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Molecular imaging using singlechain variable fragments (scFv) of antibodies targeting cancer specific antigens have been considered a non-immunogenic approach for early diagnosis in the clinic. Usually, production of proteins is performed within Escherichia coli. Recombinant proteins are either expressed in E. coli cytoplasm as insoluble inclusion bodies, that often need cumbersome denaturation and refolding processes, or secreted toward the periplasm as soluble proteins that highly reduce the overall yield. However, production of active scFvs in their native form, without any heterologous fusion, is required for clinical applications. In this study, we expressed an anti-thymocyte differentiation antigen-scFv (Thy1-scFv) as a fusion protein with a N-terminal sequence including 3 × hexa-histidines, as purification tags, together with a Trx-tag and a S-tag for enhanced-solubility. Our strategy allowed to recover ~ 35% of Thy1-scFv in the soluble cytoplasmic fraction. An enterokinase cleavage site in between Thy1-scFv and the upstream tags was used to regenerate the protein with 97.7 ± 2.3% purity without any tags. Thy1-scFv showed functionality towards its target on flow cytometry assays. Finally, in vivo molecular imaging using Thy1-scFv conjugated to an ultrasound contrast agent (MBThy1-scFv) demonstrated signal enhancement on a transgenic pancreatic ductal adenocarcinoma (PDAC) mouse model (3.1 ± 1.2 a.u.) compared to non-targeted control (0.4 ± 0.4 a.u.) suggesting potential for PDAC early diagnosis. Overall, our strategy facilitates the expression and purification of Thy1-scFv while introducing its ability for diagnostic molecular imaging of pancreatic cancer. The presented methodology could be expanded to other important eukaryotic proteins for various applications, including but not limited to molecular imaging.

The nuclear receptors liver X receptor α (LXRα) and LXRβ are lipid sensors that regulate lipid metabolism and immunity. Natural killer T (NKT) cells, a T cell subset expressing surface markers of both natural killer cells and T lymphocytes and involved in antitumor immunity, are another abundant immune cell type in the liver. The potential function of the metabolic regulators LXRα/β in hepatic NKT cells remains unknown. In this study, we examined the role of LXRα and LXRβ in NKT cells using mice deficient for LXRα and/or LXRβ, and found that hepatic invariant NKT (iNKT) cells are drastically decreased in LXRα/β-KO mice. Cytokine production stimulated by the iNKT cell activator α-galactosylceramide was impaired in LXRα/β-KO hepatic mononuclear cells and in LXRα/β-KO mice. iNKT cell-mediated antitumor effect was also disturbed in LXRα/β-KO mice. LXRα/β-KO mice transplanted with wild-type bone marrow showed decreased iNKT cells in the liver and spleen. The thymus of LXRα/β-KO mice showed a decreased population of iNKT cells. In conclusion, LXRα and LXRβ are essential for NKT cell-mediated immunity, such as cytokine production and hepatic antitumor activity, and are involved in NKT cell development in immune tissues, such as the thymus.

Salt Inducible Kinases (SIKs), of which there are 3 isoforms, are established to play roles in innate immunity, metabolic control and neuronal function, but their role in adaptive immunity is unknown. To address this gap, we used a combination of SIK knockout and kinase-inactive knock-in mice. The combined loss of SIK1 and SIK2 activity did not block T cell development. Conditional knockout of SIK3 in haemopoietic cells, driven by a Vav-iCre transgene, resulted in a moderate reduction in the numbers of peripheral T cells, but normal B cell numbers. Constitutive knockout of SIK2 combined with conditional knockout of SIK3 in the haemopoietic cells resulted in a severe reduction in peripheral T cells without reducing B cell number. A similar effect was seen when SIK3 deletion was driven via CD4-Cre transgene to delete at the DP stage of T cell development. Analysis of the SIK2/3 Vav-iCre mice showed that thymocyte number was greatly reduced, but development was not blocked completely as indicated by the presence of low numbers CD4 and CD8 single positive cells. SIK2 and SIK3 were not required for rearrangement of the TCRβ locus, or for low level cell surface expression of the TCR complex on the surface of CD4/CD8 double positive thymocytes. In the absence of both SIK2 and SIK3, progression to mature single positive cells was greatly reduced, suggesting a defect in negative and/or positive selection in the thymus. In agreement with an effect on negative selection, increased apoptosis was seen in thymic TCRbeta high/CD5 positive cells from SIK2/3 knockout mice. Together, these results show an important role for SIK2 and SIK3 in thymic T cell development.

We recently mapped a genetic susceptibility locus on chromosome 6q22.33 for type 1 diabetes (T1D) diagnosed below the age of 7 years between the PTPRK and thymocyte-selection-associated (THEMIS) genes. As the thymus plays a central role in shaping the T cell repertoire, we aimed to identify the most likely causal genetic factors behind this association using thymocyte genomic data. In four thymocyte populations, we identified 253 DNA sequence motifs underlying histone modifications. The G insertion allele of rs138300818, associated with protection from diabetes, created thymocyte motifs for multiple histone modifications and thymocyte types. In a parallel approach to identifying variants that alter transcription factor binding motifs, the same variant disrupted a predicted motif for Rfx7, which is abundantly expressed in the thymus. Chromatin state and RNA sequencing data suggested strong transcription overlapping rs138300818 in fetal thymus, while expression quantitative trait locus and chromatin conformation data associate the insertion with lower THEMIS expression. Extending the analysis to other T1D loci further highlighted rs66733041 affecting the GATA3 transcription factor binding in the AFF3 locus. Taken together, our results support a role for thymic THEMIS gene expression and the rs138300818 variant in promoting the development of early-onset T1D.

In the quest for more effective radiation treatment options that can improve both cell killing and healthy tissue recovery, combined radiation therapies are lately in the spotlight. The molecular response to a combined radiation regime where exposure to an initial low dose (priming dose) of ionizing radiation is administered prior to a subsequent higher radiation dose (challenging dose) after a given latency period have not been thoroughly explored. In this study we report on the differential response to either a combined radiation regime or a single challenging dose both in mouse in vivo and in human ex vivo thymocytes. A differential cell cycle response including an increase in the subG1 fraction on cells exposed to the combined regime was found. Together with this, a differential protein expression profiling in several pathways including cell cycle control (ATM, TP53, p21CDKN1A), damage response (γH2AX) and cell death pathways such as apoptosis (Cleaved Caspase-3, PARP1, PKCδ and H3T45ph) and ferroptosis (xCT/GPX4) was demonstrated. This study also shows the epigenetic regulation following a combined regime that alters the expression of chromatin modifiers such as DNMTs (DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3L) and glycosylases (MBD4 and TDG). Furthermore, a study of the underlying cellular status six hours after the priming dose alone showed evidence of retained modifications on the molecular and epigenetic pathways suggesting that the priming dose infers a “radiation awareness phenotype” to the thymocytes, a sensitization key to the differential response seen after the second hit with the challenging dose. These data suggest that combined-dose radiation regimes could be more efficient at making cells respond to radiation and it would be interesting to further investigate how can these schemes be of use to potential new radiation therapies.

Transgenic mice that express either a NUP98–PHF23 (NP23) or NUP98-HOXD13 (NHD13) fusion in the hematopoietic compartment develop a wide spectrum of leukemias, including myeloid, erythroid, megakaryocytic and lymphoid, at age 9–14 months. NP23-NHD13 double transgenic mice were generated by interbreeding NP23 and NHD13 mice. Remarkably, 100% of the NP23-NHD13 double transgenic mice developed acute myeloid leukemia (AML) within three months, characterized by replacement of the thymus with leukemic myeloblasts. The marked infiltration of thymus led to the intriguing hypothesis that AML generated in NP23-NHD13 mice arose in the thymus, as opposed to the bone marrow (BM). Transplantation of CD4-CD8- double negative (DN) thymocytes (which were also negative for Mac1 and Gr1) from leukemic NHD13/NP23 mice demonstrated that DN thymocytes could transmit AML, and limiting dilution studies showed that leukemia initiating cells were increased 14-fold in the thymus compared to BM. Further thymocyte fractionation demonstrated that DN1 and DN2, but not DN3 or DN4 fractions transmitted AML, and a marked expansion (100-fold) of Lineage-Sca1 + Kit + (LSK) cells in the thymus of the NP23-NHD13 mice. Taken together, these results show that the thymus of NP23-NHD13 mice acts as a reservoir for AML initiating cells and that thymic progenitors can transmit AML.

The thymus facilitates mature T cell production by providing a suitable stromal microenvironment. This microenvironment is impaired by radiation and aging which lead to immune system disturbances known as thymic involution. Young adult thymus shows thymic recovery after such involution. Although various genes have been reported for thymocytes and thymic epithelial cells in such processes, the roles of stromal transcription factors in these remain incompletely understood. MafB (v-maf musculoaponeurotic fibrosarcoma oncogene homolog B) is a transcription factor expressed in thymic stroma and its expression was induced a day after radiation exposure. Hence, the roles of mesenchymal MafB in the process of thymic regeneration offers an intriguing research topic also for radiation biology. The current study investigated whether MafB plays roles in the adult thymus. MafB/green fluorescent protein knock-in mutant (MafB+/GFP) mice showed impaired thymic regeneration after the sublethal irradiation, judged by reduced thymus size, total thymocyte number and medullary complexity. Furthermore, IL4 was induced after irradiation and such induction was reduced in mutant mice. The mutants also displayed signs of accelerated age-related thymic involution. Altogether, these results suggest possible functions of MafB in the processes of thymic recovery after irradiation, and maintenance during aging.

Arsenic is globally infamous for inducing immunosuppression associated with prevalence of opportunistic infection in exposed population, although the mechanism remains elusive. In this study, we investigate the effect of arsenic exposure on thymocyte lineage commitment and the involvement of regulatory T cells (Treg) in arsenic-induced immunosuppression. Male Balb/c mice were exposed to 0.038, 0.38 and 3.8 ppm sodium arsenite for 7, 15 and 30 days through oral gavage. Arsenic exposure promoted CD4 lineage commitment in a dose dependent manner supported by the expression of ThPOK in thymus. Arsenic also increased splenic CD4+ T cells and promoted their differentiation into Treg cells. In parallel, arsenic exposure induced immunosuppression characterized by low cytokine secretion from splenocytes and increased susceptibility to Mycobacterium fortuitum (M. fortuitum) infection. Therefore, we linked arsenic-induced rise in Treg cells with suppressed Th1 and Th2 related cytokines, which has been reversed by inhibition of Treg cells in-vivo using wortmannin. Other parameters like body weight, kidney and liver function, histoanatomy of thymus and spleen as well as thymocyte and splenocytes viability were unaltered by arsenic exposure. Taken together our findings indicated that environmentally relevant dose of arsenic enhanced differentiation of Treg cells which in turn induce immunosuppression in experimental animals.

Previous work showed that the thymus can be infected by RNA viruses as HIV and HTLV-1. We thus hypothesized that the thymus might also be infected by the Zika virus (ZIKV). Herein we provide compelling evidence that ZIKV targets human thymic epithelial cells (TEC) in vivo and in vitro. ZIKV-infection enhances keratinization of TEC, with a decrease in proliferation and increase in cell death. Moreover, ZIKV modulates a high amount of coding RNAs with upregulation of genes related to cell adhesion and migration, as well as non-coding genes including miRNAs, circRNAs and lncRNAs. Moreover, we observed enhanced attachment of lymphoblastic T-cells to infected TEC, as well as virus transfer to those cells. Lastly, alterations in thymuses from babies congenitally infected were seen, with the presence of viral envelope protein in TEC. Taken together, our data reveals that the thymus, particularly the thymic epithelium, is a target for the ZIKV with changes in the expression of molecules that are relevant for interactions with developing thymocytes.

Small numbers of hematopoietic stem cells (HSCs) balance self-renewal and differentiation to produce the diversity and abundance of cell types that make up the blood system. How nutrients are recruited to support this massive differentiation and proliferation process remains largely unknown. The unique metabolism of adult HSCs, which rely on glycolysis and glutaminolysis, suggests a potential role for the post-translational modification O-GlcNAc as a critical nutrient signal in these cells. Glutamine, glucose, and other metabolites drive the hexosamine biosynthetic pathway (HBP) ultimately leading to the O-GlcNAc modification of critical intracellular targets. Here, we used a conditional targeted genetic deletion of the enzyme that removes O-GlcNAc, O-GlcNAcase (OGA), to determine the consequences of blocked O-GlcNAc cycling on HSCs. Oga deletion in mouse HSCs resulted in greatly diminished progenitor pools, impaired stem cell self-renewal and nearly complete loss of competitive repopulation capacity. Further, early T cell specification was particularly sensitive to Oga deletion. Loss of Oga resulted in a doubling of apoptotic cells within the bone marrow and transcriptional deregulation of key genes involved in adult stem cell maintenance and lineage specification. These findings suggest that O-GlcNAc cycling plays a critical role in supporting HSC homeostasis and early thymocyte development.

The clearance of apoptotic cells is an essential process to maintain homeostasis of immune system, which is regulated by immunoregulatory cytokines such as TGFβ. We show here that Extracellular Vesicles (EVs) were highly released from apoptotic cells, and contributed to macrophage production of TGFβ in vitro and in vivo. We further elucidated mechanistically that phosphatidylserine in EVs was a key triggering-factor, and transcription factor FOXO3 was a critical mediator for apoptotic EV-induced TGFβ in macrophages. Importantly, we found that macrophages pre-exposed to EVs exhibited an anti-inflammatory phenotype. More strikingly, administration of EVs in vivo promotes Tregs differentiation and suppresses Th1 cell response, and ameliorates experimental colitis. Thus, apoptotic-EV-based treatment might be a promising therapeutic approach for human autoimmune disease.

Although natural killer T cells (NKT cells) are thought to be generated from CD4+CD8+ (DP) thymocytes, the developmental origin of CD4−CD8− (DN) NKT cells has remained unclear. In this study, we found the level of NK1.1 expression was highest in DN cells, followed by CD4 and CD8 (SP) and DP cells. The level of NK1.1 expression was highest in CD44+CD25− (DN1) cells, after that CD44+CD25+ (DN2), finally, CD44−CD25− (DN3) and CD44− CD25+ (DN4) cells. Unexpectedly, cytoplasmic CD3 was not only expressed in SP and DP thymocytes but also in most DN thymocytes at various stages. The mean fluorescence of cytoplasmic and surface CD3 in DN cells was significantly lower than in mature (SP) T and NKT cells in the thymus and spleen. Interestingly, there were more NKT cells in DN-cytoplasmic CD3 expression cells was higher than in DN-surface CD3 expression cells. There were more CD3-NKT cells in DN1 thymocytes than in TCR-β-NKT cells. NKT cells expressed higher levels of IL-7Rα which was correlated with CD44 expression in the thymus. Our data suggest that T cells and NKT cells follow similar patterns of expression with respect to cytoplasmic and surface CD3. Cytoplasmic CD3 could be used as a marker for early stage T cells. Both cytoplasmic CD3 and surface CD3 were expressed in mature T cells and immature T cells, including the immature cytoplasmic CD3+ surface CD3− and surface CD3+TCR-β− cells in DN1-NKT thymocytes. CD44 could be used as an additional marker of NKT cells which may originate from cytoplasmic CD3-positive DN thymocytes that express CD44 and IL-7Rα in mice.

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that performs multiple physiological activities. Previous studies suggest that PON1 plays an anti-inflammatory role in the cardiovascular system, although its roles in hematopoiesis and adaptive immunity have not been clarified. To investigate the impact of PON1 on the immune system, we generated PON1-knockout (PON1−/−) rats using the CRISPR/Cas9 system. The thymus was smaller in PON1−/− rats than that in wild-type (PON1+/+) rats. Furthermore, analysis of thymocyte development revealed diminished total T cell numbers and a decrease in CD4+, CD8+ and double-positive T cells in peripheral blood and thymus from PON1−/− rats. This may be due to a block in the transition of T cells from the double-negative to the double-positive stage. We also showed that the activation of p38 MAPK phosphorylation contributed to the increased apoptosis and defective T cell development in PON−/− rats. Therefore, our results indicate that PON1 functions as a novel regulator of T cell development.

Activation of the T cell receptor (TCR) leads to the generation of a network of signaling events critical to the developmental decision making and activation of T cells. Various experimental approaches continue to identify new signaling molecules, adaptor proteins, and other regulators of TCR signaling. We propose a screening strategy for the identification of small molecules affecting TCR signaling based on the uncoupling of TCR stimulation from cellular responses in developing thymocytes. We demonstrate that this strategy successfully identifies inhibitors of kinases already shown to act downstream of TCR engagement, as well as new inhibitors. The proposed strategy is easily scalable for high throughput screening and will contribute to the identification of new druggable targets in T cell activation.

Hypercholesterolemia promotes the inflammation against lipoproteins in atherosclerosis. Development of atherosclerosis is affected by the balance between pro-inflammatory effector T cells and anti-inflammatory regulatory T (Treg) cells. However, phenotype and function of T cell subpopulations in hypercholesterolemia remain to be investigated. Here, we found that cholesterol-containing diet increased the expression of the Treg cell lineage-defining transcription factor FoxP3 among thymocytes and splenocytes. Hypercholesterolemia elevated the FoxP3 expression level and population size of peripheral Treg cells, but did not prevent enhanced proliferation of stimulated T cells. Moreover, cholesterol supplementation in diet as well as in cell culture medium promoted T cell antigen receptor (TCR) signaling in CD4+ T cells. Our results demonstrate that hypercholesterolemia enhances TCR stimulation, Treg cell development as well as T cell proliferation. Thus, our findings may help to understand why hypercholesterolemia correlates with altered CD4+ T cell responses.

Semi-invariant natural killer T (NKT) cells are innate-like lymphocytes with immunoregulatory properties. NKT cell survival during development requires signal processing by activated RelA/NF-κB. Nonetheless, the upstream signal(s) integrated by NF-κB in developing NKT cells remains incompletely defined. We show that the introgression of Bcl-xL-coding Bcl2l1 transgene into NF-κB signalling-deficient IκBΔN transgenic mouse rescues NKT cell development and differentiation in this mouse model. We reasoned that NF-κB activation was protecting developing NKT cells from death signals emanating either from high affinity agonist recognition by the T cell receptor (TCR) or from a death receptor, such as tumor necrosis factor receptor 1 (TNFR1) or Fas. Surprisingly, the single and combined deficiency in PKC-θ or CARMA-1—the two signal transducers at the NKT TCR proximal signalling node—only partially recapitulated the NKT cell deficiency observed in IκBΔN tg mouse. Accordingly, introgression of the Bcl2l1 transgene into PKC-θ null mouse failed to rescue NKT cell development. Instead, TNFR1-deficiency, but not the Fas-deficiency, rescued NKT cell development in IκBΔN tg mice. Consistent with this finding, treatment of thymocytes with an antagonist of the inhibitor of κB kinase —which blocks downstream NF-κB activation— sensitized NKT cells to TNF-α-induced cell death in vitro. Hence, we conclude that signal integration by NF-κB protects developing NKT cells from death signals emanating from TNFR1, but not from the NKT TCR or Fas.

Protein malnutrition, the most deleterious cause of malnutrition in developing countries, has been considered a primary risk factor for the development of clinical visceral leishmaniasis (VL). Protein malnutrition and infection with Leishmania infantum leads to lymphoid tissue disorganization, including changes in cellularity and lymphocyte subpopulations in the thymus and spleen. Here we report that protein malnutrition modifies thymic chemotactic factors by diminishing the CCL5, CXCL12, IGF1, CXCL9 and CXCL10 protein levels in infected animals. Nevertheless, T cells preserve their migratory capability, as they were able to migrate ex vivo in response to chemotactic stimuli, indicating that malnutrition may compromise the thymic microenvironment and alter in vivo thymocyte migration. Decrease in chemotactic factors protein levels was accompanied by an early increase in the parasite load of the spleen. These results suggest that the precondition of malnutrition is affecting the cell-mediated immune response to L. infantum by altering T cell migration and interfering with the capacity of protein-deprived animals to control parasite spreading and proliferation. Our data provide evidence for a disturbance of T lymphocyte migration involving both central and peripheral T-cells, which likely contribute to the pathophysiology of VL that occurs in malnourished individuals.

The thymus is the site of T cell development in vertebrates. In general, the output of T cells is determined by the number of thymic epithelial cells (TECs) and their relative thymopoietic activity. Here, we show that the thymopoietic activity of TECs differs dramatically between individual mouse strains. Moreover, in males of some strains, TECs perform better on a per cell basis than their counterparts in females; in other strains, this situation is reversed. Genetic crosses indicate that TEC numbers and thymopoietic capacity are independently controlled. Long-term analysis of functional parameters of TECs after castration provides evidence that the number of Foxn1-expressing TECs directly correlates with thymopoietic activity. Our study highlights potential complications that can arise when comparing parameters of TEC biology across different genetic backgrounds; these could affect the interpretation of the outcomes of interventions aimed at modulating thymic activity in genetically diverse populations, such as humans.

13C-labeled glucose combined with chromatography and mass spectrometry enables us to decipher the percentage of lactate generated from various metabolic pathways. We showed that lactate derived from glycolysis, pentose phosphate pathway, Krebs cycle, and other sources accounted for 82–90%, 6.0–11%, 0.67–1.8% and 1.5–7.9%, respectively, depending on different types of cells. When using glucose isotopomers ([1-13C]-, [3-13C]-, [4-13C]-, and [6-13C]glucose) or isotopologues ([1,2-13C2]- and [1,2,3-13C3]glucose) for tracing, the ratio of lactate derived from glucose carbon 1, 2, 3 over 4, 5, 6 via glycolysis varied significantly, ranging from 1.6 (traced with [1,2-13C2]glucose) to 0.85 (traced with [6-13C]glucose), but the theoretical ratio should be 1. The odd results might be caused by intramolecular 13C, which may significantly affect lactate fragmentation under tandem mass spectrometry condition, leading to erroneous quantification. Indeed, the fragmentation efficiency of [U-13C]lactate, [2,3-13C]lactate, and [3-13C]lactate were 1.4, 1.5 and 1.2 folds higher than lactate, respectively, but [1-13C]lactate was similar to lactate, suggesting that carbon-13 at different positions could differentially influence lactate fragmentation. This observed phenomenon was inconsistent with the data based on theoretical calculation, according to which activation energies for all lactate isotopomers and isotopologues are nearly identical. The inconsistency suggested a need for further investigation. Our study suggests that calibration is required for quantifying metabolite isotopolugues and isotopomers.

Activated T cells have been shown to be able to recirculate into the thymus from the periphery. The present study was aimed to elucidate the functional consequences of thymic homing of activated T cells upon developing thymocytes and thymic epithelial cells (TEC). In the presence of activated T cells, especially CD4+ T cells, T cell development was found to be inhibited in thymic organ cultures with markedly reduced cellularity. Thymic transplantation demonstrated that the inhibitory effect was most likely due to a defective microenvironment. As the major component of the thymic stroma, the TEC compartment was severely disturbed after prolonged exposure to the activated T cells. In addition to reduced cell proliferation, TEC differentiation was heavily skewed to the mTEC lineage. Furthermore, we demonstrated that RANKL highly expressed by activated CD4+ T cells was primarily responsible for the detrimental effects. Presumably, excessive RANK signaling drove overproduction of mTECs and possibly exhaustion of epithelial progenitors, thereby facilitating the deterioration of the epithelial structures. These findings not only reveal a novel activity of activated T cells re-entering the thymus, but also provide a new perspective for understanding the mechanism underlying thymic involution.

CD4(+)CD8(+) “double positive” (DP) thymocytes differentiate into diverse αβ T cell sub-types using mechanistically distinct programs. For example, conventional αβ T cells develop from DP cells after partial-agonist T cell receptor (TCR) interactions with self-peptide/MHC, whereas unconventional αβ T cells, such as TCRαβ(+)CD8αα(+) intraepithelial lymphocytes (IELs), require full-agonist TCR interactions. Despite this, DP cells appear homogeneous, and it remains unclear how distinct TCR signalling instructs distinct developmental outcomes. Moreover, whether TCR signals at earlier stages of development, for example in CD4(−)CD8(−) double negative (DN) cells, impact on later fate decisions is presently unknown. Here, we assess four strains of mice that display altered TCR signal strength in DN cells, which correlates with altered generation of unconventional TCRαβ(+)CD8αα(+) IELs. FVB/n mice (compared to C57BL/6 animals) and mice with altered preTCRα (pTα) expression, both displayed weaker TCR signalling in DN cells, an inefficient DN-to-DP transition, and reduced contribution of TCRαβ(+)CD8αα(+) IELs to gut epithelium. Conversely, TCRαβ(+)CD8αα(+) IEL development was favoured in mice with increased TCR signal strength in DN cells. Collectively, these data suggest TCR signal strength in DN cells directly impacts on subsequent DP cell differentiation, fundamentally altering the potential of thymocyte progenitors to adopt conventional versus unconventional T cell fates.

The ordered migration of immature thymocytes through thymic microenvironments generates both adaptive MHC restricted αβT-cells and innate CD1d-restricted iNKT-cells. While several chemokine receptors and ligands control multiple stages of this process, their involvement during early thymocyte development often precludes direct analysis of potential roles during later developmental stages. For example, because of early lethality of CXCR4−/− mice, and stage-specific requirements for CXCR4 in thymus colonisation and pre-TCR mediated selection, its role in thymic positive selection is unclear. Here we have examined CXCR4-CXCL12 interactions during the maturation of CD4+CD8+ thymocytes, including downstream stages of iNKT and αβT-cell development. We show CXCL12 expression is a common feature of cortical thymic epithelial cells, indicating widespread availability throughout the cortex. Moreover, CXCR4 expression by CD4+CD8+ pre-selection thymocytes is progressively downregulated following both MHC and CD1d-restricted thymic selection events. However, using CD4Cre-mediated deletion to bypass its involvement in CD4−CD8− thymocyte development, we show CXCR4 is dispensable for the maintenance and intrathymic positioning of CD4+CD8+ thymocytes, and their ability to generate mature αβT-cells and CD1d-restricted iNKT-cells. Collectively, our data define dynamic changes in CXCR4 expression as a marker for intrathymic selection events, and show its role in T-cell development is restricted to pre-CD4+CD8+ stages.

Th1, Th2, Th9 and Th17 cells are conventional CD4+ effector T cells identified as secretors of prototypical cytokines IFNγ, IL4, IL9, and IL-17A respectively. Recently, populations of natural Th17 and Th1 cells (nTh17 and nTh1) with innate-like phenotype have been identified in the thymus that are distinct from conventional Th17 and Th1 cells. The absence of the Tec family kinase Interleukin-2 inducible T cell kinase (Itk) results in T cell immunodeficiency in mice and humans. Here we show that Itk negatively regulates the development of nTh1 cells that express IFNγ in a Tbet independent manner, and whose expansion can be enhanced by IL4. Furthermore, we show that robust induction of IL4 responses during Trichinella spiralis infection enhance the presence of nTh1 cells. We conclude T cell receptor signaling via Itk controls the development of natural Th1 cells, which are expanded by the presence of IL4.

Our previous study showed that prenatal caffeine exposure (PCE) could induce intrauterine growth retardation (IUGR) and glucocorticoid elevation in the fetus. Researchers suggested that IUGR is a risk factor for T helper cell (Th)1/Th2 deviation. However, whether PCE can induce these immune disorders and the underlying mechanisms of that induction remain unknown. This study aimed to observe the effects of PCE on the Th1/Th2 balance in offspring and further explore the developmental origin mechanisms from the perspective of glucocorticoid overexposure-induced thymocyte apoptosis. An IUGR model was established by caffeine administration from gestational day (GD) 9 to GD 18, and the offspring were immunized on postnatal day (PND) 42. The results show that maternal glucocorticoid overexposure increased fetal thymocyte apoptosis by activating both the Fas-mediated and the Bim-regulated apoptotic pathways. After birth, accelerated thymocyte apoptosis and Th1 suppression were also found in the PCE offspring at PND 14 and PND 49. Moreover, the PCE offspring showed immune disorders after immunization, manifesting as increased IgG1/IgG2a ratio and IL-4 production in the serum. In conclusion, PCE could induce fetal overexposure to maternal glucocorticoids and increase thymocyte apoptosis, which could persist into postnatal life and be implicated in Th1 inhibition and further immune disorders.

Thymocyte and T cell trafficking relies on signals initiated by G-protein coupled receptors. To address the importance of the G-proteins Gαi2 and Gαi3 in thymocyte and T cell function, we developed several mouse models. Gαi2 deficiency in hematopoietic progenitors led to a small thymus, a double negative (DN)1/DN2 thymocyte transition block, and an accumulation of mature single positive (SP) thymocytes. Loss at the double positive (DP) stage of thymocyte development caused an increase in mature cells within the thymus. In both models an abnormal distribution of memory and naïve CD4 T cells occurred, and peripheral CD4 and CD8 T cells had reduced chemoattractant responses. The loss of Gαi3 had no discernable impact, however the lack of both G-proteins commencing at the DP stage caused a severe T cell phenotype. These mice lacked a thymic medullary region, exhibited thymocyte retention, had a peripheral T cell deficiency, and lacked T cell chemoattractant responses. Yet a noteworthy population of CD4+PD-1+CXCR5+/− cells resided in the spleen of these mice likely due to a loss of regulatory T cell function. Our results delineate a role for Gαi2 in early thymocyte development and for Gαi2/3 in multiple aspects of T cell biology.

Chronic obstructive pulmonary disease (COPD) is one of the most common inflammatory diseases resulting from habitual smoking. Impaired clearance of apoptotic cell by airway macrophages contributes to lung inflammation. Milk fat globule-EGF factor 8 (MFG-E8), as a link between apoptotic cells and phagocytes, facilitates clearance of apoptotic cells and attenuates inflammation. We sought to investigate altered expression and potential role of MFG-E8 in COPD. In this study, apoptosis was increased and the level of MFG-E8 was decreased while HMGB1 expression was increased in lung tissues of CS-exposed mice. Compared with CS-exposed WT mice, more apoptotic cells were accumulated in lung tissues of CS-exposed MFG-E8 deficiency mice. Exposure of a range of macrophages to cigarette smoke extract (CSE) resulted in decreased MFG-E8 expression. Administration of rmMFG-E8 ameliorated phagocytic ability of RAW264.7 cells and suppressed inflammatory response induced by CS-exposure. 10% CSE stimulation suppressed Rac1 membrane localization in RAW264.7 cells which was restored by administration of rmMFG-E8. MFG-E8 deficiency diminished uptake of apoptotic thymocytes by peritoneal macrophages upon CSE exposure. Overall, the findings in current work provide a novel target for diagnosing and treating COPD.

The thymus is known to atrophy during infections; however, a systematic study of changes in thymocyte subpopulations has not been performed. This aspect was investigated, using multi-color flow cytometry, during oral infection of mice with Salmonella Typhimurium (S. Typhimurium). The major highlights are: First, a block in the developmental pathway of CD4−CD8− double negative (DN) thymocytes is observed. Second, CD4+CD8+ double positive (DP) thymocytes, mainly in the DP1 (CD5loCD3lo) and DP2 (CD5hiCD3int), but not DP3 (CD5intCD3hi), subsets are reduced. Third, single positive (SP) thymocytes are more resistant to depletion but their maturation is delayed, leading to accumulation of CD24hiCD3hi SP. Kinetic studies during infection demonstrated differences in sensitivity of thymic subpopulations: Immature single positive (ISP) > DP1, DP2 > DN3, DN4 > DN2 > CD4+ > CD8+. Upon infection, glucocorticoids (GC), inflammatory cytokines, e.g. Ifnγ, etc are induced, which enhance thymocyte death. Treatment with RU486, the GC receptor antagonist, increases the survival of most thymic subsets during infection. Studies with Ifnγ−/− mice demonstrated that endogenous Ifnγ produced during infection enhances the depletion of DN2-DN4 subsets, promotes the accumulation of DP3 and delays the maturation of SP thymocytes. The implications of these observations on host cellular responses during infections are discussed.

Many mathematical models have been published with the purpose of explaining aspects of T-cell development in the thymus. In this manuscript we adapted a four-compartment model of the thymus and used a range of mathematical approaches with the aim of explaining the dynamics of the four main thymocyte populations in the mouse thymus, from the emergence of the first fetal thymocyte until the death of the animal. At various pre-natal and post-natal stages we investigated experimentally the number and composition of thymocytes populations, their apoptosis and proliferation, along with data from literature, to create and validate the model. In our model the proliferation processes are characterized by decreasing proliferation rates, which allows us to model the natural involution of the thymus. The best results were obtained when different sets of parameters were used for the fetal and post-natal periods, suggesting that birth may induce a discontinuity in the modeled processes. Our model is able to model the development of both pre-natal and post-natal thymocyte populations. Also, our findings showed that the post-natal thymus is able to develop in the absence of the daily input of bone marrow progenitors, providing more evidence to support the autonomous development of the post-natal thymus.

The soluble γc protein (sγc) is a naturally occurring splice isoform of the γc cytokine receptor that is produced by activated T cells and inhibits γc cytokine signaling. Here we show that sγc expression is also highly upregulated in immature CD4+CD8+ thymocytes but then downregulated in mature thymocytes. These results indicate a developmentally controlled mechanism for sγc expression and suggest a potential role for sγc in regulating T cell development in the thymus. Indeed, sγc overexpression resulted in significantly reduced thymocyte numbers and diminished expansion of immature thymocytes, concordant to its role in suppressing signaling by IL-7, a critical γc cytokine in early thymopoiesis. Notably, sγc overexpression also impaired generation of iNKT cells, resulting in reduced iNKT cell percentages and numbers in the thymus. iNKT cell development requires IL-15, and we found that sγc interfered with IL-15 signaling to suppress iNKT cell generation in the thymus. Thus, sγc represents a new mechanism to control cytokine availability during T cell development that constrains mature T cell production and specifically iNKT cell generation in the thymus.

Matrix metalloproteinases (MMP) play critical roles in a variety of immune reactions by facilitating cell migration, and affect cell communication by processing both cytokines and cell surface receptors. Based on published data indicating that MMP-9 is upregulated upon T cell activation and also in the thymus upon the induction of negative selection, we investigated the contribution of MMP-9 into mouse T cell development and differentiation in the thymus. Our data suggest that MMP-9 deficiency does not result in major abnormalities in the development of any conventionally selected or agonist selected subsets and does not interfere with thymocyte apoptosis and clearance, and that MMP-9 expression is not induced in immature T cells at any stage of their thymic development. ispartof: Scientific Reports vol:6 issue:1 pages:29852- ispartof: location:England status: published

IL-27, as a pleiotropic cytokine, promotes the differentiation of naïve T cells to Th1, while suppressing Th2 and Th17 differentiation in the periphery. However, the role of IL-27 in the thymocyte development remains unknown. Here we showed that IL-27 was highly expressed in thymic plasmacytoid dendritic cells (pDCs) while its receptor expression was mainly detected in CD4+ single-positive (SP) thymocytes. Deletion of the p28 subunit in DCs resulted in a reduction of the most mature Qa-2+ subsets of CD4+ SP T cells. This defect was rescued by intrathymic administration of exogenous IL-27. In vitro differentiation assay further demonstrated that IL-27 alone was able to drive the maturation of the newly generated 6C10+CD69+CD4+ SP cells into Qa-2+ cells. Collectively, this study has revealed an important role of thymic DCs-derived IL-27 in the regulation of the phenotypic maturation of CD4+ SP thymocytes.

The transcription factor Runx1 has essential roles throughout hematopoiesis. Here, we demonstrate that Runx1 is critical for T cell maturation. Peripheral naïve CD4+ T cells from CD4-cre Runx1 cKO mice are phenotypically and functionally immature as shown by decreased production of TNF-α upon TCR stimulation. The loss of peripheral CD4+ T cells in CD4-cre Runx1 cKO mice is not due to defects in homeostasis or decreased expression of IL-7Rα, as transgenic expression of IL-7Rα does not rescue the loss of CD4+ T cells. Rather, immature Runx1-deficient CD4+ T cells are eliminated in the periphery by the activation and fixation of the classical complement pathway. In the thymus, there is a severe block in all aspects of intrathymic T cell maturation, although both positive and negative selection are unaltered. Thus, loss of Runx1 leads to the earliest characterized block in post-positive selection intrathymic maturation of CD4 T cells.

NF-κB inducing kinase (NIK) is a central component of the noncanonical NF-κB signaling pathway. Although NIK has been extensively studied for its function in the regulation of lymphoid organ development and B-cell maturation, the role of NIK in regulating T cell functions remains unclear and controversial. Using T cell-conditional NIK knockout mice, we here demonstrate that although NIK is dispensable for thymocyte development, it has a cell-intrinsic role in regulating the homeostasis and function of peripheral T cells. T cell-specific NIK ablation reduced the frequency of effector/memory-like T cells and impaired T cell responses to bacterial infection. The T cell-conditional NIK knockout mice were also defective in generation of inflammatory T cells and refractory to the induction of a T cell-dependent autoimmune disease, experimental autoimmune encephalomyelitis. Our data suggest a crucial role for NIK in mediating the generation of effector T cells and their recall responses to antigens. Together, these findings establish NIK as a cell-intrinsic mediator of T cell functions in both immune and autoimmune responses.

Immune activation and chronic inflammation are hallmark features of HIV infection causing T-cell depletion and cellular immune dysfunction in AIDS. Here, we addressed the issue whether HIV-1 Tat could affect T cell development and acute inflammatory response by generating a transgenic mouse expressing Tat in lymphoid tissue. Tat-Tg mice showed thymus atrophy and the maturation block from DN4 to DP thymic subpopulations, resulting in CD4+ and CD8+ T cells depletion in peripheral blood. In Tat-positive thymus, we observed the increased p65/NF-κB activity and deregulated expression of cytokines/chemokines and microRNA-181a-1, which are involved in T-lymphopoiesis. Upon LPS intraperitoneal injection, Tat-Tg mice developed an abnormal acute inflammatory response, which was characterized by enhanced lethality and production of inflammatory cytokines. Based on these findings, Tat-Tg mouse could represent an animal model for testing adjunctive therapies of HIV-1-associated inflammation and immune deregulation.