Differential Effects of Anti-CD3 Antibodies In Vivo and In Vitro on α β and γ δ T Cell Differentiation

Author(s):  
B. A. Kyewski
Blood ◽  
1999 ◽  
Vol 94 (8) ◽  
pp. 2819-2826 ◽  
Author(s):  
Rosa Sacedón ◽  
Angeles Vicente ◽  
Alberto Varas ◽  
Eva Jiménez ◽  
Juan José Muñoz ◽  
...  

In the present work, we demonstrated that both fetal liver and thymic T-cell precursors express glucocorticoid receptors (GRs) indirectly suggesting a role for glucocorticoids (GCs) in the earliest events of T-cell differentiation. To evaluate this issue, we analyzed the thymic ontogeny in the progeny of adrenalectomized pregnant rats (Adx fetuses), an in vivo experimental model, which ensures the absence of circulating GCs until the establishment of the fetal hypothalamus-pituitary-adrenal (HPA) axis. In the absence of maternal GCs, T-cell development was significantly accelerated, the process being reversed by in vivo GC replacement. Mature single positive thymocytes (both CD4 and CD8) appeared in 16-day old fetal Adx thymus when in the control fetuses, most thymocytes still remained in the double-negative (DN) CD4−CD8− cell compartment. In addition, emigration of T-cell receptor (TcR)β positive cells to the spleen also occurred earlier in Adx fetuses than in control ones. In vitro recolonization of cultured deoxiguanosine-treated mouse fetal thymus lobes with 13-day-old fetal liver cell suspensions from both Adx and control fetuses demonstrated changes in the developmental capabilities of fetal liver T-cell precursors from embryos grown in the absence of GCs. Furthermore, a precocious lymphoid colonization of the thymic primordium from Adx fetuses was evidenced by ultrastructural analysis of both Adx and Sham early thymus. Both findings accounted for the accelerated T-cell differentiation observed in Adx fetuses. Together, these results support a role for GCs not only in the thymic cell death, but also in the early steps of T-cell differentiation.


2010 ◽  
Vol 30 (20) ◽  
pp. 4877-4889 ◽  
Author(s):  
Pilar Martín ◽  
Manuel Gómez ◽  
Amalia Lamana ◽  
Arantxa Cruz-Adalia ◽  
Marta Ramírez-Huesca ◽  
...  

ABSTRACT T-cell differentiation involves the early decision to commit to a particular pattern of response to an antigen. Here, we show that the leukocyte activation antigen CD69 limits differentiation into proinflammatory helper T cells (Th17 cells). Upon antigen stimulation in vitro, CD4+ T cells from CD69-deficient mice generate an expansion of Th17 cells and the induction of greater mRNA expression of interleukin 17 (IL-17), IL 23 receptor (IL-23R), and the nuclear receptor retinoic acid-related orphan receptor γt (RORγt). In vivo studies with CD69-deficient mice bearing OTII T-cell receptors (TCRs) specific for OVA peptide showed a high proportion of antigen-specific Th17 subpopulation in the draining lymph nodes, as well as in CD69-deficient mice immunized with type II collagen. Biochemical analysis demonstrated that the CD69 cytoplasmic tail associates with the Jak3/Stat5 signaling pathway, which regulates the transcription of RORγt and, consequently, differentiation toward the Th17 lineage. Functional experiments in Th17 cultures demonstrated that the selective inhibition of Jak3 activation enhanced the transcription of RORγt. Moreover, the addition of exogenous IL-2 restored Stat5 phosphorylation and inhibited the enhanced Th17 differentiation in CD69-deficient cells. These results support the early activation receptor CD69 as an intrinsic modulator of the T-cell differentiation program that conditions immune inflammatory processes.


1986 ◽  
Vol 163 (2) ◽  
pp. 231-246 ◽  
Author(s):  
B A Kyewski ◽  
C G Fathman ◽  
R V Rouse

We present evidence for intrathymic presentation of soluble circulating antigens in vivo. Our results show that proteins of different molecular weight enter the mouse thymus rapidly after i.v. injection. The intrathymic presence of antigen was assayed by proliferation of cloned antigen-specific T helper cells, which were cocultured with purified thymic stromal cells; stromal cells were isolated and purified as lymphostromal cell complexes, which preexist in vivo. Antigen presentation copurified with non-adherent medullary dendritic cells (DC) (interdigitating cells). I-A- cortical macrophages forming thymocyte rosettes in vivo and I-A+ cortical epithelial cells forming thymic nurse cells (TNC) in vivo did not act as antigen presenting cells (APC) after antigen pulsing in vivo or in vitro. Thymic APC turn over physiologically and are rapidly replaced (within 2-5 wk) after lethal irradiation by donor bone marrow-derived cells. The frequency of thymocyte-DC interactions in vivo strictly correlates with thymic T cell differentiation, and is independent of the immune status of the animal. Fetal thymic APC seem to be secluded from antigen in the maternal circulation. Thymic DC-ROS probably represent the microenvironment where maturing T cells first encounter non-MHC antigens in the context of self-MHC antigens.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2470-2470
Author(s):  
James A Kennedy ◽  
Renata Teixeira ◽  
Sara Berthiaume ◽  
Frederic Barabe

Abstract Abstract 2470 LMO2 is overexpressed in a significant percentage of human T cell acute lymphoblastic leukemia (T-ALL) and its locus has been the target of insertional mutagenesis in gene therapy trials. In the past years, 4 X-linked severe combined immunodeficiency (X-linked SCID) and one Wiskott-Aldrich syndrome (WAS) patients who were treated by retrovirus-mediated gene therapy developed T-ALL as a result of retroviral integration in the LMO2 locus. In these patients, leukemia developed 2 to 3 years after gene therapy without prior significant haematological abnormalities. However, both the latency of disease and the finding of additional somatic mutations and/or translocations in these leukemias suggest that the overexpression of LMO2 alone is insufficient to generate leukemia, a notion that has been supported by studies in mouse. Though LMO2 is typically recognized as a T-cell oncogene, reports have shown that it is also aberrantly expressed in acute myeloid leukemias (AML), chronic myeloid leukemia (CML), B-ALL and some non-hodgkin B cell lymphomas. In order to study the impact of LMO2 overexpression on human hematopoietic stem/progenitor cells, a lentiviral vector was used to express this oncogene together with EGFP in lineage-depleted umbilical cord blood. In myeloid-promoting cultures, LMO2 had no effect on either differentiation or proliferation. Moreover, the expression of LMO2 did not modify the frequency or lineage distribution of colony forming progenitors compared to controls. However, significant differences were noted when transduced cells were assayed on OP9-Delta-Like 1 (DL1) stroma, an in vitro system that promotes T cell proliferation and differentiation. Cells overexpressing LMO2 were blocked at the double negative stage (CD4-CD8-) of differentiation and proliferated 50 to 100 times more than control cells. However, these cells were not immortalized as they proliferated for a median of 75 days, versus 50 days for controls. Immunodeficient mice transplanted with primitive human hematopoietic cells expressing LMO2 (hereafter referred as LMO2 mice) had bone marrow engraftment levels comparable to controls at 20–24 weeks post-transplant. Neither B-lymphoid nor myeloid development were affected by LMO2 overexpression. Strikingly, in the thymus, the percentage of EGFP+ cells was significantly increased in LMO2 mice compared to controls (mean of 47.7% versus 8.8%, p=0.0001), clearly indicating that expression of this oncogene enhances thymic T-cell engraftment. We next analyzed the phenotype of LMO2-expressing T cells in the thymus and peripheral blood of these mice. Surprisingly, unlike our in vitro studies, there was no evidence of a block at the DN-stage of differentiation. Instead, there were significantly less EGFP+ DN cells in the thymi of LMO2 mice compared to controls (mean of 7.5% vs 14.5%, p=0.035). These results clearly demonstrate that unlike what was observed in OP9-DL1 co-cultures, LMO2 overexpression does not induce a block in T-cell differentiation in our in vivo system. One possible explanation for this difference is the constitutive NOTCH signaling provided via DL1 on stroma compared to the in vivo setting where LMO2-expressing cells would encounter different levels and forms of NOTCH signaling throughout development. To test this hypothesis, LMO2 cells were cultured on OP9-DL1 stroma for 50 days then switched onto OP9 stroma lacking NOTCH ligand. Upon transfer, the DN cells promptly stopped proliferating and differentiated into DP (CD4+CD8+) cells expressing CD3 and TCRαβ. Thus, our results suggest that in the in vivo setting, as cells migrate through the thymus and face a decrease in NOTCH signaling, LMO2 overexpression alone can promote proliferation, but is not sufficient to maintain a differentiation block. However, constitutive NOTCH signaling can cooperate with LMO2 overexpression to block T cell differentiation at a proliferative DN stage. Thus, one can postulate that LMO2 exerts a proliferative effect on developing T-cells in thymic regions with high levels of NOTCH signaling, potentially providing a setting for the development of secondary leukemogenic events. NOTCH mutations are common in human T-ALL and can therefore allow for LMO2 overexpressing cells to become independent of the stromal niche. Taken together, our results suggest cooperation between LMO2 overexpression and NOTCH signaling in human T-cell leukemogenesis. Disclosures: No relevant conflicts of interest to declare.


2021 ◽  
Vol 5 (7) ◽  
pp. 1963-1976
Author(s):  
Alessandra Di Grande ◽  
Sofie Peirs ◽  
Paul D. Donovan ◽  
Maaike Van Trimpont ◽  
Julie Morscio ◽  
...  

Abstract B-cell lymphoma 2 (BCL-2) has recently emerged as a therapeutic target for early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL), a high-risk subtype of human T-cell ALL. The major clinical challenge with targeted therapeutics, such as the BCL-2 inhibitor ABT-199, is the development of acquired resistance. We assessed the in vivo response of luciferase-positive LOUCY cells to ABT-199 monotherapy and observed specific residual disease in the splenic microenvironment. Of note, these results were confirmed by using a primary ETP-ALL patient-derived xenograft. Splenomegaly has previously been associated with poor prognosis in diverse types of leukemia. However, the exact mechanism by which the splenic microenvironment alters responses to specific targeted therapies remains largely unexplored. We show that residual LOUCY cells isolated from the spleen microenvironment displayed reduced BCL-2 dependence, which was accompanied by decreased BCL-2 expression levels. Notably, this phenotype of reduced BCL-2 dependence could be recapitulated by using human splenic fibroblast coculture experiments and was confirmed in an in vitro chronic ABT-199 resistance model of LOUCY. Finally, single-cell RNA-sequencing was used to show that ABT-199 triggers transcriptional changes in T-cell differentiation genes in leukemic cells obtained from the spleen microenvironment. Of note, increased expression of CD1a and sCD3 was also observed in ABT199-resistant LOUCY clones, further reinforcing the idea that a more differentiated leukemic population might display decreased sensitivity toward BCL-2 inhibition. Overall, our data reveal the spleen as a site of residual disease for ABT-199 treatment in ETP-ALL and provide evidence for plasticity in T-cell differentiation as a mechanism of therapy resistance.


2017 ◽  
Vol 313 (3) ◽  
pp. L592-L601 ◽  
Author(s):  
Xiao-Ming Li ◽  
Xi Chen ◽  
Wen Gu ◽  
Yi-Jia Guo ◽  
Yi Cheng ◽  
...  

CD4+ T-cell differentiation plays an important role in allergic airway diseases. Tumor necrosis factor receptor 2 (TNFR2) has been shown to regulate CD4+ T-lymphocyte differentiation, but its role in allergic airway inflammation is not clear. Here, we investigated the role of TNFR2 in allergic airway inflammation. The mouse model was generated by immunization with ovalbumin and intranasal administration of TNFR2 antibody. Airway inflammation and CD4+ T-cell differentiation were measured in vivo and in vitro. Inhibited TNFR2 signaling aggravated airway inflammation and increased the expression of inflammatory cytokines (IL-4, IL-5, IL-17, and TNF-α) in serum and bronchoalveolar lavage fluid. Impaired TNFR2 signaling promoted Th2 and Th17 polarization but inhibited Th1 and CD4+CD25+ T-cell differentiation in vivo. Furthermore, TNFR2 signaling inhibition promoted Th2 and Th17 polarization in vitro, which may occur through the activation of TNF receptor-associated factor 2 and NF-κB signaling. Therefore, our findings indicate that impaired TNF/TNFR2 signaling enhances Th2 and Th17 polarization and aggravates allergic airway inflammation.


2019 ◽  
Author(s):  
Eliza Mari Kwesi-Maliepaard ◽  
Muhammad Assad Aslam ◽  
Mir Farshid Alemdehy ◽  
Teun van den Brand ◽  
Chelsea McLean ◽  
...  

AbstractCytotoxic T-cell differentiation is guided by epigenome adaptations but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone methyltransferase DOT1L, which marks the nucleosome core on active genes, safeguards normal differentiation of CD8+ T cells. T-cell specific ablation of Dot1L resulted in loss of naïve CD8+ T cells and premature differentiation towards a memory-like state, independent of antigen exposure and in a cell-intrinsic manner. Without DOT1L, the memory-like CD8+ cells fail to acquire full effector functions in vitro and in vivo. Mechanistically, DOT1L controlled T-cell differentiation and function by ensuring normal T-cell receptor density and signaling, and by maintaining epigenetic identity, in part by indirectly supporting the repression of developmentally-regulated genes. Through our study DOT1L is emerging as a central player in physiology of CD8+ T cells, acting as a barrier to prevent premature differentiation and supporting the licensing of the full effector potential of cytotoxic T cells.


2021 ◽  
Author(s):  
Juan Fernandez-Garcia ◽  
Fabien Franco ◽  
Sweta Parik ◽  
Antonino A Pane ◽  
Dorien Broekaert ◽  
...  

Cytotoxic T cells dynamically rewire their metabolism during the course of an immune response. While T cell metabolism has been extensively studied at phenotypic endpoints of activation and differentiation, the underlying dynamics remain largely elusive. Here, we leverage on single-cell RNA-sequencing (scRNA-seq) measurements of in vitro activated and differentiated CD8+ T cells cultured in physiological media to resolve these metabolic dynamics. We find that our scRNA-seq analysis identifies most metabolic changes previously defined in in vivo experiments, such as a rewiring from an oxidative to an anabolism-promoting metabolic program during activation to an effector state, which is later reverted upon memory polarization. Importantly, our scRNA-seq data further provide a dynamic description of these changes. In this sense, our data predict a differential time-dependent reliance of CD8+ T cells on the synthesis versus uptake of various non-essential amino acids during T cell activation, which we corroborate with additional functional in vitro experiments. We further exploit our scRNA-seq data to identify metabolic genes that could potentially dictate the outcome of T cell differentiation, by ranking them based on their expression dynamics. Among the highest-ranked hits, we find asparagine synthetase (Asns), whose expression sharply peaks for effector CD8+ T cells and further decays towards memory polarization. We then confirm that these in vitro Asns expression dynamics are representative of an in vivo situation in a mouse model of viral infection. Moreover, we find that disrupting these expression dynamics in vitro, by depleting asparagine from the culture media, delays central-memory polarization. Accordingly, we find that preventing the decay of ASNS by stable overexpression at the protein level in vivo leads to a significant increase in effector CD8+ T cell expansion, and a concomitant decrease in central-memory formation, in a mouse model of viral infection. This shows that ASNS expression dynamics dictate the fate of CD8+ T cell differentiation. In conclusion, we provide a resource of dynamic expression changes during CD8+ T cell activation and differentiation that is expected to increase our understanding of the dynamic metabolic requirements of T cells progressing along the immune response cascade.


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