Immature CD4 CD8 Research Articles

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.

Transient Differentiation-State Plasticity during Initiation of Pediatric Acute Lymphoblastic Leukemia

Introduction Leukemia is characterized by oncogenic lesions that result in a block of differentiation while at the same time intratumor phenotypic plasticity is retained. It is unclear how these two phenomena arise during leukemogenesis in humans. Here we characterized T-cell acute lymphoblastic leukemia (T-ALL), as a model disease to investigate the coherence between leukemia initiating cells (LICs), developmental arrest, and phenotypic plasticity. Methods We characterized 31 primary T-ALL samples for differentiation state heterogeneity using multi parameter flow cytometry. Subsequently, for 5 patients, phenotypic subpopulations were single cell sorted. As leukemic cells do not expand in vitro primary template-directed amplification (PTA) was used to amplify the genome of single blasts to obtain enough DNA for WGS analysis with nucleotide resolution. Shared and unique mutations were used to construct retrospective lineage trees and assess the hierarchy of phenotypically diverse blasts. Results To assess phenotypic differentiation state heterogeneity in T-ALL, we designed a 17-parameter flow cytometry panel to discriminate T-lymphoid developmental cell populations. We characterized 31 samples (median blast count 98% (IQR = 94 - 99)), and defined immunophenotypic subpopulations as populations with a size of >10% of the total CD7+ population. Differentiation state heterogeneity (> 2 subpopulations) was observed in 26 out of the 31 T-ALL patients (83.9%). Interestingly, the most immature CD4-CD8-CD3- “DN-like” differentiation state was found in all samples, suggesting that developmental hierarchy is maintained. We then questioned whether genetic determinants were driving the phenotypic diversity we observed. Therefore 5 T-ALL patients harboring translocations of oncogenes commonly seen in T-ALL were selected, namely, TLX1 (n=1) , LMO2 (n=2) , TAL1 (n=2) and TLX3 (n=1) . For each patient cells of each differentiation state were single cell and bulk sorted for single cell WGS and bulk WGS, respectively. Structural variant analysis confirmed presence of the oncogenic translocation in all single blasts, validating their (pre-)leukemic origin. Moreover, driver analysis showed that type B mutations in e.g., NOTCH1 and PHF6 were shared among all blasts. Interestingly, there were no potential genetic drivers found that were unique to a subpopulation. Neutral passenger mutations were therefore used to construct phylogenetic trees. Herein, blasts with similar phenotypic differentiation states were genetically closer related than blasts of different phenotypes, revealing the heritability of phenotypes (Fig. 1A). The biased distribution of immunophenotypes across the different branches of the phylogenetic tree was then confirmed in bulk WGS samples of sorted phenotypic populations. Suggesting that switching between phenotypes is not a frequent process. Next, the plasticity of T-ALL was assessed by identifying the phenotypic cell state of the LIC. We assessed V(D)J recombination per single blast and identified monoclonal V(D)J recombination for most of the blasts. Moreover, in TAL1 T-ALL we observed monoclonal TRG, TRB and TRA translocation. This suggested that the LIC had undergone complete V(D)J recombination and was a more mature T-cell progenitor. However, blasts with an immature CD4-CD8- (DN) phenotype had undergone the same V(D)J recombination suggesting phenotypic de-differentiation of the LIC. Additionally, in TLX1 T-ALL, the blasts with the more mature CD4+CD8+ double positive (DP) phenotype had monoclonal TRG and TRB gene recombination, but polyclonal TRA recombination, whereas cells with a more immature DN phenotype had no TRA recombination (Fig. 1B). This indicated an immature T-cell progenitor as the LIC and continued differentiation after leukemia initiation resulting in DP cells with unique TRA recombination. Conclusion In conclusion, we show immunophenotypic differentiation state heterogeneity in diagnostic primary T-ALL samples. By coupling phenotypic data with novel single cell whole genome sequencing techniques, we identified heritability of phenotypic cell states. Moreover, we reveal the ability of the LIC to differentiate and de-differentiate after leukemia initiation. Taken together our results demonstrate a transient period of plasticity during leukemia initiation where phenotypic switches appear unidirectional.

Early expression of mature αβ TCR in CD4−CD8− T cell progenitors enables MHC to drive development of T-ALL bearing NOTCH mutations

During normal T cell development in mouse and human, a low-frequency population of immature CD4-CD8- double-negative (DN) thymocytes expresses early, mature αβ T cell antigen receptor (TCR). We report that these early αβ TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αβ T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αβ TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors invivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.

Post-Irradiation Thymic Regeneration in B6C3F1 Mice Is Age Dependent and Modulated by Activation of the PI3K-AKT-mTOR Pathway.

Simple SummaryBecause children have a long life expectancy relative to adults and their tissues and organs are growing and developing rapidly, the risk of radiation carcinogenesis for children is considered higher than that for adults. However, the underlying mechanism(s) is unclear. To uncover the mechanism, we previously revealed that principal causative genes in mouse thymic lymphomas arising in irradiated infants or adults as Pten or Ikzf1, respectively, suggesting that cells with mutation in these genes might be the origin of lymphomas arising after irradiation depending on age at exposure. Here, we clarified the age-dependent differences in thymus-cell dynamics in mice during the initial post-irradiation period. Our results demonstrate that the dynamics of thymocytes during the post-irradiation period depends on the age at exposure. For irradiated infants in particular, the number of proliferating cells increase dramatically, and this correlate with activation of the PI3K-AKT-mTOR pathway. Thus, we conclude that the PI3K-AKT-mTOR pathway in infants contributed, at least in part, to thymus-cell dynamics through the modification of cell proliferation and survival after irradiation, which may be associated with the risk of Pten mutation-associated thymic lymphoma.The risk of radiation-induced carcinogenesis depends on age at exposure. We previously reported principal causative genes in lymphomas arising after infant or adult exposure to 4-fractionated irradiation as Pten or Ikzf1, respectively, suggesting that cells with mutation in these genes might be the origin of lymphomas arising after irradiation depending on age at exposure. Here, we clarified the age-dependent differences in thymus-cell dynamics in mice during the initial post-irradiation period. The thymocyte number initially decreased, followed by two regeneration phases. During the first regeneration, the proportion of phosphorylated-AKT-positive (p-AKT+) cells in cell-cycle phases S+G2/M of immature CD4−CD8− and CD4+CD8+ thymocytes and in phases G0/G1 of mature CD4+CD8− and CD4−CD8+ thymocytes was significantly greater in irradiated infants than in irradiated adults. During the second regeneration, the proportion of p-AKT+ thymocytes in phases G0/G1 increased in each of the three populations other than CD4−CD8− thymocytes more so than during the first regeneration. Finally, PI3K-AKT-mTOR signaling in infants contributed, at least in part, to biphasic thymic regeneration through the modification of cell proliferation and survival after irradiation, which may be associated with the risk of Pten mutation-associated thymic lymphoma.

Immune Thymic Profile of the MOG-Induced Experimental Autoimmune Encephalomyelitis Mouse Model.

Multiple sclerosis (MS) is a chronic, immune-mediated, demyelinating disease that affects the neurons of the central nervous system. Activated T cells, specific for myelin epitopes, cross the brain barriers, and react against the myelin sheath, leading to demyelination. Since T cells are generated within the thymus, here we explored, in mice, the alterations occurring in this organ throughout the different phases of the disease. We induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 females and sacrifice them at the onset (day 16) and chronic phases of disease (day 23), along with non-induced controls. We observed thymic atrophy in EAE mice at the onset that remained until the chronic phase of disease. This atrophy was associated with a preferential loss of the CD4+CD8+ double positive thymocytes, an intermediate population between the more immature CD4−CD8− double negative and the most mature single positive thymocytes. This was accompanied by an increase in the thymic medullary/cortical ratio and by an altered expression levels of genes important for T cell survival. During the chronic phase, the thymi remained atrophic, but reacquired the normal proportion of the main four thymocyte populations and the normal medullary/cortical ratio. Importantly, at the onset phase, and accompanying these thymic alterations, EAE animals presented an increased percentage of demyelinating lesion area in the cerebellum, and an increased expression of interferon gamma (Ifng), interleukin (Il) 12a, and Il17a. This study suggests dynamic thymic alterations occurring in response to EAE, from the induction to the chronic phase, that might help to elucidate the MS pathophysiology.

B cells regulate thymic CD8+T cell differentiation in lupus-prone mice.

Previous studies have shown that under normal physiological conditions thymic B cells play a critical function in T cell negative selection. We tested the effect of thymic B cells on thymic T-cell differentiation in autoimmune diseases including systemic lupus erythematosus (SLE). We found that thymic B cells and CD8- CD4+ and CD4-CD8+T cells increased, whereas CD4+CD8+T cells decreased in lupus-prone mice. Once B cells were reduced, the change was reversed. Furthermore, we found that B cells blocked thymic immature single positive (ISP) CD4-CD8+CD3lo/-RORγt- T cells progression into CD4+CD8+T cells. Interestingly, we found a novel population of thymic immature T cells (CD4-CD8+CD3loRORγt+) that were induced into mature CD4-CD8+CD3+RORγt+T cells by B cells in lupus-prone mice. Importantly, we found that IgG, produced by thymic B cells, played a critical role in the differentiation of thymic CD8+ISP and mature RORγt+CD8+ T cells in lupus-prone mice. In conclusion, B cells blocked the differentiation from thymic CD8+ISP and induced the differentiation of a novel immature CD4-CD8+CD3loRORγt+T cells into mature RORγt+CD8+ T cells by secreting IgG antibody in lupus-prone mice.

Microfluidic device platform for T cell fate analysis (TECH2P.907)

Abstract Microfluidic systems have enormous potential as investigational devices in biomedical research, in particular immunology. Microfluidic devices can be fabricated with the precise regulation of any number of parameters including controlled surface chemistries, geometrical dimensions, signal input and output and timing. The adjustable nature of microfluidic devices makes them an ideal platform to recreate cellular microenvironments for studying cell-cell interactions, migration, antibody and cytokine production, and differentiation in vitro. Investigations with customized microfluidic devices have the potential for elucidating the mechanism behind T lymphocyte commitment to either the αβ or γδ lineage. Both types of T cells arise from immature CD4-CD8- precursors in the thymus but diverge during development. Recent publications indicate that γδ T cell receptor (TCR) works in concert with ligand to direct progenitors to the γδ fate. We propose to develop a microfluidic device in which immature T lymphocytes can migrate through a thymus-like microenvironment allowing for αβ or γδ lineage commitment. As of yet, the microfluidic device fabrication protocol has been developed and preliminary cell culture experiments with the commonly used murine stromal cell line OP9 have success. Such a device would allow for analysis of specific developmental points in T cell development.

Elevated T Cell Receptor Signaling Identifies a Thymic Precursor to the TCRαβ+CD4−CD8β− Intraepithelial Lymphocyte Lineage

The origin and developmental pathway of intestinal T cell receptor αβ(+) CD4(-)CD8β(-) intraepithelial lymphocytes (unconventional iIELs), a major population of innate-like resident cytolytic T cells, have remained elusive. By cloning and expressing several TCRs isolated from unconventional iIELs, we identified immature CD4(lo)CD8(lo)(DP(lo))CD69(hi)PD-1(hi) thymocytes as the earliest postsignaling precursors for these cells. Although these precursors displayed multiple signs of elevated TCR signaling, a sizeable fraction of them escaped deletion to selectively engage in unconventional iIEL differentiation. Conversely, TCRs cloned from DP(lo)CD69(hi)PD-1(hi) thymocytes, a population enriched in autoreactive thymocytes, selectively gave rise to unconventional iIELs upon transgenic expression. Thus, the unconventional iIEL precursor overlaps with the DP(lo) population undergoing negative selection, indicating that, concomitant with the downregulation of both CD4 and CD8 coreceptors, a balance between apoptosis and survival signals results in outcomes as divergent as clonal deletion and differentiation to the unconventional iIEL lineage.

Transgenic Expression of MicroRNA-181d Augments the Stress-Sensitivity of CD4+CD8+ Thymocytes

Physiological stress resulting from infections, trauma, surgery, alcoholism, malnutrition, and/or pregnancy results in a substantial depletion of immature CD4+CD8+ thymocytes. We previously identified 18 distinct stress-responsive microRNAs (miRs) in the thymus upon systemic stress induced by lipopolysaccharide (LPS) or the synthetic glucocorticoid, dexamethasone (Dex). MiRs are short, non-coding RNAs that play critical roles in the immune system by targeting diverse mRNAs, suggesting that their modulation in the thymus in response to stress could impact thymopoiesis. MiR-181d is one such stress-responsive miR, exhibiting a 15-fold down-regulation in expression. We utilized both transgenic and gene-targeting approaches to study the impact of miR-181d on thymopoiesis under normal and stress conditions. The over-expression of miR-181d in developing thymocytes reduced the total number of immature CD4+CD8+ thymocytes. LPS or Dex injections caused a 4-fold greater loss of these cells when compared with the wild type controls. A knockout mouse was developed to selectively eliminate miR-181d, leaving the closely spaced and contiguous family member miR-181c intact. The targeted elimination of just miR-181d resulted in a thymus stress-responsiveness similar to wild-type mice. These experiments suggest that one or more of three other miR-181 family members have overlapping or compensatory functions. Gene expression comparisons of thymocytes from the wild type versus transgenic mice indicated that miR-181d targets a number of stress, metabolic, and signaling pathways. These findings demonstrate that selected miRs enhance stress-mediated thymic involution in vivo.

C‐Jun NH2‐terminal kinase is a critical node in the death of CD4+CD8+ thymocytes during Salmonella enterica serovar Typhimurium infection

Thymic atrophy, due to the depletion of CD4(+) CD8(+) thymocytes, is observed during infections with numerous pathogens. Several mechanisms, such as glucocorticoids and inflammatory cytokines, are known to be involved in this process; however, the roles of intracellular signaling molecules have not been investigated. In this study, the functional role of c-Jun NH2 -terminal kinase (JNK) during infection-induced thymic atrophy was addressed. The levels of phosphorylated JNK in immature CD4(+) CD8(+) thymocytes from C57BL/6 (Nramp-deficient) and 129/SvJ (Nramp-sufficient) mice were increased upon oral infection of mice with Salmonella enterica serovar Typhimurium (S. typhimurium). Furthermore, inhibition of JNK signaling, but not ERK or p38 MAPK, prevented the in vitro death of infected thymocytes. Importantly, the in vivo inhibition of JNK signaling with SP600125 protected C57BL/6 CD4(+) CD8(+) thymocytes from depletion via multiple mechanisms as follows: lower intracellular ROS, inflammatory cytokines, Bax and caspase 3 activity, increase in Bcl-xL amounts, and prevention of the loss in mitochondrial membrane potential. Notably, thymic architecture was preserved in infected mice treated with SP600125. Overall, this study identifies a novel role for JNK as a crucial regulator of the death of CD4(+) CD8(+) thymocytes during S. typhimurium infection.

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.

T-cell-specific deletion of Mof blocks their differentiation and results in genomic instability in mice

Ataxia telangiectasia patients develop lymphoid malignancies of both B- and T-cell origin. Similarly, ataxia telangiectasia mutated (Atm)-deficient mice exhibit severe defects in T-cell maturation and eventually develop thymomas. The function of ATM is known to be influenced by the mammalian orthologue of the Drosophila MOF (males absent on the first) gene. Here, we report the effect of T-cell-specific ablation of the mouse Mof (Mof) gene on leucocyte trafficking and survival. Conditional Mof(Flox/Flox) (Mof (F/F)) mice expressing Cre recombinase under control of the T-cell-specific Lck proximal promoter (Mof(F/F)/Lck-Cre(+)) display a marked reduction in thymus size compared with Mof(F/F)/Lck-Cre(-) mice. In contrast, the spleen size of Mof(F/F)/Lck-Cre(+) mice was increased compared with control Mof(F/F)/Lck-Cre(-) mice. The thymus of Mof(F/F)/Lck-Cre(+) mice contained significantly reduced T cells, whereas thymic B cells were elevated. Within the T-cell population, CD4(+)CD8(+) double-positive T-cell levels were reduced, whereas the immature CD4(-)CD8(-) double-negative (DN) population was elevated. Defective T-cell differentiation is also evident as an increased DN3 (CD44(-)CD25(+)) population, the cell stage during which T-cell receptor rearrangement takes place. The differentiation defect in T cells and reduced thymus size were not rescued in a p53-deficient background. Splenic B-cell distributions were similar between Mof(F/F)/Lck-Cre(+) and Mof(F/F)/Lck-Cre(-) mice except for an elevation of the κ light-chain population, suggestive of an abnormal clonal expansion. T cells from Mof(F/F)/Lck-Cre(+) mice did not respond to phytohaemagglutinin (PHA) stimulation, whereas LPS-stimulated B cells from Mof(F/F)/Lck-Cre(+) mice demonstrated spontaneous genomic instability. Mice with T-cell-specific loss of MOF had shorter lifespans and decreased survival following irradiation than did Mof(F/F)/Lck-Cre(-) mice. These observations suggest that Mof plays a critical role in T-cell differentiation and that depletion of Mof in T cells reduces T-cell numbers and, by an undefined mechanism, induces genomic instability in B cells through bystander mechanism. As a result, these mice have a shorter lifespan and reduced survival after irradiation.

Immature CD4+CD8+ Thymocytes Are Preferentially Infected by Measles Virus in Human Thymic Organ Cultures

Cells of the human immune system are important target cells for measles virus (MeV) infection and infection of these cells may contribute to the immunologic abnormalities and immune suppression that characterize measles. The thymus is the site for production of naïve T lymphocytes and is infected during measles. To determine which populations of thymocytes are susceptible to MeV infection and whether strains of MeV differ in their ability to infect thymocytes, we used ex vivo human thymus organ cultures to assess the relative susceptibility of different subpopulations of thymocytes to infection with wild type and vaccine strains of MeV. Thymocytes were susceptible to MeV infection with the most replication in immature CD4+CD8+ double positive cells. Susceptibility correlated with the level of expression of the MeV receptor CD150. Wild type strains of MeV infected thymocytes more efficiently than the Edmonston vaccine strain. Thymus cultures from children ≥3 years of age were less susceptible to MeV infection than cultures from children 5 to 15 months of age. Resistance in one 7 year-old child was associated with production of interferon-gamma suggesting that vaccination may result in MeV-specific memory T cells in the thymus. We conclude that immature thymocytes are susceptible to MeV infection and thymocyte infection may contribute to the immunologic abnormalities associated with measles.

ShcA and c-Abl provide a new signaling axis regulating beta-selection (63.8)

Abstract Progression of immature CD4-CD8- thymocytes through β selection checkpoint is determined by signals from the pre-T cell receptor (pre-TCR) along with signals from Notch. Knockout of the adapter protein ShcA, or transgenic expression of a phosphorylation-defective mutant of ShcA (ShcFFF) leads to a severe block at β selection. Our studies to mechanistically understand the ShcA function showed that ShcA acts upstream of ERK, and the transcription factors Egr-1 and Egr-3 during β selection; however, phospho-ShcA also appeared to be linked to other signaling pathways. We performed a yeast three-hybrid screen, using as bait ShcA that was phosphorylated by Lck. This screen identified binding of c-Abl (via its SH2 domain) to phosphorylated ShcA. During pre TCR dependent signaling, phosphorylation of ShcA was required for optimal c-Abl phosphorylation. Using mice deficient either in c-Abl and/or its homologue Arg, we show that ShcA and c-Abl regulate DN thymocyte proliferation, and the proper localization of DN thymocytes near the thymic cortex. Ectopic expression of a constitutive active c-Abl allowed thymocytes to resume proliferation, differentiation, and CXCR4 dependent migration to SDF-1α. Collectively, these data reveal a previously unappreciated ShcA::c-Abl signaling axis that appears to critically influence thymic β selection.

TNF-α Is Involved in the Abnormal Thymocyte Migration during Experimental Trypanosoma cruzi Infection and Favors the Export of Immature Cells

Previous studies revealed a significant production of inflammatory cytokines together with severe thymic atrophy and thymocyte migratory disturbances during experimental Chagas disease. Migratory activity of thymocytes and mature T cells seem to be finely tuned by cytokines, chemokines and extracellular matrix (ECM) components. Systemic TNF-α is enhanced during infection and appears to be crucial in the response against the parasite. However, it also seems to be involved in disease pathology, since it is implicated in the arrival of T cells to effector sites, including the myocardium. Herein, we analyzed the role of TNF-α in the migratory activity of thymocytes in Trypanosoma cruzi (T. cruzi) acutely-infected mice. We found increased expression and deposition of TNF-α in the thymus of infected animals compared to controls, accompanied by increased co-localization of fibronectin, a cell migration-related ECM molecule, whose contents in the thymus of infected mice is also augmented. In-vivo studies showed an enhanced export of thymocytes in T. cruzi-infected mice, as ascertained by intrathymic injection of FITC alone or in combination with TNF-α. The increase of immature CD4+CD8+ T cells in secondary lymphoid organs was even more clear-cut when TNF-α was co-injected with FITC. Ex-vivo transmigration assays also revealed higher number of migrating cells when TNF-α was added onto fibronectin lattices, with higher input of all thymocyte subsets, including immature CD4+CD8+. Infected animals also exhibit enhanced levels of expression of both mRNA TNF-α receptors in the CD4+CD8+ subpopulation. Our findings suggest that in T. cruzi acute infection, when TNF-α is complexed with fibronectin, it favours the altered migration of thymocytes, promoting the release of mature and immature T cells to different compartments of the immune system. Conceptually, this work reinforces the notion that thymocyte migration is a multivectorial biological event in health and disease, and that TNF-α is a further player in the process.

The role of the gut as a primary lymphoid organ: CD8αα intraepithelial T lymphocytes in euthymic mice derive from very immature CD44+ thymocyte precursors.

Intestinal CD8αα intraepithelial T lymphocytes (T-IELs) have a key role in mucosal immunity and, unlike other T cells, were proposed to differentiate locally. In apparent contradiction, these cells were also shown to originate from a wave of thymus migrants colonizing the gut in the first 3 weeks after birth. We here identify previously uncharacterized very immature CD4(-)CD8(-)CD3(-)CD44(+)CD25(int) thymocytes, which have not yet rearranged their T-cell antigen receptor (TCR), as having the capacity to leave the thymus, migrate to the blood, colonize the gut, and reconstitute CD8αα T-IEL, and show that this cell set is fully responsible for the generation of the CD8αα T-IEL pool. Thus, although the thymus may be fundamental for efficient T-cell commitment, CD8αα T-IEL' complete TCR rearrangements and TCR-αβ/γδ lineage commitment must occur in the gut. These results demonstrate a major role of the gut environment as a primary lymphoid organ.

Role of endogenous Annexin-A1 in the regulation of thymocyte positive and negative selection

We have recently shown that endogenous Annexin-A1 (AnxA1) plays a homeostatic regulatory role in mature T cells by modulating the strength of TCR signaling. In this study we investigated the role of endogenous AnxA1 in thymocyte maturation. Analysis of AnxA1-/- thymocyte populations at the immature CD4-CD8- double negative (DN) stage showed a proportional decrease in the DN1 and an increase in the DN3 subsets compared to control littermates. There were no significant differences in thymocyte numbers or proportions of CD4+ and CD8+ single positive (SP) populations between Anx1-/- and AnxA1+/+ mice. However, when we crossed AnxA1-/- mice onto HY-TCR transgenic mice, we observed an increase in CD4+CD8+ double positive (DP) and CD4 SP cells in male AnxA1-/-/HY-TCR compared to AnxA1+/+/HY-TCR. Conversely, female AnxA1-/-/HY-TCR mice showed an increase in DP and a decrease in CD8 (SP) cells compared to female AnxA1+/+/HY-TCR. Biochemical analysis of the signaling pathways responsible for these effects showed a decrease in anti-CD3-induced Erk phosphorylation and NF-κB activation in AnxA1-/- thymocytes compared to control littermates. Together these findings demonstrate a role for endogenous AnxA1 in regulating both positive and negative selection of the TCR repertoire. These results suggest that targeting AnxA1 expression or function in T cells could represent a useful approach for the development of novel therapies for the treatment of autoimmune diseases.

A Novel TCR Transgenic Model Reveals That Negative Selection Involves an Immediate, Bim-Dependent Pathway and a Delayed, Bim-Independent Pathway

A complete understanding of negative selection has been elusive due to the rapid apoptosis and clearance of thymocytes in vivo. We report a TCR transgenic model in which expression of the TCR during differentiation occurs only after V(D)J-like recombination. TCR expression from this transgene closely mimics expression of the endogenous TCRα locus allowing for development that is similar to wild type thymocytes. This model allowed us to characterize the phenotypic changes that occurred after TCR-mediated signaling in self-reactive thymocytes prior to their deletion in a highly physiological setting. Self-reactive thymocytes were identified as being immature, activated and CD4loCD8lo. These cells had upregulated markers of negative selection and were apoptotic. Elimination of Bim reduced the apoptosis of self-reactive thymocytes, but it did not rescue their differentiation and the cells remained at the immature CD4loCD8lo stage of development. These cells upregulate Nur77 and do not contribute to the peripheral T cell repertoire in vivo. Remarkably, development past the CD4loCD8lo stage was possible once the cells were removed from the negatively selecting thymic environment. In vitro development of these cells occurred despite their maintenance of high intracellular levels of Nur77. Therefore, in vivo, negatively selected Bim-deficient thymocytes are eliminated after prolonged developmental arrest via a Bim-independent pathway that is dependent on the thymic microenvironment. These data newly reveal a layering of immediate, Bim-dependent, and delayed Bim-independent pathways that both contribute to elimination of self-reactive thymocytes in vivo.

Characterization, function and targeting of dendritic cell associated c-type lectin-2 (DCAL-2) (135.54)

Abstract Dendritic Cell (DC) Associated C-type Lectin Receptors (CLRs) play important roles in immunity through their abilities in antigen capturing and presentation, modulation of TLR signaling, co-stimulation and adhesion. Murine DCAL-2 (mDCAL-2/ mMICL/ mKLRL-1) is a type-II CLR with a cytosolic immunoreceptor tyrosine inhibitory motif (ITIM). Using a mAb we developed, we found that mDCAL-2 is expressed at high levels on plasmacytoid DCs (pDCs), CD8α+ DCs and a subset of CD8α- DCs, at moderate levels on immature CD4-CD8- T cells and low levels on a subset of B-cells, but not on mature T cells and NK cells. DCAL-2 expression subdivides CD8α- DCs into mDCAL-2High33D1- and mDCAL-2Low33D1+ populations. These two subsets of DCs express different levels of TLRs. In addition, these subsets have significantly distinct cytokine profiles in response to TLR ligands. Whereas mDCAL-2High33D1- produced high levels of TNFα, IL-6, IL-10 and IL-12, mDCAL-2Low33D1+ DCs produced extremely low levels of these cytokines. Thus, we hypothesize that mDCAL-2High DC subsets induce T-cell responses that are distinct from that of mDCAL-2Low DCs. To investigate what type of T-cell responses are induced through mDCAL-2 in an Ag specific manner, we developed an anti-mDCAL-2 single chain fragment variable (scFv) fused with antigens (OVA or HEL). We found that anti-mDCAL-2 scFv successfully targets mDCAL-2+ DC subsets in vivo. What type of T cell responses are induced by OVA-anti-mDCAL-2 scFv injection in OT-II transgenic mice will be presented. This study is supported by NIH grants AI52203, DE16381, and RR00166

LEF-1 Negatively Controls Interleukin-4 Expression through a Proximal Promoter Regulatory Element

Lymphoid enhancer-binding factor 1 (LEF-1) and T cell factor (TCF-1) are downstream effectors of the Wnt signaling pathway and are involved in the regulation of T cell development in the thymus. LEF-1 and TCF-1 are also expressed in mature peripheral primary T cells, but their expression is down-regulated following T cell activation. Although the decisive roles of LEF-1 and TCF-1 in the early stages of T cell development are well documented, the functions of these factors in mature peripheral T cells are largely unknown. Recently, LEF-1 was shown to suppress Th2 cytokines interleukin-4 (IL-4), -5, and -13 expression from the developing Th2 cells that overexpress LEF-1 through retrovirus gene transduction. In this study, we further investigated the expression and functions of LEF-1 and TCF-1 in peripheral CD4+ T cells and revealed that LEF-1 is dominantly expressed in Th1 but not in Th2 cells. We identified a high affinity LEF-1-binding site in the negative regulatory element of the IL-4 promoter. Knockdown LEF-1 expression by LEF-1-specific small interfering RNA resulted in an increase in the IL-4 mRNA expression. This study further confirms a negative regulatory role of LEF-1 in mature peripheral T cells. Furthermore, we found that IL-4 stimulation possesses a negative effect on the expressions of LEF-1 and TCF-1 in primary T cells, suggesting a positive feedback effect of IL-4 on IL4 gene expression.