Development of Thymic Microenvironments In Vitro Is Oxygen-dependent and Requires Permanent Presence of T-cell Progenitors

Development of a mature T-cell repertoire in the thymus depends on lympho-stromal interaction between thymocytes and stromal cells. To facilitate intercellular contact, the epithelium in the thymus has differentiated into a unique three-dimensionally (3D)-oriented network. Here we analyze factors influencing induction and maintenance of the 3D configuration of the epithelial network in fetal thymic lobes in vitro. We show that the 3D configuration of the thymic stroma depends on (a) the oxygen pressure in vitro and (b) permanent physical contact between stromal cells and developing thymocytes. This latter feature is demonstrated by incubation of fetal thymic lobes with deoxyguanosine (d-Guo), inducing a 2D-organized thymic stroma, with thymic cysts appearing. Reconstitution of d-Guo-treated lobes with a limited number of flow-sorted T-cell progenitors restores the 3D configuration of the thymic epithelium, but only at high oxygen pressure. This study underlines the plasticity of thymic epithelium and shows that the unique organization of the thymic epithelium is dependent on both oxygen and crosstalk signals derived from developing thymocytes.

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Notch ligands Delta1 and Jagged1 transmit distinct signals to T-cell precursors

Blood ◽

10.1182/blood-2004-08-3257 ◽

2005 ◽

Vol 105 (4) ◽

pp. 1440-1447 ◽

Cited By ~ 119

Author(s):

Sophie M. Lehar ◽

James Dooley ◽

Andrew G. Farr ◽

Michael J. Bevan

Keyword(s):

T Cell ◽

Stromal Cells ◽

Notch Pathway ◽

Cell Lineage ◽

Thymocyte Development ◽

Thymic Development ◽

T Cell Progenitors ◽

Notch Ligands ◽

Immature Thymocytes

AbstractSignaling through the Notch pathway plays an essential role in inducing T-lineage commitment and promoting the maturation of immature thymocytes. Using an in vitro culture system, we show that 2 different classes of Notch ligands, Jagged1 or Delta1, transmit distinct signals to T-cell progenitors. OP9 stromal cells expressing either Jagged1 or Delta1 inhibit the differentiation of DN1 thymocytes into the B-cell lineage, but only the Delta1-expressing stromal cells promote the proliferation and maturation of T-cell progenitors through the early double-negative (DN) stages of thymocyte development. Whereas the majority of bone marrow–derived stem cells do not respond to Jagged1 signals, T-cell progenitors respond to Jagged1 signals during a brief window of their development between the DN1 and DN3 stages of thymic development. During these stages, Jagged1 signals can influence the differentiation of immature thymocytes along the natural killer (NK) and γδ T-cell lineages.

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762 A combination of functional biomarkers improves identification of the tumor-specific reactive T cell repertoire

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0762 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A810-A810

Author(s):

Arianna Draghi ◽

Katja Harbst ◽

Inge Svane ◽

Marco Donia

Keyword(s):

T Cells ◽

T Cell ◽

De Novo ◽

Autologous Tumor ◽

T Cell Repertoire ◽

Cell Repertoire ◽

Simple Method ◽

Functional Biomarkers

BackgroundDetecting the entire repertoire of tumor-specific reactive T cells is essential for investigating the broad range of T cell functions in the tumor-microenvironment. At present, assays identifying tumor-specific functional activation measure either upregulation of specific surface molecules, de novo production of the most common antitumor cytokines or mobilization of cytotoxic granules.MethodsIn this study, we combined transcriptomic analyses of tumor-specific reactive tumorinfiltrating lymphocytes (TILs), TIL-autologous tumor cell co-cultures and commonly used established detection protocols to develop an intracellular flow cytometry staining method encompassing simultaneous detection of intracellular CD137, de novo production of TNF and IFNy and extracellular mobilization of CD107a.ResultsThis approach enabled the identification of a larger fraction of tumor-specific reactive T cells in vitro compared to standard methods, revealing the existence of multiple distinct functional clusters of tumor-specific reactive TILs. Publicly available datasets of fresh tumor single-cell RNA-sequencing from four cancer types were investigated to confirm that these functional biomarkers identified distinct functional clusters forming the entire repertoire of tumor-specific reactive T cells in situ.ConclusionsIn conclusion, we describe a simple method using a combination of functional biomarkers that improves identification of the tumor-specific reactive T cell repertoire in vitro and in situ.

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Evaluation of human Cd69+ T-cell phenotypes after co-culture with genetically-modified pig mesenchymal stromal cells: An in vitro xenotransplantation model

International Journal of Surgery ◽

10.1016/j.ijsu.2014.07.029 ◽

2014 ◽

Vol 12 ◽

pp. S14

Author(s):

Ali Malik ◽

Oleg Andreyev ◽

David K.C. Cooper ◽

Mohamed Ezzelarab

Keyword(s):

T Cell ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Genetically Modified ◽

T Cell Phenotypes ◽

Cell Phenotypes ◽

Xenotransplantation Model

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Thymic stroma is required for the development of human T cell lineages in vitro

International Immunology ◽

10.1093/intimm/1.5.471 ◽

1989 ◽

Vol 1 (5) ◽

pp. 471-478 ◽

Cited By ~ 22

Author(s):

Antonio de la Hera ◽

Wendy Marston ◽

Crisanto Aranda ◽

Marie-Luisa Toribio ◽

Carlos Martinez-A

Keyword(s):

T Cell ◽

Cell Lineages ◽

Human T Cell ◽

Thymic Stroma

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Immunotherapeutic strategies: the melanoma example

Immunotherapy ◽

10.2217/imt.09.20 ◽

2009 ◽

Vol 1 (4) ◽

pp. 679-690

Author(s):

Annelies Jorritsma ◽

Ton NM Schumacher ◽

John BAG Haanen

Keyword(s):

T Cell ◽

Adoptive Immunotherapy ◽

Low Frequency ◽

Cell Repertoire ◽

Very High Frequency ◽

In Vitro Expansion ◽

Clinical Responses ◽

Application Potential ◽

T Cell Transfer

T-cell-based immunotherapy can be induced by nonspecific activation, by antigen-specific immunization, or by adoptive immunotherapy. In this review, progress in these areas is discussed as based on data from clinical trials for the treatment of metastatic melanoma. Nonspecific immunotherapy has been shown to result in low, but in some cases significant, levels of objective tumor responses, and is often associated with autoimmune reactions. Antigen-specific targeting of tumors via vaccination has only resulted in low to very low levels of objective responses, and these strategies seem to have most value when the T-cell repertoire is not affected by tolerance. Finally, adoptive immunotherapy can be applied by in vitro expansion of autologous lymphocytes that have escaped tolerance or by genetic transfer of allogeneic T-cell receptors (TCRs). Autologous adoptive T-cell transfer has resulted in a very high frequency of clinical responses when combined with chemotherapy and IL-2 administration in single-center studies. Although TCR gene transfer has, until now, only resulted in a low frequency of clinical responses, it does have a broader application potential, and optimization of this strategy is likely to improve its efficacy.

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Stromal Cells Protect B-CLL Cells from Killing by Antigen-Specific Cytotoxic T Cells.

Blood ◽

10.1182/blood.v108.11.2814.2814 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2814-2814

Author(s):

Katja Zirlik ◽

Meike Burger ◽

Philipp Brantner ◽

Gabriele Prinz ◽

Maike Buchner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Line ◽

Cd8 T Cells ◽

Stromal Cells ◽

Stromal Cell ◽

Cytotoxic T Cells ◽

Protective Effects ◽

Stromal Cell Line

Abstract B-cell malignancy-derived immunoglobulin (idiotype) and survivin, a member of the inhibitor of apoptosis gene family and a shared tumor-associated antigen, are expressed by B-CLL cells. Idiotype- and survivin-specific cytotoxic T cells (CTLs), capable of lysing primary autologous B-CLL cells, can be induced in patients with B-CLL. However, the leukemia cell microenvironment was shown to protect B-CLL cells from apoptosis. The protective effects of stromal cells can be reversed by CXCR4 antagonists in vitro and resensitize CLL cells to spontaneous and chemotherapy-induced apoptosis. The aim of the present study is to investigate whether stromal cell contact impairs CLL killing by CTLs raised against immunoglobulin- or survivin-derived peptides and whether the addition of CXCR4 inhibitors enhances T cell mediated cytotoxicity. To analyze the T cell response, we isolated CD8+ T cells and PBMCs from HLA-A2+ healthy donors. PBMCs were differentiated into dendritic cells (DCs) and CD40-activated B cells. CD8+ T cells were primarily stimulated with peptide-pulsed DCs and then restimulated weekly with peptide-pulsed CD40-activated B cells. Heteroclitic framework region (FR−), heteroclitic complementarity-determining region (CD−) derived peptides, and native and heteroclitic survivin-derived peptides were used for CTL induction. As expected, heteroclitic peptide modifications increased the binding affinity to HLA-A*0201 compared to the native peptide as predicted by the Parker Score (Median change of predicted half-time of dissociation to HLA class I molecules 1429 minutes) and measured by the T2 binding assay (Fluorescence Index (FI) native 0.2; FI heteroclitic 0.9). Cytotoxicity of T cells was assessed by chromium release assay and by flow cytometry against CFSE-labelled CLL cells alone and in co-culture with unlabelled stromal cells in the absence or presence of CXCR4 blocking agents. The induced CTLs efficiently lysed allogenic HLA-A2+ CLL cells (mean cytotoxicity at 30:1, 10:1, 3:1 effector-to-target (E:T) ratio: 15,5%+/−2,8; 7,5%+/−2,8; and 1,9%+/− 0,6), but not HLA-A2 negative CLL cells. Co-culture of CLL cells with the murine stromal cell line M2-10B4 resulted in protection of CLL cells from lysis by antigen-specific cytotoxic T cells in vitro, indeed suggesting a protective role of the microenvironment (mean cytotoxicity at 30:1, 10:1, 3:1 E:T ratio: 5,2%+/−4,1; 0,4%+/−1,6; 1,2%+/−2,0). In contrast to apoptosis induced by fludarabine, CXCR4 blocking agents did not reverse the protective effects of the stromal cell line on T cell mediated cytotoxicity (mean cytotoxicity 30:1, 10:1, 3:1 E:T ratio: 3,1%+/−2,4; 0,8%+/−2,5; 2,3%+/−1,6). These data indicate that the microenvironment may exert protective effects against immunotherapeutic strategies in CLL. However, the protective interaction is not entirely mediated by the CXCR4 - CXCL12 axis. Additional cell-cell interactions appear to play a role and need to be identified as therapeutic targets in order to effectively interrupt the protective effect of the microenvironment on T cell mediated cytotoxicity of B-CLL cells.

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Donor Dual TCR T Cells Preferentially Expand and Mediate Pathologic Alloreactivity in Acute Graft Versus Host Disease

Blood ◽

10.1182/blood.v118.21.1972.1972 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1972-1972

Author(s):

Gerald P. Morris ◽

Geoffrey L Uy ◽

David L Donermeyer ◽

Paul M Allen ◽

John F. DiPersio

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Thymic Selection ◽

Cell Repertoire ◽

Host Disease ◽

Graft Versus Host

Abstract Abstract 1972 The nature of the T cell repertoire mediating pathologic in vivo alloreactivity is an important question for understanding the development of acute graft-versus-host disease (aGvHD) following clinical allogeneic transplantation. We have previously demonstrated that the small proportion of T cells that naturally express 2 T cell receptors (TCR) as a consequence of incomplete TCRa allelic exclusion during thymic development contribute disproportionately to the alloreactive T cell repertoire, both in vitro and in vivo in a mouse model of graft versus host disease (GvHD) (J. Immunol., 182:6639, 2009). Here, we extend these findings to human biology, examining dual TCR T cells from healthy volunteer donors (n = 12) and patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT) (n = 19). Peripheral blood was collected at day 30 post-HSCT or at the time of presentation with symptomatic acute GvHD. Dual TCR T cells were measured in peripheral blood by pair-wise staining with 3 commercially-available and 2 novel TCRa mAbs. Dual TCR T cells were consistently and significantly expanded in patients with symptomatic aGvHD, representing 5.3±3.8 % of peripheral T cells, compared to 1.7±0.8 % of T cells in healthy controls (p < 0.005) (Figure 1). There was no correlation between dual TCR T cell frequency and GvHD severity. Furthermore, sequential analysis of peripheral blood in 2 patients demonstrated expansion of dual TCR T cells concurrent with the development of aGvHD (Figure 2). Dual TCR T cells from patients with symptomatic aGvHD demonstrated increased expression of CD69 as compared to T cells expressing a single TCR, indicative of preferential activation of dual TCR T cells during aGvHD. Similarly, dual TCR T cells isolated from patients with symptomatic aGvHD demonstrate increased production of IFN-g ex vivo, indicative of the ability to mediate pathogenic alloreactive responses. Dual TCR T cell clones isolated from healthy donors and patients post-HSCT by single cell FACS sorting demonstrate alloreactive responses against a range of allogeneic cell lines in vitro. We propose that the increased alloreactivity of dual TCR T cells results from the less stringent thymic selection for secondary TCR, and thus provides a link between thymic selection, the TCR repertoire, and alloreactivity. These findings may lead to simple ways of phenotypically identifying specific T cells predisposed to inducing aGvHD for subsequent examination of T cell repertoires and functional studies. Furthermore, these data suggest that dual TCR T cells represent a potential predictive biomarker for aGvHD and a potential target for selective T cell depletion in HSCT. Disclosures: No relevant conflicts of interest to declare.

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In vitro correlate for a clonal deletion mechanism of immune response gene-controlled nonresponsiveness.

Journal of Experimental Medicine ◽

10.1084/jem.157.3.998 ◽

1983 ◽

Vol 157 (3) ◽

pp. 998-1005 ◽

Cited By ~ 20

Author(s):

N Ishii ◽

Z A Nagy ◽

J Klein

Keyword(s):

T Cells ◽

T Cell ◽

Immune Response Gene ◽

Mouse Strains ◽

Single Chain ◽

Cell Repertoire ◽

Clonal Deletion ◽

Class Ii Mhc ◽

Antigen Presenting

We used T cell-antigen-presenting cell (APC) combinations from two pairs of recombinant mouse strains, B10.A(4R)-B10.A(2R) and B10.S(7R)-B10.S(9R) (abbreviated 4R, 2R, 7R, 9R, respectively), which differ from each other only in the nonexpression vs. expression of cell-surface E molecules, to study the mechanism of the Ir gene-controlled (E-restricted) response to the terpolymer poly(glu51lys34tyr15) (GLT). No response to GLT occurred when the APC were from E-nonexpressor strains 4R and 7R. When APC from E-expressor strains were used and alloreactivity against the incompatible E molecules was removed by BUdR + light treatment, 7R T cells responded to GLT presented by 9R APC, but 4R T cells failed to respond to GLT presented by 2R APC. However, 4R T cells mounted a proliferative response to GLT presented by fully allogeneic 5R or 9R APC. The latter response was completely abolished by the depletion of cells alloreactive against 2R and 5R or 2R and 9R. Since removal of alloreactivity against 5R plus 9R did not affect the response of 4R T cells to GLT presented by either 5R or 9R cells, we conclude that the 4R T cells generated in response to GLT cross-react with the additional incompatibility presented by 2R cells, that is, the Ek beta chain. In contrast, 7R T cells recognizing GLT presented by 9R APC do not cross-react with Ek beta. These results demonstrate that "blind spots" in the T cell repertoire produced by depletion of cells alloreactive against a single chain of a class II MHC molecule can render a strain nonresponsive to a synthetic polypeptide antigen, and that this nonresponsiveness corresponds to that attributed to the MHC-linked Ir genes.

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Allogeneic Adipose-Derived Mesenchymal Stromal Cells Ameliorate Experimental Autoimmune Encephalomyelitis by Regulating Self-Reactive T Cell Responses and Dendritic Cell Function

Stem Cells International ◽

10.1155/2017/2389753 ◽

2017 ◽

Vol 2017 ◽

pp. 1-15 ◽

Cited By ~ 18

Author(s):

Per Anderson ◽

Elena Gonzalez-Rey ◽

Francisco O’Valle ◽

Francisco Martin ◽

F. Javier Oliver ◽

...

Keyword(s):

T Cell ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Cell Function ◽

Multipotent Mesenchymal Stromal Cells ◽

Brain Inflammation ◽

Inos Inhibitor ◽

Cox 1

Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising therapy for autoimmune diseases, including multiple sclerosis (MS). Administration of MSCs to MS patients has proven safe with signs of immunomodulation but their therapeutic efficacy remains low. The aim of the current study has been to further characterize the immunomodulatory mechanisms of adipose tissue-derived MSCs (ASCs) in vitro and in vivo using the EAE model of chronic brain inflammation in mice. We found that murine ASCs (mASCs) suppress T cell proliferation in vitro via inducible nitric oxide synthase (iNOS) and cyclooxygenase- (COX-) 1/2 activities. mASCs also prevented the lipopolysaccharide- (LPS-) induced maturation of dendritic cells (DCs) in vitro. The addition of the COX-1/2 inhibitor indomethacin, but not the iNOS inhibitor L-NAME, reversed the block in DC maturation implicating prostaglandin (PG) E2 in this process. In vivo, early administration of murine and human ASCs (hASCs) ameliorated myelin oligodendrocyte protein- (MOG35-55-) induced EAE in C57Bl/6 mice. Mechanistic studies showed that mASCs suppressed the function of autoantigen-specific T cells and also decreased the frequency of activated (CD11c+CD40high and CD11c+TNF-α+) DCs in draining lymph nodes (DLNs). In summary, these data suggest that mASCs reduce EAE severity, in part, through the impairment of DC and T cell function.

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