-7 signaling phosphorylates STAT5 and ERK; induces Foxo1, Klf2, and S1pr1; and supports the differentiation of classic CD8 T cells. IL-4 activates STAT6 and ERK and supports the differentiation of CD8intPD-L1hiCD44hiEOMES+ innate CD8 T cells. IL-21 is produced by thymic epithelial cells and the IL-21 receptor-α is strongly induced on DP thymocytes undergoing positive selection. IL-21 signaling phosphorylates STAT3 and STAT5, but not ERK, and does not support CD8 T-cell differentiation. However, IL-21 has a unique ability to up-regulate BCL-6, expand DP thymocytes undergoing positive selection, and increase the production of mature T cells. Our data suggest that injection of recombinant IL-21 might enhance thymic output in subjects with age- or disease-related thymic atrophy.

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Identification of a CD11b+/Gr-1+/CD31+ myeloid progenitor capable of activating or suppressing CD8+T cells

Blood ◽

10.1182/blood.v96.12.3838 ◽

2000 ◽

Vol 96 (12) ◽

pp. 3838-3846 ◽

Cited By ~ 372

Author(s):

Vincenzo Bronte ◽

Elisa Apolloni ◽

Anna Cabrelle ◽

Roberto Ronca ◽

Paolo Serafini ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Cell Function ◽

Interleukin 4 ◽

Double Positive ◽

Gm Csf ◽

Macrophage Colony ◽

Immunocompromised Mice

Abstract Apoptotic death of CD8+ T cells can be induced by a population of inhibitory myeloid cells that are double positive for the CD11b and Gr-1 markers. These cells are responsible for the immunosuppression observed in pathologies as dissimilar as tumor growth and overwhelming infections, or after immunization with viruses. The appearance of a CD11b+/Gr-1+ population of inhibitory macrophages (iMacs) could be attributed to high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in vivo. Deletion of iMacs in vitro or in vivo reversed the depression of CD8+ T-cell function. We isolated iMacs from the spleens of immunocompromised mice and found that these cells were positive for CD31, ER-MP20 (Ly-6C), and ER-MP58, markers characteristic of granulocyte/monocyte precursors. Importantly, although iMacs retained their inhibitory properties when cultured in vitro in standard medium, suppressive functions could be modulated by cytokine exposure. Whereas culture with the cytokine interleukin 4 (IL-4) increasediMac inhibitory activity, these cells could be differentiated into a nonadherent population of fully mature and highly activated dendritic cells when cultured in the presence of IL-4and GM-CSF. A common CD31+/CD11b+/Gr-1+ progenitor can thus give rise to cells capable of either activating or inhibiting the function of CD8+ T lymphocytes, depending on the cytokinemilieu that prevails during antigen-presenting cell maturation.

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Role of CD4 in thymocyte selection and maturation.

Journal of Experimental Medicine ◽

10.1084/jem.169.6.2085 ◽

1989 ◽

Vol 169 (6) ◽

pp. 2085-2096 ◽

Cited By ~ 51

Author(s):

J C Zuñiga-Pflücker ◽

S A McCarthy ◽

M Weston ◽

D L Longo ◽

A Singer ◽

...

Keyword(s):

T Cells ◽

Mhc Class Ii ◽

Cd8 T Cells ◽

Decisive Role ◽

Class Ii ◽

Double Positive ◽

Adult Mice

We examined the possible role of CD4 molecules during in vivo and in vitro fetal thymic development. Our results show that fetal thymi treated with intact anti-CD4 mAbs fail to generate CD4 single-positive T cells, while the generation of the other phenotypes remains unchanged. Most importantly, the use of F(ab')2 and Fab anti-CD4 mAb gave identical results, i.e., failure to generate CD4+/CD8- T cells, with no effect on the generation of CD4+/CD8+ T cells. Since F(ab')2 and Fab anti-CD4 fail to deplete CD4+/CD8- in adult mice, these results strongly argue that the absence of CD4+/CD8- T cells is not due to depletion, but rather, is caused by a lack of positive selection, attributable to an obstructed CD4-MHC class II interaction. Furthermore, we also observed an increase in TCR/CD3 expression after anti-CD4 (divalent or monovalent) mAb treatment. The TCR/CD3 upregulation occurs in the double-positive population, and may result from CD4 signaling after mAb engagement, or may be a consequence of the blocked CD4-class II interactions. One proposed model argues that the CD3 upregulation occurs in an effort to compensate for the reduction in avidity or signaling that is normally provided by the interaction of the CD4 accessory molecule and its ligand. As a whole, our findings advocate that CD4 molecules play a decisive role in the differentiation of thymocytes.

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Generation of Melanoma Antigen Specific T Cells From Human Ips Cells Using a Feeder Free System.

Blood ◽

10.1182/blood.v120.21.2292.2292 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2292-2292

Author(s):

Raul Vizcardo ◽

Kyoko Masuda ◽

Daisuke Yamada ◽

Tomokatsu Ikawa ◽

Kanako Shimizu ◽

...

Keyword(s):

T Cells ◽

Cell Therapy ◽

Cd8 T Cells ◽

Es Cells ◽

Cytotoxic T Cells ◽

Ips Cells ◽

Antigen Specificity ◽

Double Positive ◽

Melanoma Antigen

Abstract Abstract 2292 iPS cells can be induced from various types of somatic cells by reprogramming them using Yamanaka factors (Oct4, Sox2, Klf4, and Myc). They are reported to be very similar to ES cells in many respects, such as gene expression pattern and multipotency. They are expected to be used as a cell source for production of various types of cells to be used in regeneration medicine. In this study, we planned to use iPS cells as a progenitor source for immune cell therapy. For this purpose, iPS cells derived from a lymphocyte that shows a certain antigen-specificity are preferable, because antigen specificity is inherited to iPS cells made from the lymphocyte. For example, if iPS cell are produced from cytotoxic T cells specific to a tumor antigen, T cells generated from these T-iPS can be used in cell therapy for patients bearing cancer. We have succeeded in establishing iPS cells from human mature T cells (T-iPS cells), namely from whole CD3+ cells or CD4−CD8+ cells of cord blood as well of adult peripheral blood. These T-iPS cells were confirmed to bear productively rearranged TCRβ chain gene. These T-iPS cells were differentiated to CD4+CD8+ double positive T cells and eventually into CD8+ single positive T cells expressing αβTCR in an in vitro co-culture system using OP9-DL1 stromal cells. Such T cell induction occurred more efficiently from T-iPS cells than from human ES cells or from iPS cells derived from other cell types. Sequence analysis of their TCRβ chain suggests that these T cells preserved their original antigen specificity. To confirm that T-iPS cells can generate antigen specific T cells we established T-iPS cells from mature cytotoxic T cells specific to MART-1 (Melanoma antigen recognized by T cells 1) epitope. These MART-1 T-iPS cells were differentiated in vitro until CD4+CD8+ double positive T cells. By stimulation with anti-CD3 antibody, DP cells generated in vitro from MART1-T-iPS cells turned into a large number of CD8+ T cells specific to MART-1, which produced a substantial amount of IFNg upon TCR stimulation. The present study thus provides a novel method for cloning and expanding CD8+ T cells specific to a given antigen, which can be potentially applied for cell therapy against cancer. Disclosures: No relevant conflicts of interest to declare.

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T cell receptor ligation induces interleukin (IL) 2R beta chain expression in rat CD4,8 double positive thymocytes, initiating an IL-2-dependent differentiation pathway of CD8 alpha+/beta- T cells.

Journal of Experimental Medicine ◽

10.1084/jem.177.2.541 ◽

1993 ◽

Vol 177 (2) ◽

pp. 541-546 ◽

Cited By ~ 12

Author(s):

J H Park ◽

R Mitnacht ◽

N Torres-Nagel ◽

T Hünig

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cd8 T Cells ◽

Cell Receptor ◽

Beta Chain ◽

Double Positive ◽

Alpha Chain ◽

Alpha Beta

The role of interleukin (IL)2 in intrathymic T cell development is highly controversial, and nothing is known about IL-2R expression on thymocytes of the T cell receptor (TCR) alpha/beta lineage undergoing TCR-driven differentiation events. We analyze here IL-2R alpha and beta mRNA expression in an in vitro system where newly generated rat CD4,8 double positive (DP) thymocytes respond to TCR ligation plus IL-2 (but not to either stimulus alone) with rapid differentiation to functional CD8 single positive T cells (Hünig, T., and R. Mitnacht. 1991. J. Exp. Med. 173:561). TCR ligation induced expression of IL-2R beta (but not alpha) chain mRNA in DP thymocytes. Addition of IL-2 then lead to functional maturation and expression of the IL-2R alpha chain. To investigate if the CD8 T cells generated via this IL-2R beta-driven pathway in vitro correspond to the bulk of CD8 T cells seeding peripheral lymphoid organs in vivo, we compared their phenotype to that of lymph node CD8 T cells. Surprisingly, analysis of CD8 cell surface expression using a novel anti-CD8 monoclonal antibody specific for the alpha/beta heterodimeric isoform, and of CD8 alpha and beta chain mRNA revealed that T cells generated by TCR ligation plus IL-2 resemble thymus-independent rather than thymus-derived CD8 cells in that they express CD8 alpha without beta chains. These findings demonstrate that TCR crosslinking induces functional IL-2R on immature DP rat thymocytes. In addition, they show that at least in vitro, CD8 alpha/alpha T cells are generated from TCR-stimulated DP thymocytes (which express the CD8 alpha/beta in the heterodimeric isoform) along an IL-2-driven pathway of T cell differentiation.

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Identification of a CD11b+/Gr-1+/CD31+ myeloid progenitor capable of activating or suppressing CD8+T cells

Blood ◽

10.1182/blood.v96.12.3838.h8003838_3838_3846 ◽

2000 ◽

Vol 96 (12) ◽

pp. 3838-3846 ◽

Cited By ~ 4

Author(s):

Vincenzo Bronte ◽

Elisa Apolloni ◽

Anna Cabrelle ◽

Roberto Ronca ◽

Paolo Serafini ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Cell Function ◽

Interleukin 4 ◽

Double Positive ◽

Gm Csf ◽

Macrophage Colony ◽

Immunocompromised Mice

Apoptotic death of CD8+ T cells can be induced by a population of inhibitory myeloid cells that are double positive for the CD11b and Gr-1 markers. These cells are responsible for the immunosuppression observed in pathologies as dissimilar as tumor growth and overwhelming infections, or after immunization with viruses. The appearance of a CD11b+/Gr-1+ population of inhibitory macrophages (iMacs) could be attributed to high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in vivo. Deletion of iMacs in vitro or in vivo reversed the depression of CD8+ T-cell function. We isolated iMacs from the spleens of immunocompromised mice and found that these cells were positive for CD31, ER-MP20 (Ly-6C), and ER-MP58, markers characteristic of granulocyte/monocyte precursors. Importantly, although iMacs retained their inhibitory properties when cultured in vitro in standard medium, suppressive functions could be modulated by cytokine exposure. Whereas culture with the cytokine interleukin 4 (IL-4) increasediMac inhibitory activity, these cells could be differentiated into a nonadherent population of fully mature and highly activated dendritic cells when cultured in the presence of IL-4and GM-CSF. A common CD31+/CD11b+/Gr-1+ progenitor can thus give rise to cells capable of either activating or inhibiting the function of CD8+ T lymphocytes, depending on the cytokinemilieu that prevails during antigen-presenting cell maturation.

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