Immature Thymocytes Research Articles

Tracking migration during human T cell development.

Specialized microenvironments within the thymus are comprised of unique cell types with distinct roles in directing the development of a diverse, functional, and self-tolerant T cell repertoire. As they differentiate, thymocytes transit through a number of developmental intermediates that are associated with unique localization and migration patterns. For example, during one particular developmental transition, immature thymocytes more than double in speed as they become mature T cells that are among the fastest cells in the body. This transition is associated with dramatic changes in the expression of chemokine receptors and their antagonists, cell adhesion molecules, and cytoskeletal components to direct the maturing thymocyte population from the cortex to medulla. Here we discuss the dynamic changes in behavior that occur throughout thymocyte development, and provide an overview of the cell-intrinsic and extrinsic mechanisms that regulate human thymocyte migration.

Neonatal surgical castration of male pigs reduces thymic growth but has moderate consequences on thymocytes1

Thymus integrates numerous signals from the neuroendocrine-immune system, including sex steroids, glucocorticoids, and catecholamines. Neonatal surgical castration, commonly practiced in pig husbandry, modifies thymic hormonal environment, for example, sex steroids and probably glucocorticoids and catecholamines, which are important modulators of thymic function. This study aimed at investigating, in pubescent male pigs, the consequences of neonatal suppression of testicular hormones on thymic T cell differentiation and hormonal control of thymocyte proliferation. A total of 34 male pigs were allocated to 2 experimental groups: control (CT) intact males and males surgically castrated (SC) at 5 or 6 d of age. At slaughter, thymus was weighed and thymic samples were collected to determine fat content and distribution of thymocyte subsets by identification of CD1, CD4, CD8, and γδ T cell receptor (TCR) cell surface markers and to measure thymocyte proliferation in presence of cortisol, norepinephrine, and sex steroids. Results showed that absolute and relative thymus weights were greater (P < 0.01 and P < 0.01, respectively) whereas thymic fat content was less (P < 0.01) in CT than in SC pigs. Surgical castration did not change the frequency of CD1+ immature thymocytes. The proportion of γδ T cells tended to be greater in CT than in SC pigs (P < 0.1) but the proportions of CD4+, CD8+, and CD4+CD8+ thymocytes were similar in both groups (P > 0.1) indicating that the Tαβ lineage was not influenced by early castration. Proliferation of thymocytes in response to concanavalin A (ConA) was greater in SC than in CT pigs (P < 0.05). Cortisol and norepinephrine decreased the ConA-induced proliferation in CT and SC pigs (P < 0.05). In addition, proliferation of thymocytes was less inhibited by norepinephrine in SC than in CT males (P < 0.05). The greatest concentration of testosterone (25 ng/mL) increased (SC males, P < 0.05) or tended to increase (CT males, P < 0.1) the proliferative responsiveness to ConA but the lowest dose (2.5 ng/mL) and the greatest dose of testosterone combined with estradiol had no significant effect (P > 0.1). Overall, our data show little effect of neonatal castration on thymocyte differentiation as well as of sex hormones on thymocyte proliferation. However, thymic cells seem to be more sensitive to the inhibitory influence of norepinephrine in CT than in CS pigs. The significance of such difference for animal health remains to be explored.

Thymic Atrophy during Infection as a Host Response to Avoid Tolerance to Persistent Pathogens

The immune system consists in part of a functionally competent T -cell repertoire that is reactive to foreign antigens but tolerant to self -antigens. The repertoire of T cells is primarily formed in the thymusthrough positive and negative selection of developing thymocytes that are critical for establishing central tolerance. One of the features of the thymus is to sense stress hormones produced in pathophysiological conditions. Increased levels of these hormones are associated with infections and are able to induce thymic atrophy. We have shown that in acuteTrypanosoma cruziinfections, the atrophic thymus is a consequence of increased thymocyte apoptosis and premature export of immature thymocytes to secondary lymph nodes. Thisatrophy does not necessarily result in dysfunction of the thymus since the organ micro architecture is preserved and maintains negative selection, thus avoiding the development of tolerance to the pathogen during the establishment of protective immunity. However, in chronic infections, the dissemination of invading pathogens able to target the thymus i

Toward a Molecular Understanding of Adaptive Immunity: A Chronology, Part III.

Early reports on T cell antigen receptor (TCR) signaling uncovered a rapid increase in intracellular calcium concentration and the activation of calcium-dependent protein kinase as necessary for T cell activation. Cytolytic T cell clones were instrumental in the discovery of intracellular cytolytic granules, and the isolation of the perforin and granzyme molecules as the molecular effectors of cell-mediated lysis of target cells via apoptosis. Cytolytic T cell clones and TCR cDNA clones were also instrumental for the generation of TCR transgenic animals, which provided definitive evidence for negative selection of self-reactive immature thymocytes. In addition, studies of TCR complex signaling of immature thymocytes compared with mature T cells were consistent with the interpretation that negative selection occurs as a consequence of the incapacity of immature cells to produce IL-2, resulting in cytokine deprivation apoptosis. By comparison, taking advantage of cloned TCRs derived from T cell clones reactive with male-specific molecules, using TCR transgenic mice it was possible to document positive selection of female thymocytes when the male-specific molecules were absent. Focusing on the molecular mechanisms of T cell “help” for the generation of antibody-forming cells following the path opened by the elucidation of the IL-2 molecule, several groups were successful in the identification, isolation, and characterization of three new interleukin molecules (IL-4, IL-5, and IL-6) that promote the proliferation and differentiation of B cells. In addition, the identification of a B cell surface molecule (CD40) that augmented B cell antigen receptor-stimulated proliferation and differentiation led to the discovery of a T cell activation surface molecule that proved to be the CD40-ligand, thus finally providing a molecular explanation for “linked or cognate” recognition when T cells and B cells interact physically. Accordingly, the decade after the generation of the first T cell clones saw the elucidation of the molecular mechanisms of T cell cytotoxicity and T cell help, thereby expanding the number of molecules responsible for adaptive T cell immunity.

Diacylglycerol metabolism attenuates T-cell receptor signaling and alters thymocyte differentiation

Diacylglycerol (DAG) metabolism has a critical function in Ras-regulated functions in mature T cells, but causal data linking defects in DAG-based signals with altered thymus development are missing. To study the effect of increased DAG metabolism in T-cell development, we engineered a membrane-targeted constitutive active version of DAG kinase-α (DGKα). We show that transgenic expression of constitutive active DGK leads to developmental defects in T cells, with a marked accumulation of immature CD8 thymocytes and a reduction in positive selected populations. These alterations are reflected in the periphery by a CD4/CD8 cell imbalance and general T-cell lymphopenia. The results link DAG metabolism to T-cell homeostasis, and show that correctly controlled generation and consumption of this lipid at the plasma membrane ensure T-cell passage through quality-control checkpoints during differentiation.

Redox balance of mouse medullary CD4 single‐positive thymocytes

After positive selection, the newly differentiated single-positive (SP) thymocytes undergo negative selection to eliminate autoreactive T cells, functional maturation to acquire immunocompetence and egress capability. To investigate whether the intracellular reduction/oxidation (redox) balance has an important role on SP maturation, the levels of intracellular reactive oxygen species (ROS) and the expression of proteins that regulate ROS were compared among the four subsets of mouse TCRαβ(+)CD4(+)CD8(-) thymocytes (SP1-SP4) that represent sequential stages of SP differentiation program. A gradual increase of ROS and a gradual decrease of thioredoxin were revealed along the SP maturation process. The high ROS level at the mature SP stage did not result from a specific enrichment at this stage of natural regulatory T cells and SP thymocytes undergoing negative selection (Helios positive). An increase of ROS in the most mature SP4 cells resulted in enhanced cytokine production upon stimulation, whereas an early increase of ROS in the immature SP1 thymocytes resulted in enhanced apoptosis. Aire(-/-) mice that have defects in negative selection and a developmental blockage at the SP3-SP4 transition showed significantly less ROS in SP3 thymocytes. Thymic epithelial cells that have been shown to promote SP maturation in vitro also increased the ROS level of SP thymocytes. These results suggest that ROS may be involved in promoting the functional maturation of CD4(+) SPs and thymic medullary microenvironment contributes to the pro-oxidant shift of SP thymocytes.

Keratin 8 Is Required for the Maintenance of Architectural Structure in Thymus Epithelium

Keratins (Ks), the intermediate filament (IF) proteins of epithelia, are coordinately expressed as pairs in a cell-lineage and differentiation manner. Cortical thymic epithelial cells (cTECs) predominantly express the simple epithelium keratin 8/18 (K8/K18) pair, whereas medullary thymic epithelial cells (mTECs) express the stratified epithelium K5/K14 pair, with TECs exhibiting K5 and K8 at the cortico-medullary junction in mature thymus. In the work reported here, we used wild-type (WT) and K8-knockout (K8-null) mice to address the contribution of K8/K18 IFs in the maintenance of the thymic epithelial structure. K8-null thymus maintained the differential cell segregation at the cortex versus the medulla observed in WT thymus, and the distribution of immature thymocytes at the cortex. The K8/K18 loss did not affect thymocyte development. However, it massively perturbed the TEC morphology both at the cortex and the medulla, along with a prominent depletion of cTECs. Such tissue alterations coincided with an increase in apoptosis and a reduced expression of Albatross (Fas-binding factor-1), also known for its capacity to bind K8/18 IFs. In addition, the K8/K18 loss affected the distribution of K5/K14-positive mTECs, but not their differentiation status. Together, the results indicate that K8/K18 IFs constitute key promoters of the thymic epithelium integrity.

Defective thymic progenitor development and mature T‐cell responses in a mouse model for Down syndrome

In addition to archetypal cognitive defects, Down syndrome (DS) is characterized by altered lymphocyte development and function, including premature thymic involution and increased incidence of infections. However, the potential mechanisms for these changes have not been fully elucidated. The current study used the Ts65Dn mouse model of DS to assess deficiencies in T-cell development and possible molecular alterations. Ts65Dn mice exhibited premature thymic involution and a threefold to fourfold decrease in the number and proportion of immature, double-negative thymocyte progenitors. In addition, there were twofold fewer double-positive and CD4 single-positive thymocytes in Ts65Dn thymuses. Reflecting this deficient thymic function, there were fewer naive T cells in the spleen and polyclonal stimulation of peripheral T cells exhibited a marked reduction in proliferation, suggesting a senescent phenotype. In contrast, B-cell progenitors were unchanged in the bone marrow of Ts65Dn mice, but in the spleen, there were decreased transitional and follicular B cells and these cells proliferated less upon antigen receptor stimulus but not in response to lipopolysaccharide. As a potential mechanism for diminished thymic function, immature thymocyte populations expressed diminished levels of the cytokine receptor interleukin-7Rα, which was associated with decreased proliferation and increased apoptosis. Increased oxidative stress and inhibition of the Notch pathway were identified as possible mediators of decreased interleukin-7Rα expression in Ts65Dn mice. The data suggest that immature thymocyte defects underlie immune dysfunction in DS and that increased oxidative stress and reduced cytokine signalling may alter lymphocyte development in Ts65Dn mice.

Cell of origin in radiation‐induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele

Bcl11b is a haploinsufficient tumor suppressor, mutations or deletion of which has been found in 10-16% of T-cell acute lymphoblastic leukemias. Bcl11b(KO) (/+) heterozygous mice are susceptible to thymic lymphomas, a model of T-cell acute lymphoblastic leukemia, when γ-irradiated, and irradiated Bcl11b(KO) (/+) mice generate clonally expanding or premalignant thymocytes before thymic lymphoma development. Cells with radiation-induced DNA damages are assumed to be the cells of origin in tumors; however, which thymocyte is the tumor cell origin remains obscure. In this study we generated Bcl11b(flox/+) ;Lck-Cre and Bcl11b(flox/+) ;CD4-Cre mice; in the former, loss of one Bcl11b allele occurs in thymocytes at the immature CD4(-) CD8(-) stage, whereas in the latter the loss occurs in the more differentiated CD4(+) CD8(+) double-positive stage. We examined clonal expansion and differentiation of thymocytes in mice 60days after 3Gy γ-irradiation. Half (9/18) of the thymuses in the Bcl11b(flox/+) ;Lck-Cre group showed limited rearrangement sites at the T-cell receptor-β (TCRβ) locus, indicating clonal cell expansion, but none in the Bcl11b(flox/+) ;CD4-Cre group did. This indicates that the origin of the premalignant thymocytes is not in double-positive cells but immature thymocytes. Interestingly, those premalignant thymocytes underwent rearrangement at various different sites of the TCRα locus and the majority showed a higher expression of TCRβ and CD8, and more differentiated phenotypes. This suggests the existence of a subpopulation of immature cells within the premalignant cells that is capable of proliferating and continuously producing differentiated thymocytes.

Identification of TMEM131L as a Novel Regulator of Thymocyte Proliferation in Humans

In this study, we identify transmembrane protein 131-like (TMEM131L) as a novel regulator of thymocyte proliferation and demonstrate that it corresponds to a not as yet reported inhibitor of Wnt signaling. Short hairpin RNA-mediated silencing of TMEM131L in human CD34(+) hematopoietic progenitors, which were then grafted in NOD-SCID/IL-2rγ(null) mice, resulted in both thymocyte hyperproliferation and multiple pre- and post-β-selection intrathymic developmental defects. Consistent with deregulated Wnt signaling, TMEM131L-deficient thymocytes expressed Wnt target genes at abnormally high levels, and they displayed both constitutive phosphorylation of Wnt coreceptor LRP6 and β-catenin intranuclear accumulation. Using T cell factor reporter assays, we found that membrane-associated TMEM131L inhibited canonical Wnt/β-catenin signaling at the LRP6 coreceptor level. Whereas membrane-associated TMEM131L did not affect LRP6 expression under basal conditions, it triggered lysosome-dependent degradation of its active phosphorylated form following Wnt activation. Genetic mapping showed that phosphorylated LRP6 degradation did not depend on TMEM131L cytoplasmic part but rather on a conserved extracellular domain proximal to the membrane. Collectively, these data indicate that, during thymopoiesis, stage-specific surface translocation of TMEM131L may regulate immature single-positive thymocyte proliferation arrest by acting through mixed Wnt-dependent and -independent mechanisms.

Gfi1 couples maturation of DP thymocytes and generation of nTreg cells (P4418)

Abstract Double-positive (DP) thymocytes respond to intrathymic TCR signaling by undergoing positive selection and lineage differentiation. Concomitant with these well-characterized events is the acquisition of mature T-cell gene-expression programs, notably the upregulation of effector molecules that are essential for their proper survival and trafficking, a process that is much less understood. nTreg cells also develop from immature thymocytes, but how their development is linked to the thymocyte maturation remains unclear. Here we report that Gfi1 has a central role in coupling DP cell maturation and thymic generation of nTreg cells. Using a conditional Gfi1 deletion system, we observed that specific ablation of Gfi1 in DP cells led to premature expression of mature effector genes and transcription factors, thereby facilitating developmental maturation. Associated with this “expedited” maturation of DP cells was expansion of nTreg cells. Mechanistically, we identified that elevated IL-2 signaling in the absence of Gfi1 drove the expansion of nTreg cells via a non-cell-autonomous mechanism. Finally, we provide evidence that deficiency of Gfi1 dampened anti-tumor immunity. Our results establish a Gfi1-dependent mechanism that actively maintains DP cell fate for proper developmental maturation and generation of nTreg cells.

Introduction of Exogenous T-cell Receptors Into Human Hematopoietic Progenitors Results in Exclusion of Endogenous T-cell Receptor Expression

Current tumor immunotherapy approaches include the genetic modification of peripheral T cells to express tumor antigen-specific T-cell receptors (TCRs). The approach, tested in melanoma, has led to some limited success of tumor regression in patients. Yet, the introduction of exogenous TCRs into mature T cells entails an underlying risk; the generation of autoreactive clones due to potential TCR mispairing, and the lack of effective negative selection, as these peripheral cells do not undergo thymic selection following introduction of the exogenous TCR. We have successfully generated MART-1-specific CD8 T cells from genetically modified human hematopoietic stem cells (hHSC) in a humanized mouse model. The advantages of this approach include a long-term source of antigen specific T cells and proper T-cell selection due to thymopoiesis following expression of the TCR. In this report, we examine the molecular processes occurring on endogenous TCR expression and demonstrate that this approach results in exclusive cell surface expression of the newly introduced TCR, and the exclusion of endogenous TCR cell surface expression. This suggests that this stem cell based approach can provide a potentially safer approach for anticancer immunotherapy due to the involvement of thymic selection.

The role of pTα/pre-TCR in susceptibility to autoimmunity (P1464)

Abstract Autoimmune diseases result from a breakdown in self-tolerance, with a major contribution of pathogenic self-reactive T cells. Individuals with a particular autoimmune disease are often at risk of developing additional autoimmunity directed towards other target tissues. These observations suggest that there might be a mechanism leading to a general defect in self-tolerance, resulting in increased susceptibility to autoimmune disease. Tolerance towards self is initially established in the thymus during the process of thymic selection. Developing thymocytes are first immature CD4 and CD8 double negative thymocytes. Cells with a productive TCRβ gene rearrangement will express a pre-TCR/CD3 complex on the cell surface by combining a TCRβ chain with the pTα chain. Signaling through the pre-TCR, β-selection, plays a crucial role in the generation αβT cells, by enabling further maturation to the CD4 and CD8 double positive stage. The crucial role of pTα at β-selection together with the genetic localization of the ptcra gene and the fact that variations in pTα will affect all developing T cells, suggests that pTα could be a common factor leading to a general defect in self-tolerance. We have discovered differences in the isoform expression pattern and amino acid sequence of pTa between NOD and B6 mice. These findings are consistent with the hypothesis that alterations in the pre-TCR might affect selection, giving a modified TCR repertoire and increased susceptibility to autoimmunity.

Abstract 3598: Cell of origin in radiation-induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele.

Abstract Mouse thymic lymphoma (TL) has been used for study of radiation carcinogenesis because of its high incidence. The lymphoma or leukemia is a mouse model of human T-cell acute lymphoblastic leukemias (T-ALL), which is a malignant clonal expansion of thymocytes that accounts for about 15% of ALL cases. Ionizing radiation can damage DNA, and the cells with damaged DNA are assumed to be the cells of origin in tumors. However, which cell in the thymus or the bone marrow leads to TL remains obscure. Bcl11b gene encodes zinc-finger transcription proteins, mutations or deletion of which has been found in 10-16% of T-ALL. Bcl11bKO/+ heterozygous mice are susceptible to thymic lymphomas when γ-irradiated, and irradiated Bcl11bKO/+ mice generate clonally expanding or premalignant thymocytes within atrophic thymus. Resembling clonal expansion of thymocytes was reported in the T-ALL model overexpressing LMO2 oncogene. Interestingly, LMO2-induced clonally expanding thymocytes persist long-term and comprise cells with the stem cell-like self-renewal property that function for the bone marrow stem cells before TL development. In this study, we developed Bcl11bflox/+;Lck-Cre and Bcl11bflox/+;CD4-Cre mice, in which loss of one Bcl11b allele occurs in thymocytes at the immature CD4−CD8− stage without expression of CD4 and CD8 cell surface markers and at the CD4+CD8+ double-positive (DP) stage, respectively. Those mice were subjected to γ-irradiation of 3Gy at 8 weeks of age and their thymocytes were characterized of clonal expansion, differentiation and cell number. The clonal expansion was observed in only the former mice, suggesting the origin of the premaligant thymocytes not in the DP cells, the cell type seen in a majority of TL. The clonally expanded thymocytes possessed common rearrangement sites at the TCRβ locus but they underwent rearrangement at various different sites at the TCRα locus, and their majority showed mature phenotypes, a higher expression of TCRβ and the CD8 expression. This suggests the presence of a subpopulation of immature thymocytes that is capable to rearrange DNA at the TCRα locus in DP cells to continuously produce further differentiated thymocytes. We also found the decrease in thymocyte number in irradiated Bcl11bflox/+;Lck-Cre mice. This probably reflects the reduced proliferation of bone marrow-derived progenitors, and this reduction may weaken the competition in niche with premaligant thymocytes and help for them to survive and proliferate. Citation Format: Ryo Kominami, Rieka Go, Satoshi Hirose, Yukio Mishima. Cell of origin in radiation-induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3598. doi:10.1158/1538-7445.AM2013-3598

Immunomodulatory effects of black cohosh (Actaea racemosa) extract in female B6C3F1/N mice

Black cohosh extracts (BCE; Actaea racemosa) are being used worldwide as an alternative to hormone replacement therapy for the management of menstrual and menopausal symptoms, yet the effects of BCE on the immune system are largely unknown. Female B6C3F1/N mice were treated daily with BCE (0, 62.5, 125, 250, 500, or 1000mg/kg) for 28 days by oral gavage. Liver weights were significantly increased (26–32%) at the 1000mg/kg dose. Dose-related increases in mean corpuscular volume and mean corpuscular hemoglobin were observed. Decreasing trends were observed in all thymic T cell populations, with the most notable dose-responsive effects on immature thymocytes. In the spleen, dose-related decreases were observed in all cell phenotypes evaluated, reaching the level of statistical significance at the 1000mg/kg BCE dose. Splenic natural killer (NK) cell numbers were significantly decreased at all BCE doses, with the exception of absolute NK numbers at the 125mg/kg dose. No effects were observed on T-dependent antibody responses of the humoral immune system, including the antibody-forming cell response to sheep erythrocytes (sRBC) and IgM antibody levels to both sRBC and keyhole limpet hemocyanin. Cytotoxic T cell (TCTL) activity was increased, as was the mixed leukocyte response in one of two studies. Anti-CD3 mediated proliferation and the delayed-type hypersensitivity response were unaffected. No effects were observed on innate immunity or on bone marrow cellularity and colony-forming units. Overall, BCE exposure in B6C3F1/N mice for 28 days at doses up to 1000mg/kg had minimal immune effects, with the exception of an increased TCTL response.

Dosage-dependent tumor suppression by histone deacetylases 1 and 2 through regulation of c-Myc collaborating genes and p53 function

AbstractHistone deacetylases (HDACs) are epigenetic erasers of lysine-acetyl marks. Inhibition of HDACs using small molecule inhibitors (HDACi) is a potential strategy in the treatment of various diseases and is approved for treating hematological malignancies. Harnessing the therapeutic potential of HDACi requires knowledge of HDAC-function in vivo. Here, we generated a thymocyte-specific gradient of HDAC-activity using compound conditional knockout mice for Hdac1 and Hdac2. Unexpectedly, gradual loss of HDAC-activity engendered a dosage-dependent accumulation of immature thymocytes and correlated with the incidence and latency of monoclonal lymphoblastic thymic lymphomas. Strikingly, complete ablation of Hdac1 and Hdac2 abrogated lymphomagenesis due to a block in early thymic development. Genomic, biochemical and functional analyses of pre-leukemic thymocytes and tumors revealed a critical role for Hdac1/Hdac2-governed HDAC-activity in regulating a p53-dependent barrier to constrain Myc-overexpressing thymocytes from progressing into lymphomas by regulating Myc-collaborating genes. One Myc-collaborating and p53-suppressing gene, Jdp2, was derepressed in an Hdac1/2-dependent manner and critical for the survival of Jdp2-overexpressing lymphoma cells. Although reduced HDAC-activity facilitates oncogenic transformation in normal cells, resulting tumor cells remain highly dependent on HDAC-activity, indicating that a critical level of Hdac1 and Hdac2 governed HDAC-activity is required for tumor maintenance.

Porcine γδ T Lymphocytes Can Be Categorized into Two Functionally and Developmentally Distinct Subsets according to Expression of CD2 and Level of TCR

Porcine γδ T cells have two levels of TCRγδ expression. Whereas TCRγδ(med) cells are mostly CD2(+)CD8(-) and CD2(+)CD8(+), TCRγδ(hi) cells are highly enriched for CD2(-)CD8(-). This distribution is independent of bacterial colonization and it is already established in the thymus prior to export of γδ cells to the periphery. Sorting and cultivation experiments revealed that CD2(-)CD8(-) γδ cells are unable to acquire CD2 and CD8, whereas CD2(+) subsets can gain or loose CD8. There is also differential susceptibility for proliferation between CD2(+) and CD2(-) γδ cells. Although CD2(-)CD8(-) almost do not proliferate, proliferation of CD2(+)CD8(-) and CD2(+)CD8(+) is substantial. Population of CD2(-) γδ cells is also absent in CD1(+) immature thymocytes. Additionally, subpopulations of CD2(+) and CD2(-) γδ cells in the thymus differ in expression of auxiliary surface molecules such as CD25, CD45RA/RC, and MHC class II. Moreover, TCRγδ(hi) cells can generate TCRγδ(med) cells but never the opposite. The only exception is the thymus, where a few TCRγδ(med) cells can be induced to TCRγδ(hi) but only under IL-2 influence. The repertoire of TCRδ is polyclonal in all subsets, indicating that there is the same extent of diversification and equal capability of immune responses. Results collectively indicate that CD2 expression determines two lineages of γδ cells that differ in many aspects. Because CD2(-) γδ cells are missing in the blood of humans and mice but are obvious in other members of γδ-high species such as ruminants and birds, our findings support the idea that circulating CD2(-) γδ T cells are a specific lineage.

Trans-sialidase from Trypanosoma cruzi enhances the adhesion properties and fibronectin-driven migration of thymocytes

In experimental Trypanosoma cruzi infections, severe thymic atrophy leads to release of activated CD4+CD8+ double-positive (DP) T cells to the periphery. In humans, activated DP T cells are found in the blood in association with severe cardiac forms of human chronic Chagas disease. The mechanisms underlying the premature thymocyte release during the chagasic thymic atrophy remain elusive. We tested whether the migratory properties of intrathymic thymocytes are modulated by the parasite trans-sialidase (TS). We found that TS affected the dynamics of thymocytes undergoing intrathymic maturation, and these changes were accompanied by an increase in the number of recent DP thymic emigrants in the peripheral lymphoid organs. We demonstrated that increased percentages of blood DP T cell subsets were associated with augmented antibody titers against TS in chagasic patients with chronic cardiomyopathy. In vitro studies showed that TS was able to activate the MAPK pathway and actin filament mobilization in thymocytes. These effects were correlated with its ability to modulate the adhesion of thymocytes to thymic epithelial cells and their migration toward extracellular matrix. These findings point to effects of TS that could influence the escape of immature thymocytes in Chagas disease.

Effect of a probiotic fermented milk on the thymus in Balb/c mice under non-severe protein–energy malnutrition

Protein–energy malnutrition (PEM) causes a significant impairment of the immune system, the thymus being one of the most affected organs. It has been demonstrated that the administration of probiotic fermented milk (PFM) recovered the intestinal barrier, histological alterations and mucosal and systemic immune functions in a non-severe malnutrition model using BALB/c mice. The aim of the present study was to evaluate, in the same model of malnutrition, the effect of a PFM added to a re-nutrition diet on the recovery of the thymus, analysing histological and functional alterations caused by malnutrition. Mice were undernourished and divided into three groups according to the dietary supplement received during re-nutrition: milk, PFM or its bacterial-free supernatant (BFS). They were compared with well-nourished and malnourished mice. PFM was the most effective re-nutrition supplement to improve the histology of the thymus, decreasing cellular apoptosis in this organ and recovering the percentage of CD4þ/CD82 single-positive thymocytes. Immature doublepositive thymocytes were increased in the malnourished control (MC). The production of different cytokines in the thymus was increased in mice given PFM, compared with the mice that received other dietary supplements and MC. Mice given the BFS presented an improvement in the thymus similar to those that received milk. We demonstrated the importance of the whole PFM supplementation on the histological and functional recovery of the thymus in a non-severe PEM model.

RET/GFRα Signals Are Dispensable for Thymic T Cell Development In Vivo

Identification of thymocyte regulators is a central issue in T cell biology. Interestingly, growing evidence indicates that common key molecules control neuronal and immune cell functions. The neurotrophic factor receptor RET mediates critical functions in foetal hematopoietic subsets, thus raising the possibility that RET-related molecules may also control T cell development. We show that Ret, Gfra1 and Gfra2 are abundantly expressed by foetal and adult immature DN thymocytes. Despite the developmentally regulated expression of these genes, analysis of foetal thymi from Gfra1, Gfra2 or Ret deficient embryos revealed that these molecules are dispensable for foetal T cell development. Furthermore, analysis of RET gain of function and Ret conditional knockout mice showed that RET is also unnecessary for adult thymopoiesis. Finally, competitive thymic reconstitution assays indicated that Ret deficient thymocytes maintained their differentiation fitness even in stringent developmental conditions. Thus, our data demonstrate that RET/GFRα signals are dispensable for thymic T cell development in vivo, indicating that pharmacological targeting of RET signalling in tumours is not likely to result in T cell production failure.