CD8 Single-positive Thymocytes Research Articles

Decoding mutational hotspots in human disease through the gene modules governing thymic regulatory T cells.

Computational strategies to extract meaningful biological information from multiomics data are in great demand for effective clinical use, particularly in complex immune-mediated disorders. Regulatory T cells (Tregs) are essential for immune homeostasis and self-tolerance, controlling inflammatory and autoimmune processes in many diseases with a multigenic basis. Here, we quantify the Transcription Factor (TF) differential occupancy landscape to uncover the Gene Regulatory Modules governing lineage-committed Tregs in the human thymus, and show that it can be used as a tool to prioritise variants in complex diseases. We combined RNA-seq and ATAC-seq and generated a matrix of differential TF binding to genes differentially expressed in Tregs, in contrast to their counterpart conventional CD4 single-positive thymocytes. The gene loci of both established and novel genetic interactions uncovered by the Gene Regulatory Modules were significantly enriched in rare variants carried by patients with common variable immunodeficiency, here used as a model of polygenic-based disease with severe inflammatory and autoimmune manifestations. The Gene Regulatory Modules controlling the Treg signature can, therefore, be a valuable resource for variant classification, and to uncover new therapeutic targets. Overall, our strategy can also be applied in other biological processes of interest to decipher mutational hotspots in individual genomes.

T cell-intrinsic PKD3 fine-tunes differentiation into CD8+ central memory T cells and CD8 single positive thymocyte development.

Members of the Protein kinases D (PKD) family are described as regulators of T cell responses. From the two T cell-expressed isoforms PKD2 and PKD3, so far mainly the former was thoroughly investigated and is well understood. Recently, we have investigated also PKD3 using conventional as well as conditional T cell-specific knockout models. These studies suggested PKD3 to be a T cell-extrinsic regulator of the cells' fate. However, these former model systems did not take into account possible redundancies with the highly homologous PKD2. To overcome this issue and thus properly unravel PKD3's T cell-intrinsic functions, here we additionally used a mouse model overexpressing a constitutively active isoform of PKD3 specifically in the T cell compartment. These transgenic mice showed a slightly higher proportion of central memory T cells in secondary lymphoid organs and blood. This effect could not be explained via differences upon polyclonal stimulation in vitro, however, may be connected to the observed developmental aberrances in the CD8 single positive compartment during thymic development. Lastly, the observed alterations in the CD8+ T cell compartment did not impact proper immune response upon immunization with ovalbumin or in a subcutaneous tumour model suggesting only a small to absent biological relevance. Taking together the knowledge of all our published studies on PKD3 in the T cell compartment, we now conclude that T cell-intrinsic PKD3 is a fine-tuner of central memory T cell as well as CD8 single positive thymocyte development.

Fam49b dampens TCR signal strength to regulate survival of positively selected thymocytes and peripheral T cells.

The fate of developing T cells is determined by the strength of T cell receptor (TCR) signal they receive in the thymus. This process is finely regulated through the tuning of positive and negative regulators in thymocytes. The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in vitro in cultured T cell lines. However, the contribution of Fam49b to the thymic development of T cells is unknown. To investigate this important issue, we generated a novel mouse line deficient in Fam49b (Fam49b-KO). We observed that Fam49b-KO double positive (DP) thymocytes underwent excessive negative selection, whereas the positive selection stage was unaffected. Fam49b deficiency impaired the survival of single positive thymocytes and peripheral T cells. This altered development process resulted in significant reductions in CD4 and CD8 single-positive thymocytes as well as peripheral T cells. Interestingly, a large proportion of the TCRγδ+ and CD8αα+TCRαβ+ gut intraepithelial T lymphocytes were absent in Fam49b-KO mice. Our results demonstrate that Fam49b dampens thymocytes TCR signaling in order to escape negative selection during development, uncovering the function of Fam49b as a critical regulator of the selection process to ensure normal thymocyte development and peripheral T cells survival.

Transcriptomic profiling of thymic dysregulation and viral tropism after neonatal roseolovirus infection.

Herpesviruses, including the roseoloviruses, have been linked to autoimmune disease. The ubiquitous and chronic nature of these infections have made it difficult to establish a causal relationship between acute infection and subsequent development of autoimmunity. We have shown that murine roseolovirus (MRV), which is highly related to human roseoloviruses, induces thymic atrophy and disruption of central tolerance after neonatal infection. Moreover, neonatal MRV infection results in development of autoimmunity in adult mice, long after resolution of acute infection. This suggests that MRV induces durable immune dysregulation. In the current studies, we utilized single-cell RNA sequencing (scRNAseq) to study the tropism of MRV in the thymus and determine cellular processes in the thymus that were disrupted by neonatal MRV infection. We then utilized tropism data to establish a cell culture system. Herein, we describe how MRV alters the thymic transcriptome during acute neonatal infection. We found that MRV infection resulted in major shifts in inflammatory, differentiation and cell cycle pathways in the infected thymus. We also observed shifts in the relative number of specific cell populations. Moreover, utilizing expression of late viral transcripts as a proxy of viral replication, we identified the cellular tropism of MRV in the thymus. This approach demonstrated that double negative, double positive, and CD4 single positive thymocytes, as well as medullary thymic epithelial cells were infected by MRV in vivo. Finally, by applying pseudotime analysis to viral transcripts, which we refer to as "pseudokinetics," we identified viral gene transcription patterns associated with specific cell types and infection status. We utilized this information to establish the first cell culture systems susceptible to MRV infection in vitro. Our research provides the first complete picture of roseolovirus tropism in the thymus after neonatal infection. Additionally, we identified major transcriptomic alterations in cell populations in the thymus during acute neonatal MRV infection. These studies offer important insight into the early events that occur after neonatal MRV infection that disrupt central tolerance and promote autoimmune disease.

A Bcl11bN797K variant isolated from an immunodeficient patient inhibits early thymocyte development in mice.

BCL11B is a transcription factor with six C2H2-type zinc-finger domains. Studies in mice have shown that Bcl11b plays essential roles in T cell development. Several germline heterozygous BCL11B variants have been identified in human patients with inborn errors of immunity (IEI) patients. Among these, two de novo mis-sense variants cause asparagine (N) to lysine (K) replacement in distinct zinc-finger domains, BCL11BN441K and BCL11BN807K. To elucidate the pathogenesis of the BCL11BN807K variant, we generated a mouse model of BCL11BN807K by inserting the corresponding mutation, Bcl11bN797K, into the mouse genome. In Bcl11b+/N797K mice, the proportion of immature CD4-CD8+ single-positive thymocytes was increased, and the development of invariant natural killer cells was severely inhibited in a T-cell-intrinsic manner. Under competitive conditions, γδT cell development was outcompeted by control cells. Bcl11bN797K/N797K mice died within one day of birth. Recipient mice reconstituted with Bcl11bN797K/N797K fetal liver cells nearly lacked CD4+CD8+ double-positive thymocytes, which was consistent with the lack of their emergence in culture from Bcl11bN797K/N797K fetal liver progenitors. Interestingly, Bcl11bN797K/N797K progenitors gave rise to aberrant c-Kit+ and CD44+ cells both in vivo and in vitro. The increase in the proportion of immature CD8 single-positive thymocytes in the Bcl11bN797K mutants is caused, in part, by the inefficient activation of the Cd4 gene due to the attenuated function of the two Cd4 enhancers via distinct mechanisms. Therefore, we conclude that immunodeficient patient-derived Bcl11bN797K mutant mice elucidated a novel role for Bcl11b in driving the appropriate transition of CD4-CD8- into CD4+CD8+ thymocytes.

Controlled WASp activity regulates the proliferative response for Treg cell differentiation in the thymus.

The Wiskott-Aldrich syndrome protein (WASp) regulates actin cytoskeletal dynamics and function of hematopoietic cells. Mutations in the WAS gene lead to two different syndromes; Wiskott-Aldrich syndrome (WAS) caused by loss-of-function mutations, and X-linked neutropenia (XLN) caused by gain-of-function mutations. We previously showed that WASp-deficient mice have a decreased number of regulatory T (Treg) cells in the thymus and the periphery. We here evaluated the impact of WASp mutations on Treg cells in the thymus of WAS and XLN mouse models. Using in vitro Treg differentiation assays, WAS CD4 single-positive thymocytes have decreased differentiation to Treg cells, despite normal early signaling upon IL-2 and TGF-β stimulation. They failed to proliferate and express CD25 at high levels, leading to poor survival and a lower number of Foxp3+ Treg cells. Conversely, XLN CD4 single-positive thymocytes efficiently differentiate into Foxp3+ Treg cells following a high proliferative response to IL-2 and TGF-β, associated with high CD25 expression when compared with WT cells. Altogether, these results show that specific mutations of WASp affect Treg cell development differently, demonstrating a critical role of WASp activity in supporting Treg cell development and expansion.

CCK2-receptor deficiency impairs immune balance by influencing CD4+ T cells development by inhibiting cortical-thymic-epithelial-cells.

Immune balance is crucial for an organism's survival and is inseparable from the regulation of the nervous system. Accumulating evidence indicates that cholecystokinin (CCK) plays an important role in mediating the immune response through the activation of cholecystokinin receptors (CCKRs). However, it remains unclear whether CCKRs deficiency may impair immune balance. Here, we showed that CCK2R-deficient adult mice were immunocompromised and had an increased risk of shock and even death in an endotoxemia (ETM)/endotoxin shock (ES) model. In addition, in both adult and juvenile mice, CCK2R deficiency not only influenced the development of CD4 single-positive (SP) thymocytes in thymic positive selection but also decreased the population of CD3+ CD4+ T cells in the spleen. More importantly, CCK2R deficiency inhibited the expression of major histocompatibility complex class II (MHC II) and CD83 on cortical thymic epithelial cells (cTECs) in juvenile and adult mice. Overall, our study suggests that CCK2R is essential for maintaining CD4+ T cell development in the thymus and reveals that CCK2R plays an important role in maintaining immune balance.

Thymocyte Development of Humanized Mice Is Promoted by Interactions with Human-Derived Antigen Presenting Cells upon Immunization.

Immune responses in humanized mice are generally inefficient without co-transplantation of human thymus or HLA transgenes. Previously, we generated humanized mice via the intra-bone marrow injection of CD133+ cord blood cells into irradiated adult immunodeficient mice (IBMI-huNSG mice), which could mount functional immune responses against HTLV-1, although the underlying mechanisms were still unknown. Here, we investigated thymocyte development in IBMI-huNSG mice, focusing on the roles of human and mouse MHC restriction. IBMI-huNSG mice had normal developmental profiles but aberrant thymic structures. Surprisingly, the thymic medulla-like regions expanded after immunization due to enhanced thymocyte expansion in association with the increase in HLA-DR+ cells, including CD205+ dendritic cells (DCs). The organ culture of thymus from immunized IBMI-huNSG mice with a neutralizing antibody to HLA-DR showed the HLA-DR-dependent expansion of CD4 single positive thymocytes. Mature peripheral T-cells exhibited alloreactive proliferation when co-cultured with human peripheral blood mononuclear cells. Live imaging of the thymus from immunized IBMI-huNSG mice revealed dynamic adhesive contacts of human-derived thymocytes and DCs accompanied by Rap1 activation. These findings demonstrate that an increase in HLA-DR+ cells by immunization promotes HLA-restricted thymocyte expansion in humanized mice, offering a unique opportunity to generate humanized mice with ease.

Severe CD8+ T Lymphopenia in WHIM Syndrome Caused by Selective Sequestration in Primary Immune Organs.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is an ultra-rare combined primary immunodeficiency disease caused by heterozygous gain-of-function mutations in the chemokine receptor CXCR4. WHIM patients typically present with recurrent acute infections associated with myelokathexis (severe neutropenia due to bone marrow retention of mature neutrophils). Severe lymphopenia is also common, but the only associated chronic opportunistic pathogen is human papillomavirus and mechanisms are not clearly defined. In this study, we show that WHIM mutations cause more severe CD8 than CD4 lymphopenia in WHIM patients and WHIM model mice. Mechanistic studies in mice revealed selective and WHIM allele dose-dependent accumulation of mature CD8 single-positive cells in thymus in a cell-intrinsic manner due to prolonged intrathymic residence, associated with increased CD8 single-positive thymocyte chemotactic responses invitro toward the CXCR4 ligand CXCL12. In addition, mature WHIM CD8+ T cells preferentially home to and are retained in the bone marrow in mice in a cell-intrinsic manner. Administration of the specific CXCR4 antagonist AMD3100 (plerixafor) in mice rapidly and transiently corrected T cell lymphopenia and the CD4/CD8 ratio. After lymphocytic choriomeningitis virus infection, we found no difference in memory CD8+ T cell differentiation or viral load between wild-type and WHIM model mice. Thus, lymphopenia in WHIM syndrome may involve severe CXCR4-dependent CD8+ T cell deficiency resulting in part from sequestration in the primary lymphoid organs, thymus, and bone marrow.

DNA demethylation fine-tunes IL-2 production during thymic development and promotes Regulatory T cell differentiation

Abstract Regulatory T (T reg) cells developing in the thymus are essential to maintain tolerance and prevent fatal autoimmunity in mice and humans. Expression of the T reg lineage-defining transcription factor FoxP3, is critically dependent upon T cell receptor (TCR) and interleukin-2 (IL-2) signaling. Here, we report that ten-eleven translocation (Tet) enzymes, which are DNA demethylases, are required early during double positive (DP) thymic T cell differentiation and prior to the upregulation of FoxP3 in CD4 single-positive (SP) thymocytes, to promote Treg differentiation. We show that Tet enzymes selectively control the development of CD25 −FoxP3 loCD4SPTreg cell precursors and are critical for optimal TCR-dependent IL-2 production, which drive chromatin remodeling at the FoxP3 locus as well as other Treg-effector gene loci in an autocrine/paracrine manner. Together, our results demonstrate a novel role for DNA demethylation in regulating the TCR response and promoting Treg cell differentiation. These findings highlight a novel epigenetic pathway to promote the generation of endogenous Treg cells for mitigation of autoimmune responses.

Distinct populations of eosinophils in the human thymus with capacity to modulate thymocyte maturation.

Local differentiation of eosinophil precursors occurs in the human thymus. Thymic eosinophils are often positioned in the corticomedullary junction between the CD4+ CD8+ double-positive (DP) thymocytes and the CD4+ or CD8+ single-positive (SP) thymocytes. The aims of this study were to (1) determine if there are distinct thymic eosinophil populations that differ from the blood eosinophil populations and (2) evaluate the capacity of thymic eosinophils to promote the development of SP thymocytes from DP thymocytes. Thymic and blood eosinophils from thymectomized infants (n=7) were compared regarding the expression of 34 molecules using cytometry by time-of-flight (CyTOF). In addition, FACS-sorted thymic eosinophils were co-cultured with autologous CD3/CD28-stimulated DP, CD4 SP, and CD8 SP thymocytes and analysed by flow cytometry and CyTOF. X-shift clustering analysis and viSNE dimensionality reduction were performed. Seven eosinophil populations were identified within the blood and thymus, respectively, five of which were specific for either tissue. Whereas the blood eosinophil populations varied between individuals, the thymic eosinophil populations were more uniform. The eosinophil-thymocyte co-cultures resulted in (1) an increase in CD4 SP thymocytes when eosinophils were cultured with DP thymocytes, (2) decreased frequency of CD8 SP thymocytes when these were cultured with eosinophils, and (3) a more mature thymic phenotype when eosinophils were cultured with CD4 SP thymocytes. Thymic eosinophils are a specialized population of eosinophils with a distinct phenotype that separates them from their blood counterparts, and in vitro they appear to favour CD4 SP thymocyte development to the detriment of CD8 SP thymocytes.

IGF-1 increases survival of CD4+ lineage in a 2D model of thymocyte/thymic stromal cell co-culture.

Insulin-like growth factor-1 (IGF-1), in addition to its classic effects on cell proliferation and organism growth, has pleiotropic actions on the immune system, particularly on the thymus. Thus, the objective of this study was to evaluate the influence of IGF-1 on molecules involved in the survival of thymocytes in vitro using a co-culture system with thymic stromal cells obtained from C57BL/6 mice. The obtained thymic stroma has contained thymic epithelial cells, macrophages, dendritic cells, fibroblasts, and preserved the expression of the major histocompatibility complex (MHC) molecules. Fresh thymocytes were added to these cultures and the co-culture were treated daily with IGF-1 (100ng/mL) for 3days. In this scheme, the viability of the thymocytes was about 70%, either in the control (non-treated cells) or in the IGF-1-treated cultures. It was found that IGF-1 was able to increase the percentage of thymocytes from the CD4+ single-positive (SP) subset. This result was accompanied by an increase in the MHC II expression on thymic stromal cells and an augment on the interleukin-7 receptor (CD127) on the surface of the CD4 SP thymocytes after treatment with IGF-1. Finally, IGF-1 treatment increased the expression of the ThPOK encoding gene Zbtb7b, which is involved in the differentiation of CD4+ SP thymocytes. Our study demonstrates the participation of IGF-1 in the thymocyte/thymic stroma interactions, especially in the extended survival of the CD4+ lineage in the thymus.

Tracking Regulatory T Cell Development in the Thymus Using Single-Cell RNA Sequencing/TCR Sequencing.

Recent studies have demonstrated that regulatory T cells (Tregs) develop in the thymus via two pathways involving distinct Treg progenitors (TregP): CD25+FOXP3- (CD25+ TregP) and CD25-FOXP3lo (FOXP3lo TregP) Treg progenitors. To examine this process in more detail, we carried out single-cell RNA sequencing (scRNA-Seq) and TCR-Seq on sorted murine CD4+CD8+ double-positive (DP) thymocytes, CD4+ single-positive (CD4SP) thymocytes, CD25+FOXP3-CD73- TregP, CD25-FOXP3loCD73- TregP, newly generated mature CD25+FOXP3+CD73- Tregs, and FOXP3+CD73+ recirculating/long-term resident Tregs (RT-Tregs). Sorted populations were individually hashtagged and then combined into one scRNA-Seq/TCR-Seq library before sequencing and subsequent analysis. We found that both CD25+ TregP and FOXP3lo TregP arise via an initial agonist-activated state that gives rise to a second transitional stage before differentiating into mature Tregs Using both scRNA-Seq and bulk RNA-Seq on sorted thymocyte subsets, we demonstrate that CD25+ TregP are significantly enriched for Il2 production, suggesting that they are the major source of IL-2 needed to convert TregP into mature Tregs Using TCR-Seq, we found that several TCRs were clearly biased in favor of the conventional or Treg lineages, but that a large fraction of TCRs were found in both these lineages. Finally, we found that RT-Tregs in the thymus are not monomorphic but are composed of multiple distinct subsets and that these RT-Tregs express the most diverse TCR repertoire of all CD4SP thymocytes. Thus, our studies define multiple stages of Treg differentiation within the murine thymus and serve as a resource for future studies on CD4+ thymocyte development and Treg differentiation.

Chromatin organizer SATB1 controls the cell identity of CD4+ CD8+ double-positive thymocytes by regulating the activity of super-enhancers

CD4+ and CD8+ double-positive (DP) thymocytes play a crucial role in T cell development in the thymus. DP cells rearrange the T cell receptor gene Tcra to generate T cell receptors with TCRβ. DP cells differentiate into CD4 or CD8 single-positive (SP) thymocytes, regulatory T cells, or invariant nature kill T cells (iNKT) in response to TCR signaling. Chromatin organizer SATB1 is highly expressed in DP cells and is essential in regulating Tcra rearrangement and differentiation of DP cells. Here we explored the mechanism of SATB1 orchestrating gene expression in DP cells. Single-cell RNA sequencing shows that Satb1 deletion changes the cell identity of DP thymocytes and down-regulates genes specifically and highly expressed in DP cells. Super-enhancers regulate the expressions of DP-specific genes, and our Hi-C data show that SATB1 deficiency in thymocytes reduces super-enhancer activity by specifically decreasing interactions among super-enhancers and between super-enhancers and promoters. Our results reveal that SATB1 plays a critical role in thymocyte development to promote the establishment of DP cell identity by globally regulating super-enhancers of DP cells at the chromatin architectural level.

The circadian clock sets a spatial-temporal window for recent thymic emigrants.

The diurnal timing system regulates multiple functions of lymphocytes in peripheral lymphoid organs. Whether T-cell development in the thymus and T-cell egress from the thymus are affected by the circadian clock is not clear. Herein, we used flow cytometry to examine the cell number and percentage of total thymocytes and various thymocyte subsets from Zeitgeber time (ZT) 1 to ZT21. CD4 and CD8 single-positive (SP) thymocytes, in particular, the mature CD4 SP4 thymocyte subset with emigration capability and P-phycoerythrin+ CD4 SP thymocytes in the perivascular space of the thymus, exhibited robust circadian oscillations. The diurnal expression of sphingosine-1-phosphate receptor-1 (S1PR1) and CCR2 on SP thymocytes and the rhythmic sphingosine-1-phosphate (S1P) and CCL2 gradient formed between peripheral blood and thymus likely promoted SP thymocyte egress in a circadian pattern. Switching the daylight cycle disturbed the rhythm of S1PR1 and CCR2 expression and subsequent thymocyte output. We further demonstrated that the core clock molecule BMAL1 had rhythmic binding of the promoters of Klf2, S1pr1 and Sphk2. Together, we elucidated the circadian dynamic characteristics of mature thymocyte egress, which coordinated with the diurnal changes in T-cell homing to the lymph nodes. The core rhythmic molecule BMAL1 likely promoted thymocyte emigration through transcriptional regulation of emigration-related molecules.

Transcriptional coregulator Ess2 controls survival of post-thymic CD4+ T cells through the Myc and IL-7 signaling pathways

Ess2, also known as Dgcr14, is a transcriptional co-regulator of CD4+ T cells. Ess2 is located in a chromosomal region, the loss of which has been associated with 22q11.2 deletion syndrome (22q11DS), which causes heart defects, skeletal abnormalities, and immunodeficiency. However, the specific association of Ess2 with 22q11DS remains unclear. To elucidate the role of Ess2 in T-cell development, we generated Ess2 floxed (Ess2fl/fl) and CD4+ T cell–specific Ess2 KO (Ess2ΔCD4/ΔCD4) mice using the Cre/loxP system. Interestingly, Ess2ΔCD4/ΔCD4 mice exhibited reduced naïve T-cell numbers in the spleen, while the number of thymocytes (CD4−CD8−, CD4+CD8+, CD4+CD8−, and CD4−CD8+) in the thymus remained unchanged. Furthermore, Ess2ΔCD4/ΔCD4 mice had decreased NKT cells and increased γδT cells in the thymus and spleen. A genome-wide expression analysis using RNA-seq revealed that Ess2 deletion alters the expression of many genes in CD4 single-positive thymocytes, including genes related to the immune system and Myc target genes. In addition, Ess2 enhanced the transcriptional activity of c-Myc. Some genes identified as Ess2 targets in mice show expressional correlation with ESS2 in human immune cells. Moreover, Ess2ΔCD4/ΔCD4 naïve CD4+ T cells did not maintain survival in response to IL-7. Our results suggest that Ess2 plays a critical role in post-thymic T-cell survival through the Myc and IL-7 signaling pathways.

Zbtb20 identifies and controls a thymus-derived population of regulatory T cells that play a role in intestinal homeostasis.

The expression of BTB-ZF transcription factors such as ThPOK in CD4+ T cells, Bcl6 in T follicular helper cells, and PLZF in natural killer T cells defines the fundamental nature and characteristics of these cells. Screening for lineage-defining BTB-ZF genes led to the discovery of a subset of T cells that expressed Zbtb20. About half of Zbtb20+ T cells expressed FoxP3, the lineage-defining transcription factor for regulatory T cells (Tregs). Zbtb20+ Tregs were phenotypically and genetically distinct from the larger conventional Treg population. Zbtb20+ Tregs constitutively expressed mRNA for interleukin-10 and produced high levels of the cytokine upon primary activation. Zbtb20+ Tregs were enriched in the intestine and specifically expanded when inflammation was induced by the use of dextran sodium sulfate. Conditional deletion of Zbtb20 in T cells resulted in a loss of intestinal epithelial barrier integrity. Consequently, knockout (KO) mice were acutely sensitive to colitis and often died because of the disease. Adoptive transfer of Zbtb20+ Tregs protected the Zbtb20 conditional KO mice from severe colitis and death, whereas non-Zbtb20 Tregs did not. Zbtb20 was detected in CD24hi double-positive and CD62Llo CD4 single-positive thymocytes, suggesting that expression of the transcription factor and the phenotype of these cells were induced during thymic development. However, Zbtb20 expression was not induced in "conventional" Tregs by activation in vitro or in vivo. Thus, Zbtb20 expression identified and controlled the function of a distinct subset of Tregs that are involved in intestinal homeostasis.

Depletion of Ift88 in thymic epithelial cells affects thymic synapse and T-cell differentiation in aged mice.

Primary cilia are ubiquitous hair-like organelles, usually projecting from the cell surface. They are essential for the organogenesis and homeostasis of various physiological functions, and their dysfunction leads to a plethora of human diseases. However, there are few reports on the role of primary cilia in the immune system; therefore, we focused on their role in the thymus that nurtures immature lymphocytes to full-fledged T cells. We detected primary cilia on the thymic epithelial cell (TEC) expressing transforming growth factor β (TGF-β) receptor in the basal body, and established a line of an intraflagellar transport protein 88 (Ift88) knockout mice lacking primary cilia in TECs (Ift88-TEC null mutant) to clarify their precise role in thymic organogenesis and T-cell differentiation. The Ift88-TEC null mutant mice showed stunted cilia or lack of cilia in TECs. The intercellular contact between T cells and the "thymic synapse" of medullary TECs was slightly disorganized in Ift88-TEC null mutants. Notably, the CD4- and CD8-single positive thymocyte subsets increased significantly. The absence or disorganization of thymic cilia downregulated the TGF-β signaling cascade, increasing the number of single positive thymocytes. To our knowledge, this is the first study reporting the physiological role of primary cilia and Ift88 in regulating the differentiation of the thymus and T cells.

Human CD4 T Cells From Thymus and Cord Blood Are Convertible Into CD8 T Cells by IL-4.

Commitment to the CD4+ or CD8+ T cell lineages is linked to the acquisition of a functional program broadly defined by helper and cytotoxic properties, respectively. The mechanisms underlying these processes in the human thymus remain largely unclear. Moreover, recent thymic emigrants are thought to have some degree of plasticity, which may be important for the shaping of the immune system and adjustment to specific peripheral needs. We show here that IL-4 induces proliferation-independent de novo synthesis of CD8αβ in human CD4 single-positive (SP) thymocytes, generating a stable CD8SP population that features a diverse TCRαβ repertoire, CD4 expression shut-down and ThPOK downregulation. IL-4 also promotes an innate-like program in both CD4SP and CD8SP thymocytes, characterized by Eomes upregulation in the absence of T-bet, in line with its recognized role in the generation of thymic innate-like CD8+ T cells. The clinical relevance of these findings is further supported by the profile of IL-4 production and IL-4 receptor expression that we identified in the human thymus. Importantly, human cord blood CD4+ T cells preserve the ability to generate Eomes+ CD8+ T cells in the presence of IL-4, with implications in neonatal immunity. Our results support a role for IL-4 in the dynamic regulation of human thymocyte plasticity and identify novel strategies to modulate immune responses.

Expression of Human Endogenous Retroviruses in the Human Thymus Along T Cell Development

Human Endogenous Retroviruses (HERVs) constitute up to 8% of the human genome and have been emerging as important modulators of the immune system, being associated with cancer, autoimmunity and infectious diseases. Here, we investigated the expression of three HERV families in the human thymus. HERV-K, -W, and -R envelope (env) and HERV-Kgagtranscriptional levels were quantified in the main thymocyte subsets, thymic epithelial cells (TECs), B cells and myeloid populations, and Env protein expression was studied in thymic tissue. We found that HERV mRNA decreased with T cell development, which was in agreement with the identification of HERV-K Env protein in CD3 negative cortical cells. These results suggest a distinct regulation of HERV expression along T cell development, prompting us to evaluate the interplay with host restriction factors and potential underlying pathways. The transcriptional levels of some HERVs were found to positively correlate with the expression of the host restriction factors APOBEC3G and SLFN11, and, conversely, a negative correlation was found with SAMHD1. Moreover, IFN-α and IFN-γ induced the upregulation of HERV-Kenvandgagin purified CD4 single-positive thymocytes. Additionally, we found high levels of HERV mRNAs in TECs. Overall, our data support a tight regulation of HERV expression during human T cell development, with possible implications for the process of T cell selection.