Fig 1 - uploaded by Hisse M van Santen
Content may be subject to copyright.
Reduction in size of thymic populations and impaired DN3a-to-DN3b transition in L19A mice. (A) Quantification of the total number of thymocytes (Upper). Each dot shows the total number of thymocytes in an individual mouse. Quantification of the number (Middle) and percentage (Lower) of thymocytes in the major populations defined according to expression of CD4 and CD8. Results and statistical analysis shown are based on pooled data from 67 WT and 72 L19A mice from 10 independent experiments. (B) Representative plots of the DN (CD4 À CD8 À ) subpopulations defined by the expression of CD44 and CD25 (Upper) and quantification of the percentage (Middle) and cell number (Lower) of these populations in WT, L19A, and Cd3z À/À mice. The DN population was pregated on the lineage negative (Lin À ) population (CD4 À , CD8 À , CD19 À , B220 À , GR1 À , NK1.1 À , TCRγδ À , CD11b À , CD11c À , TER119 À ). IEL precursors, identified as Lin À CD24 lo CD5 hi TCRβ hi CD44 lo cells, were excluded from the quantifications. Graphs show the averages calculated on the basis of data obtained from three independent experiments with 12 WT, 12 L19A, and 5 Cd3z À/À mice. (C) Representative flow cytometry plots of Lin À DN populations (defined as above) in WT, L19A, and Cd3e À/À thymi according to CD44 and CD25 markers (Upper; CD25 +/lo CD44 À DN3 and CD25 À CD44 À DN4 gates indicated) and identification of DN3a and DN3b subpopulations within the DN3 gate according to forward scatter (FSC) and CD3ζ-GFP expression (Lower). (C, Right) Quantification based on pooled data from six independent experiments showing the percentage of the DN3a, DN3b, and DN4 populations. (D) Representative histograms (Left) and quantification (Right) of the intracellular TCRβ expression levels in the DN3a (CD44 À CD25 + ), DN3b (CD44 À CD25 lo ), and DN4 (CD44 À CD25 À ) subpopulations. Thymocytes from Rag1-deficient mice were used as a control for intracellular staining and used as background control in all three overlays. Dashed lines indicate signal intensity obtained in Rag1 À/À thymocytes. Quantification for one out of four experiments is shown. (E) Representative histograms (Left) and quantification (Right) of extracellular TCRβ expression. Data represent quantification for one out of four experiments. All quantifications are presented as the mean ± SEM. P values were calculated using unpaired two-tailed Student's t tests with 95% CI (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). e.c., extracellular; i.c., intracellular.
Source publication
Significance
The ability of the T cell receptor (TCR) to convey signals of different intensity is essential for the generation of a diverse, protecting, and self-tolerant T cell repertoire. We provide evidence that pre-TCR signaling during the first stage of T cell differentiation, thought to only check for in-frame rearrangement of TCRβ gene segme...
Contexts in source publication
Context 1
... mice to a Cd3z À/À background (15) so that the GFP-CD3ζ chains were the only CD3ζ chains expressed in these mice. Immunoblot analysis of whole lysates from total thymus and purified double-negative (DN) thymocytes showed the presence of ∼100-kDa covalently associated homodimers reactive with both CD3ζ-and GFP-specific antibodies (SI Appendix, Fig. S1 A and B). Homogeneous expression of the transgenic CD3ζ-GFP fusion proteins was detected by flow cytometry in all thymic subsets tested (SI Appendix, Fig. S1C). Mice on this background will be referred to as WT and L19A mice. We pooled the data of the two independent lines analyzed of each genotype, as we did not observe phenotypic ...
Context 2
... total thymus and purified double-negative (DN) thymocytes showed the presence of ∼100-kDa covalently associated homodimers reactive with both CD3ζ-and GFP-specific antibodies (SI Appendix, Fig. S1 A and B). Homogeneous expression of the transgenic CD3ζ-GFP fusion proteins was detected by flow cytometry in all thymic subsets tested (SI Appendix, Fig. S1C). Mice on this background will be referred to as WT and L19A mice. We pooled the data of the two independent lines analyzed of each genotype, as we did not observe phenotypic differences between them (SI Appendix, Fig. ...
Context 3
... of the transgenic CD3ζ-GFP fusion proteins was detected by flow cytometry in all thymic subsets tested (SI Appendix, Fig. S1C). Mice on this background will be referred to as WT and L19A mice. We pooled the data of the two independent lines analyzed of each genotype, as we did not observe phenotypic differences between them (SI Appendix, Fig. ...
Context 4
... isolated from L19A mice contained two-to threefold fewer cells than thymi from WT mice ( Fig. 1 A, Upper). The double-positive (DP), 4SP, and 8SP populations were reduced in number whereas there was a relative increase in DN thymocytes, indicating a defect in thymocyte differentiation that was occurring at the DN stage in L19A mice ( Fig. 1 A, Middle and Lower). ...
Context 5
... isolated from L19A mice contained two-to threefold fewer cells than thymi from WT mice ( Fig. 1 A, Upper). The double-positive (DP), 4SP, and 8SP populations were reduced in number whereas there was a relative increase in DN thymocytes, indicating a defect in thymocyte differentiation that was occurring at the DN stage in L19A mice ( Fig. 1 A, Middle and Lower). Subdivision of the DN population into the DN1 to DN4 subsets using the CD25 and CD44 markers (16) revealed similar percentages and numbers of CD44 + CD25 À DN1 and CD44 + CD25 + DN2 cells in L19A and WT thymi but a relative accumulation of CD44 À CD25 + DN3 cells and a relative and absolute reduction of CD44 À ...
Context 6
... population into the DN1 to DN4 subsets using the CD25 and CD44 markers (16) revealed similar percentages and numbers of CD44 + CD25 À DN1 and CD44 + CD25 + DN2 cells in L19A and WT thymi but a relative accumulation of CD44 À CD25 + DN3 cells and a relative and absolute reduction of CD44 À CD25 À DN4 cells in L19A thymi as compared with WT thymi (Fig. 1B). Thus, the earliest observable defect in αβT cell differentiation in L19A mice occurred at the transition from the DN3 to DN4 stage. The absolute numbers of DN4 thymocytes and the percentages of DN3 and DN4 mutant thymocytes were significantly different from those of CD3ζ-deficient mice (Fig. 1B), which present an almost complete loss ...
Context 7
... DN4 cells in L19A thymi as compared with WT thymi (Fig. 1B). Thus, the earliest observable defect in αβT cell differentiation in L19A mice occurred at the transition from the DN3 to DN4 stage. The absolute numbers of DN4 thymocytes and the percentages of DN3 and DN4 mutant thymocytes were significantly different from those of CD3ζ-deficient mice (Fig. 1B), which present an almost complete loss of the DN4 population, indicating that the L19A CD3ζ chain could at least partially overcome defects imposed by Cd3z deficiency on pre-TCR ...
Context 8
... quantified the percentage of DN3a and DN3b thymocytes in the DN population of L19A and WT mice using as a reference DN3 thymocytes from Cd3e-deficient mice, which can rearrange their Tcrb locus but cannot express the pre-TCR and do not reach the DN3b stage (19). L19A thymi contained significantly more DN3a and fewer DN3b thymocytes than WT thymi (Fig. 1C). This lag in T cell development was not due to the inability of L19A thymocytes to rearrange their Tcrb locus, as intracellular staining for TCRβ in the DN3b and DN4 populations of WT and L19A thymi showed an equal intensity (Fig. 1D). Moreover, cell-surface expression of the pre-TCR on the DN3b-enriched CD44 À CD25 lo population and ...
Context 9
... reach the DN3b stage (19). L19A thymi contained significantly more DN3a and fewer DN3b thymocytes than WT thymi (Fig. 1C). This lag in T cell development was not due to the inability of L19A thymocytes to rearrange their Tcrb locus, as intracellular staining for TCRβ in the DN3b and DN4 populations of WT and L19A thymi showed an equal intensity (Fig. 1D). Moreover, cell-surface expression of the pre-TCR on the DN3b-enriched CD44 À CD25 lo population and CD44 À CD25 À DN4 cells, measured by extracellular staining with an antibody against the TCRβ chain, was indistinguishable between L19A and WT mice (Fig. 1E). Likewise, cellsurface expression of CD3ε and pTα and expression of the ...
Context 10
... for TCRβ in the DN3b and DN4 populations of WT and L19A thymi showed an equal intensity (Fig. 1D). Moreover, cell-surface expression of the pre-TCR on the DN3b-enriched CD44 À CD25 lo population and CD44 À CD25 À DN4 cells, measured by extracellular staining with an antibody against the TCRβ chain, was indistinguishable between L19A and WT mice (Fig. 1E). Likewise, cellsurface expression of CD3ε and pTα and expression of the transgenic CD3ζ-GFP chain were equal for WT and L19A DN3b and DN4 thymocytes (SI Appendix, Fig. S2 A-D). We found that cell-surface expression and stoichiometry of the cell surface-displayed complexes of the pre-TCR were not affected by the L19A mutation in cell ...
Context 11
... and did not fuse or split during a time period of at least 5 s were analyzed. The average number of particles detected at the cell surface of L19A DN thymocytes was significantly lower than observed for WT DN thymocytes (Fig. 3B), even though no difference in the total level of expression of pre-TCRs was observed for these populations ( Fig. 1E and SI Appendix, Fig. S2 A-D). L19A pre-TCR particles had an increased diffusion rate as compared with WT particles (Fig. 3C). There was no significant difference in distribution between confined, free, and directed diffusion of mobile particles (Fig. 3D) excluding a shift in diffusion mode as the mechanism underlying the overall ...
Context 12
... a recent report strongly suggests that nanoclustering of the TCR indeed occurs in these cells and is relevant for the outcome of positive and negative selection (44). Analysis of the 4SP-to-8SP ratio in WT and L19A thymi also provides support for this notion, with L19A thymi having a lower ratio than WT thymi (data underlying Fig. 1A; 1.41 ± 0.67 vs. 2.07 ± 1.13; P < 0.05, unpaired two-tailed Student's t test). Lineage choice is determined by TCR signaling strength at the CD8 + CD4 low stage of selection with a higher signaling capacity necessary for the CD4 lineage choice (49). It could be expected that TCRs with higher self-pMHC affinity would be allowed to ...
Context 13
... male founder mice for each transgenic construct (WT-F10, WT-F19, L19A-F13, and L19A-F23), initially identified via Southern blot using a GFPspecific probe, were selected and backcrossed once with C57BL/6J females to check for germ-line transmission and further backcrossed to a Cd3z tm1Lov knockout background (15). Relative copy numbers of the transgenes, as determined by qPCR analysis of genomic DNA, were three-to fivefold higher for the L19A lines as compared with the WT lines, which displayed similar copy numbers (SI Appendix, Fig. S1E). Mice backcrossed for two to five generations to the C57BL/6J background, homozygous for the Cd3z tm1Lov allele, and always carrying the transgene in hemizygosity were used for the experiments described in this manuscript. ...
Context 14
... Microscopy. Pooled thymocyte suspensions from two or three WT or L19A mice were obtained and Lin À thymocytes were negatively selected using Ly6c-, F4/80-, CD11b-, CD11c-, CD19-, B220-, NK1.1-, CD4-, and CD8-specific antibodies and subsequently sheep anti-rat Dynabeads (Invitrogen, 11035). A second round of selection using a FACSAria Fusion cell sorter was performed on Lin À thymocytes to obtain DN CD44 À thymocytes (DN3 + DN4 populations) that express GFP levels under 10 3 relative fluorescence units. ...
Similar publications
As an immune checkpoint molecule, CD200 serves a foundational role in regulating immune homeostasis and promoting self-tolerance. While CD200 expression occurs in various immune cell subsets and normal tissues, its aberrant expression patterns in hematologic malignancies and solid tumors have been linked to immune evasion and cancer progression und...
Investments in the country's actual economy and its capital market will increase as a result of globalization. Because of this, the capital market requires members of Generation Z to have a high level of self-awareness. The purpose of this study is to examine how generation Z investors' levels of investing expertise, risk aversion, and confidence i...
In autoimmune rheumatic diseases, immune hyperactivity and chronic inflammation associate with immune dysregulation and the breakdown of immune self-tolerance. A continued, unresolved imbalance between effector and regulatory immune responses further exacerbates inflammation that ultimately causes tissue and organ damage. Many treatment modalities...
Systemic lupus erythematosus (SLE) is a chronic immune-inflammatory disease characterized by multiorgan affectation and lowered self-tolerance. Additionally, epigenetic changes have been described as playing a pivotal role in SLE. This work aims to assess the effects of oleacein (OLA), one of the main extra virgin olive oil secoiridoids, when used...
Scleroderma is a rare, potentially fatal, clinically heterogeneous, systemic autoimmune connective tissue disorder that is characterized by progressive fibrosis of the skin and visceral organs, vasculopathy and immune dysregulation. The more severe form of the disease, diffuse cutaneous scleroderma (dcSSc), has no cure and limited treatment options...
Citations
Non-mutated FVIII-specific CD4 T cell epitopes have been recently found to contribute to the development of inhibitors in patients with hemophilia A (HA), while auto-reactive CD4 T cells specific to FVIII circulate in the blood of healthy individuals at a frequency close to the foreign protein ovalbumin. Thus, although FVIII is a self-protein, the central tolerance raised against FVIII appears to be low. In this study, we conducted a comprehensive analysis of the FVIII CD4 T cell repertoire in 29 healthy donors. Sequencing of the CDR3β TCR region from isolated FVIII-specific CD4 T cells revealed a limited usage and pairing of TRBV and TRBJ genes as well as a mostly hydrophobic composition of the CDR3β region according to their auto-reactivity. The FVIII repertoire is dominated by a few clonotypes, with only 13 clonotypes accounting for half of the FVIII response. Through a large-scale epitope mapping of the full-length FVIII sequence, we identified 18 immunodominant epitopes located in the A1, A3, C1, and C2 domains and covering half of the T cell response. These epitopes exhibited a broad specificity for HLA-DR or DP molecules or both. T cell priming with this reduced set of peptides revealed that highly expanded clonotypes specific to these epitopes were responsible individually for up to 32% of the total FVIII repertoire. These FVIII T cell epitopes and clonotypes were shared among HLA-unrelated donors tested and previously reported HA patients. Our study highlights the role of the auto-reactive T cell response against FVIII in HA and its similarity to the response observed in healthy individuals. Thus, it provides valuable insights for the development of new tolerance induction and deimmunization strategies.
Introduction. Glycosphingolipids are compounds composed of hydrophilic sugar structures and hydrophobic ceramides. These molecules form lipid rafts or microdomains in the cell membrane together with cholesterol, sphingomyelin, glycosylphosphatidylinositol and molecules, which determines their properties.
Aim. To systematize data on the structure of lipid rafts, their involvement in the functioning of immunocompetent cells and the development of the immune response, and the mechanisms of SARS-CoV-2 viral invasion.
Materials and methods. From these positions, literary sources for 1981-2023 are analyzed. Literature search was carried out in information systems: PubMed and Google Scholar.
Results. There are separate works that reflect the role of lipid rafts as mediators of signal transduction in the development of innate and adaptive immune responses. Other studies describe their importance in pathogen-host interaction and avoidance of immune control. Recently, studies have appeared on the effect of lipid microdomains of the cell membrane on viral invasion, including that caused by SARS-CoV-2.
Conclusion. This review makes a significant contribution to understanding the role of lipid rafts in the functioning of the immune system and viral invasion, which determines the prospects for further research and the possibility of their use as therapeutic targets in the development of immunomodulatory drugs.
A critical stage of T cell development is β-selection; at this stage, the T cell receptor β (TCRβ) chain is generated, and the developing T cell starts to acquire antigenic specificity. Progression through β-selection is assisted by low-affinity interactions between the nascent TCRβ chain and peptide presented on stromal major histocompatibility complex and cues provided by the niche. In this study, we identify a cue within the developing T cell niche that is critical for T cell development. E-cadherin mediates cell-cell interactions and influences cell fate in many developmental systems. In developing T cells, E-cadherin contributed to the formation of an immunological synapse and the alignment of the mitotic spindle with the polarity axis during division, which facilitated subsequent T cell development. Collectively, these data suggest that E-cadherin facilitates interactions with the thymic niche to coordinate the β-selection stage of T cell development.
There is a long-standing assumption that naive CD4⁺ and CD8⁺ T cells are largely homogeneous populations despite the extraordinary diversity of their T cell receptors (TCR). The self-immunopeptidome plays a key role in the selection of the naive T cell repertoire in the thymus, and self-peptides are also an important driver of differences between individual naive T cells with regard to their subsequent functional contributions to an immune response. Accumulating evidence suggests that as early as the β-selection stage of T cell development, when only one of the recombined chains of the mature TCR is expressed, signaling thresholds may be established for positive selection of immature thymocytes. Stochastic encounters subsequently made with self-ligands during positive selection in the thymus imprint functional biases that a T cell will carry with it throughout its lifetime, although ongoing interactions with self in the periphery ensure a level of plasticity in the gene expression wiring of naive T cells. Identifying the sources of heterogeneity in the naive T cell population and which functional attributes of T cells can be modulated through post-thymic interventions versus those that are fixed during T cell development, could enable us to better select or generate T cells with particular traits to improve the efficacy of T cell therapies.
