CD8 Single-positive Cells Research Articles

Selection of Thymocytes Expressing Transgenic TCR Specific for a Minor Histocompatibility Antigen, H60.

Minor histocompatibility antigens are MHC-bound peptides and contribute to the generation of allo-responses after allogeneic transplantation. H60 is a dominant minor H antigen that induces a strong CD8 T-cell response in MHC-matched allogeneic transplantation settings. Here, we report establishment of a TCR transgenic mouse line named J15, wherein T cells express TCRs specific for H60 in complex with H-2Kb, and different fates of the thymocytes expressing J15 TCRs in various thymic antigenic environments. Thymocytes expressing the J15 TCRs were positively selected and differentiated into CD8+ single positive (SP) cells in the thymus of C57BL/6 mice, wherein the cognate antigen H60 is not expressed. However, thymocytes were negatively selected in thymus tissue where H60 was transgenically expressed under the control of the actin promoter, with double-positive stages of cells being deleted. Despite the ability of the H60H peptide (LTFHYRNL) variant to induce cytotoxic activity from H60-specific CTL lines at ~50% of the activity induced by normal H60 peptides (LTFNYRNL), J15-expressing thymocytes were positively selected in the thymus where the variant H60H was transgenically expressed. These results demonstrate that a single amino-acid change in the H60 epitope peptide influences the fate of thymocytes expressing the cognate TCR.

Synergism Between HOXA9 and Mutant JAK3 (M511I) Leads to Rapid Leukemia Development within an in Vivo Murine Bone Marrow Transplant Model

Activation mutations in JAK3 occur in 16% of T-cell acute lymphoblastic leukemia (T-ALL) cases, and co-occur frequently with HOXA cluster rearrangement. Genomic rearrangement of the HOXA cluster results in increased expression of HOXA9 and HOXA10. However it remains unclear if either HOXA9 or HOXA10 can cooperate with activating JAK3 mutations during oncogenic transformation and leukemogenesis. We have previously shown that JAK3 mutations lead to cell transformation and cause a long latency T-ALL in vivo using a mouse bone marrow transplant model. In this study we demonstrate that co-expression of the activating JAK3(M511I) protein with HOXA9 cooperate to develop leukemia within 30 days of transplant using an in vivo bone marrow transplant model. In our cooperative model, murine hematopoietic stem / progenitor cells were co-transduced with either both retroviral vectors encoding JAK3(M511I)/GFP and HOXA9/mCherry or each individually and then injected into sub-lethally irradiated recipient mice. Mice transplanted with bone marrow cells expressing JAK3(M511I) mutant alone developed T-ALL in 120 to 150 days. In sharp contrast, mice transplanted with cells expressing both JAK3(M511I) and HOXA9 showed rapid leukemia development within 30 days after transplant. Leukemia development was characterized by the rapid and specific increase in GFP-mCherry double positive cells. These animals showed high WBC, and splenomegaly and accumulation of immature CD8 single positive cells in the thymus. Similar experiments with HOXA10 did not show cooperation suggesting that HOXA9 is the more important oncogene in HOXA rearranged leukemias when a JAK3 activating mutation is present. To determine the underlying genetic mechanism for cooperation between HOXA9 and JAK3(M511I) the single positive JAK3 and double positive JAK3/HOXA9 expressing cells were isolated from thymi of leukemic mice for both epigenomic profiling using ATAC-seq and gene expression profiling. These analyses identified genetic pathways activated by the co-expression of HOXA9 and JAK3(M511I) mutation and provide mechanistic insight into the synergistic interaction between these two factors in driving leukemia development. Treatment of the animals with a JAK kinase inhibitor resulted in delayed leukemia development, confirming that the leukemia cells remain sensitive to the JAK inhibitor. This mouse model provides insight in the cooperation between oncogenes in leukemia development and provides a model for the study of targeted agents in this setting. DisclosuresNo relevant conflicts of interest to declare.

Bcl11b prevents the intrathymic development of innate CD8 T cells in a cell intrinsic manner.

If Bcl11b activity is compromised, CD4(+)CD8(+) double-positive (DP) thymocytes produce a greatly increased fraction of innate CD8(+) single-positive (SP) cells highly producing IFN-γ, which are also increased in mice deficient of genes such as Itk, Id3 and NF-κB1 that affect TCR signaling. Of interest, the increase in the former two is due to the bystander effect of IL-4 that is secreted by promyelocytic leukemia zinc finger-expressing NKT and γδT cells whereas the increase in the latter is cell intrinsic. Bcl11b zinc-finger proteins play key roles in T cell development and T cell-mediated immune response likely through TCR signaling. We examined thymocytes at and after the DP stage in Bcl11b (F/S826G) CD4cre, Bcl11b (F/+) CD4cre and Bcl11b (+/S826G) mice, carrying the allele that substituted serine for glycine at the position of 826. Here we show that Bcl11b impairment leads to an increase in the population of TCRαβ(high)CD44(high)CD122(high) innate CD8SP thymocytes, together with two different developmental abnormalities: impaired positive and negative selection accompanying a reduction in the number of CD8SP cells, and developmental arrest of NKT cells at multiple steps. The innate CD8SP thymocytes express Eomes and secrete IFN-γ after stimulation with PMA and ionomycin, and in this case their increase is not due to a bystander effect of IL-4 but cell intrinsic. Those results indicate that Bcl11b regulates development of different thymocyte subsets at multiple stages and prevents an excess of innate CD8SP thymocytes.

Zfat-Deficiency Results in a Loss of CD3ζ Phosphorylation with Dysregulation of ERK and Egr Activities Leading to Impaired Positive Selection

The human ZFAT gene was originally identified as a susceptibility gene for autoimmune thyroid disease. Mouse Zfat is a critical transcriptional regulator for primitive hematopoiesis and required for peripheral T cell homeostasis. However, its physiological roles in T cell development remain poorly understood. Here, we generated Zfat f/f-LckCre mice and demonstrated that T cell-specific Zfat-deletion in Zfat f/f-LckCre mice resulted in a reduction in the number of CD4+CD8+double-positive (DP) cells, CD4+single positive cells and CD8+single positive cells. Indeed, in Zfat f/f-LckCre DP cells, positive selection was severely impaired. Defects of positive selection in Zfat-deficient thymocytes were not restored in the presence of the exogenous TCR by using TCR-transgenic mice. Furthermore, Zfat-deficient DP cells showed a loss of CD3ζ phosphorylation in response to T cell antigen receptor (TCR)-stimulation concomitant with dysregulation of extracellular signal-related kinase (ERK) and early growth response protein (Egr) activities. These results demonstrate that Zfat is required for proper regulation of the TCR-proximal signalings, and is a crucial molecule for positive selection through ERK and Egr activities, thus suggesting that a full understanding of the precise molecular mechanisms of Zfat will provide deeper insight into T cell development and immune regulation.

Zinc Finger Protein 148 Is Dispensable for Primitive and Definitive Hematopoiesis in Mice

Hematopoiesis is regulated by transcription factors that induce cell fate and differentiation in hematopoietic stem cells into fully differentiated hematopoietic cell types. The transcription factor zinc finger protein 148 (Zfp148) interacts with the hematopoietic transcription factor Gata1 and has been implicated to play an important role in primitive and definitive hematopoiesis in zebra fish and mouse chimeras. We have recently created a gene-trap knockout mouse model deficient for Zfp148, opening up for analyses of hematopoiesis in a conventional loss-of-function model in vivo. Here, we show that Zfp148-deficient neonatal and adult mice have normal or slightly increased levels of hemoglobin, hematocrit, platelets and white blood cells, compared to wild type controls. Hematopoietic lineages in bone marrow, thymus and spleen from Zfp148 gt/gt mice were further investigated by flow cytometry. There were no differences in T-cells (CD4 and CD8 single positive cells, CD4 and CD8 double negative/positive cells) in either organ. However, the fraction of CD69- and B220-positive cells among lymphocytes in spleen was slightly lower at postnatal day 14 in Zfp148 gt/gt mice compared to wild type mice. Our results demonstrate that Zfp148-deficient mice generate normal mature hematopoietic populations thus challenging earlier studies indicating that Zfp148 plays a critical role during hematopoietic development.

NBR1 in the regulation of T cell development and differentiation (P1000)

Abstract NBR1 (neighbor of BRCA1 gene 1) is a newly discovered signaling molecule containing a Phox and Bem1 (PB1) domain. PB1-domains are protein interaction modules. PB1 domain-containing molecules participate in diverse biological processes, which also include p62, Par-6, MEK5a, MEKK3, PKCζ and PKC λ/ι besides NBR1. We have recently reported that NBR1 regulates Th2 function and allergic airway inflammation. However, the role of NBR1 in the T cell development and in the regulation of other CD4+ T cell subsets remains to be elucidated. With the use of CD4+ T cell specific conditional NBR1-deficient mice, we found that NBR1 did not affect T cell development. Thymocyte subpopulations (double negative, double positive, CD4 or CD8 single positive cells) were similar between conditional NBR1-deficient mice and their WT littermates. CD4+ and CD8+ cells in the spleen and blood also did not differ significantly between these two groups. Furthermore, NBR1-deficiency did not affect the development of natural Treg in vivo and the differentiation of induced Treg in vitro. However, NBR1 was found selectively regulate Th2 function without affect Th1 cells. Interestingly, our preliminary data indicated that NBR1-deficiency might affect Th17 differentiation in vitro. These data suggest that NBR1 has no effect on T cell development, whereas it may selectively affect some CD4+ T cell subsets, especially Th2 and Th17. Further study is needed to elucidate the underlying mechanisms.

The Hedgehog Receptor Patched1 in T Cells Is Dispensable for Adaptive Immunity in Mice

Hedgehog (Hh) signaling modulates T cell development and function but its exact role remains a matter of debate. To further address this issue we made use of conditional knock-out mice in which the Hh receptor Patched1 (Ptch) is inactivated in the T cell lineage. Thymocyte development was moderately compromised by the deletion of Ptch as characterized by reduced numbers of CD4 and CD8 single-positive cells. In contrast, peripheral T cells were not affected. Proliferation and IFNγ secretion by Ptch-deficient T cells were indistinguishable from controls irrespectively of whether we used strong or suboptimal conditions for stimulation. Analysis of CTL and Treg cell functions did not reveal any differences between both genotypes, and T cell apoptosis induced by glucocorticoids or γ-irradiation was also similar. Surprisingly, absence of Ptch did not lead to an activation of canonic Hh signaling in peripheral T cells as indicated by unaltered expression levels of Gli1 and Gli2. To test whether we could uncover any role of Ptch in T cells in vivo we subjected the mutant mice to three different disease models, namely allogeneic bone marrow transplantation mimicking graft-versus-host disease, allergic airway inflammation as a model of asthma and growth of adoptively transferred melanoma cells as a means to test tumor surveillance by the immune system. Nonetheless, we were neither able to demonstrate any difference in the disease courses nor in any pathogenic parameter in these three models of adaptive immunity. We therefore conclude that the Hh receptor Ptch is dispensable for T cell function in vitro as well as in vivo.

Cutting Edge: Ly9 (CD229), a SLAM Family Receptor, Negatively Regulates the Development of Thymic Innate Memory-like CD8+ T and Invariant NKT Cells

Signaling lymphocytic activation molecule family receptors and the specific adapter signaling lymphocytic activation molecule-associated protein modulate the development of innate-like lymphocytes. In this study, we show that the thymus of Ly9-deficient mice contains an expanded population of CD8 single-positive cells with the characteristic phenotype of innate memory-like CD8(+) T cells. Moreover, the proportion of these innate CD8(+) T cells increased dramatically postinfection with mouse CMV. Gene expression profiling of Ly9-deficient mice thymi showed a significant upregulation of IL-4 and promyelocytic leukemia zinc finger. Analyses of Ly9(-/-)IL4ra(-/-) double-deficient mice revealed that IL-4 was needed to generate the thymic innate CD8(+) T cell subset. Furthermore, increased numbers of invariant NKT cells were detected in Ly9-deficient thymi. In wild-type mice, IL-4 levels induced by α-galactosylceramide injection could be inhibited by a mAb against Ly9. Thus, Ly9 plays a unique role as an inhibitory cell surface receptor regulating the size of the thymic innate CD8(+) T cell pool and the development of invariant NKT cells.

The Role of IL7R in the Induction and Maintenance of T-Cell Leukemia/Lymphoma in Vivo

AbstractAbstract 116Interleukin 7 (IL-7) and its receptor, a heterodimer constituted by IL-7Rα (hereafter referred to as IL7R) and γc subunits, are essential for normal T-cell development and homeostasis. While it is known for long that IL7R genetic inactivation leads to severe combined immunodeficiency, only recently we and others have found IL7R gain-of-function mutations in T-cell acute lymphoblastic leukemia (T-ALL). In agreement with an oncogenic role for deregulated IL-7/IL7R signaling, IL-7 transgenic mice develop lymphomas and we have shown that xenotransplant models of human T-ALL rely on microenvironmental IL-7 for tumor acceleration. These studies have highlighted the importance of keeping IL7R-mediated signaling within physiological levels. Indeed, throughout normal T-cell development IL7R expression at the cell surface is tightly controlled and there is correlative evidence, from studies in AKR/J mice, that high IL7R levels in developing thymocytes can promote the development of T-cell leukemia. However, no studies have yet directly evaluated the impact of IL7R on T-cell oncogenesis.In the present study, we used a mouse model in which expression of an IL7R transgene is controlled by a tetracycline responsive promotor (TreIL7R rtTAhuCD2Il7r−/− mice) to analyze the role of IL7R-mediated signaling in T-cell leukemogenesis. Continuous expression of TreIL7R upon doxycycline feeding induced thymus hypertrophy and hyperplasia due to increased size and hyperproliferation of T-cells, which subsequently infiltrated lymph nodes, spleen and bone marrow, ultimately leading to leukemia/lymphoma-associated death. Adoptive transfer of thymic TreIL7R cells to immunodeficient mice confirmed their malignant origin. Surprisingly, tumors developed in recipient animals even in the absence of doxycycline administration, indicating that they eventually become independent of continuous IL7R expression. Remarkably, the tumors mimicked several features of human T-ALL. First, their immunophenotype varied considerably between animals (from CD4 CD8 double-negative to double positive to CD4 or CD8 single positive cells), reflecting the heterogeneity of human disease. Second, similar to the majority of primary T-ALL cases, most tumors displayed hyperactivation of PI3K/Akt pathway, which sometimes associated with absence or decreased PTEN protein expression. Third, the cell cycle inhibitor p27Kip1 was frequently downregulated, a molecular characteristic associated with some T-ALLs.We next sought to determine the contribution of TCR diversity to IL7R-mediated tumorigenesis by crossing mice to the F5 TCR transgenic background. Tumor development in these mice occurred with similar timing and incidence, suggesting that the tumors arise irrespectively of whether a clonal or polyclonal TCR repertoire exists. Finally, the role of Rag recombinase dependent genomic instability in tumor development was assessed by further crossing mice F5 TreIL-7R rtTAhuCD2Il7r−/− mice to Rag1−/− background. Tumor development occurred independently of Rag1 expression, indicating a dominant role for IL7R elicited signals in tumorigenesis.Altogether, our results reveal that continuous IL7R-mediated signaling promotes T-cell tumorigenesis in vivo, providing further indication that IL7R can act as a critical T-cell oncogene. Disclosures:No relevant conflicts of interest to declare.

Reciprocal Control of G1-Phase Progression Is Required for Th-POK/Runx3–Mediated CD4/8 Thymocyte Cell Fate Decision

After receiving a TCR-mediated differentiation signal, CD4 and CD8 double-positive thymocytes diverge into CD4 or CD8 single-positive T cells, for which Th-POK and Runx3 have been identified as pivotal transcription factors, respectively. The cross-antagonistic regulation of Th-POK and Runx3 seems to be essential for CD4/8 thymocyte lineage commitment. However, the process for determining which pivotal factor acts dominantly has not been established. To explore the determining process, we used an in vitro culture system in which CD4 or CD8 single-positive cells are selectively induced from CD4/8 double-positive cells. Surprisingly, we found that control of G(1) cell cycle phase progression is critical for the determination. In the CD4 pathway, sustained TCR signal, as well as Th-POK, induces G(1)-phase extension and represses CD8 expression in a G(1) extension-dependent manner. In the CD8 pathway, after receiving a transient TCR signal, the IL-7R signal, as well as Runx3, antagonizes TCR signal-mediated G(1) extension and CD8 repression. Importantly, forced G(1) extension cancels the functions of Runx3 to repress Th-POK and CD4 and to reactivate CD8. In contrast, it is suggested that forced G(1) progression inhibits Th-POK function to repress CD8. Collectively, Th-POK and Runx3 are reciprocally involved in the control of G(1)-phase progression, on which they exert their functions dependently. These findings may provide novel insight into how CD4/CD8 cell lineages are determined by Th-POK and Runx3.

FTY720 Markedly Increases Alloengraftment but Does Not Eliminate Host Anti-Donor T Cells that Cause Graft Rejection on Its Withdrawal

The immunomodulator FTY720 (FTY) is beneficial in models of graft-versus-host disease, solid organ transplantation, and autoimmunity and has been approved for use in patients with multiple sclerosis. FTY modifies the homing and migration of many cell types. We report that FTY has profound positive and negative effects on allogeneic bone marrow (BM) engraftment in sublethally irradiated recipients. FTY increased donor hematopoietic progenitors in the BM, resulting in high donor engraftment in the B cell, myeloid cell, and natural killer cell, but not T cell, lineages. Donor T cell progenitors within the thymus of FTY-treated recipients were dramatically reduced, resulting in a lack of donor T cell reconstitution. In addition to preventing the ingress of donor (and host) T cell progenitors, FTY prevented the egress of fully functional host CD4+CD8- and CD4-CD8+ thymocytes that on cessation of FTY administration were able to exit from the thymus and contribute to a rapid and complete rejection of a well-established donor BM graft. When used in combination with anti-CD40L mAbs to block the CD40L:CD40 costimulatory pathway, FTY markedly enhanced anti-CD40L mAb-mediated alloengraftment promotion. In contrast to FTY alone, the combination of anti-CD40L mAb and FTY resulted in a surprisingly stable, multilineage, long-term donor chimerism. These data illustrate FTY's profound migration modulating effects and suggest a use in combinatorial therapy in achieving stable alloengraftment under nonmyeloablative conditions.

T cell-specific deletion of EFNB2 minimally affects T cell development and function

BackgroundEph kinases and their ephrin ligands (EFN) are all cell surface molecules, capable of transmitting signals in both directions (1, 2). Such bidirectional signaling is called forward (from EFNs to Ephs) and reverse (from Ephs to EFNs) signaling. Eph family kinases have 15 members, divided into A and B subfamilies. Ephrin ligands have 9 members, also classified into A and B families. Ephs and ephrins interact promiscuously, but EphAs mainly interact with EFNAs, and EphBs with EFNBs. EphB family kinases and their ephrin ligands (EFN) are expressed in the T cell compartment. ResultsIn this study, using mice with T cell-specific EFNB2 gene knockout (EFNB2 KO mice), we investigated T cell development and function after EFNB2 deletion. EFNB2 KO mice presented normal thymus weight and cellularity. Their thymocyte subpopulations, such as CD4CD8 double positive cells and CD4 and CD8 single positive cells, were normally distributed, but there was a significant relative increase of CD4CD8 double negative cells. Flow cytometry analysis revealed that there was a moderate increase in the DN3 subpopulation. This augmented percentage of DN cells was further confirmed in competitive repopulation chimeras, suggesting that EFNB2 is involved in thymocyte development. The EFNB2 KO mice had normal T cell numbers and percentages in the spleen, and the T cells were able to be activated and to proliferate normally upon TCR ligation. Further, EFNB2 KO naïve CD4 cells were capable of differentiating into Th1, Th2, Th17 and Treg cells similar to WT naïve CD4 cells. ConclusionsOur results suggest the involvement of EFNB2 in thymocyte development. However, heavy redundancy among Eph/EFN family members prevents the occurrence of detrimental phenotypes in the T cell compartment caused by T cell-specific EFNB2 gene null mutation.

Impairment in differentiation and cell cycle of thymocytes by loss of a Bcl11b tumor suppressor allele that contributes to leukemogenesis

Genetic changes in T-ALL are classified into type A abnormalities leading to arrest at a specific stage of T-cell differentiation and type B abnormalities that target cellular processes including cell cycle regulation. Mutations and deletion of a BCL11B haploinsuffiecient tumor suppressor allele have been found in 10–16% of T-ALL subgroups. Analysis of Bcl11bKO/+ mice revealed impaired T-cell differentiation at two different stages and attenuation of γ-ray induced cell-cycle arrest at S/G2/M phase in immature CD8 single positive cells. Hence, those phenotypes provided by loss of a Bcl11b allele favor that Bcl11b mutation belongs to type B abnormalities.

Regulation of selection and autoimmunity by miR-181 family miRNAs (123.36)

Abstract T cell sensitivity to antigens is regulated during development to ensure proper immunity and tolerance. We previously showed that miR-181a regulates the strength and threshold of T cell receptor signaling by targeting negative feedback molecules. Here we used miR-181 knockout mice to examine the role of miR-181 miRNAs in selection and T cell reactivity to antigens. We found that loss of mir-181ab1 caused an increase in the percent of positively selected CD8 single-positive (SP) cells in the thymi of female HY TCR transgenic mice. However, loss of miR-181 had no effect on the deletion CD8 SP cells in the thymi of male HY mice. Moreover, we showed that loss of mir-181ab1 resulted in two-fold more self-reactive T cells in the periphery after immunization with a self-peptide derived from myelin oligodendrocyte glycoprotein (MOG). Paradoxically, knockout mice exhibited a delayed disease onset in a MOG-dependent mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). In particular, the central nervous systems of knockout mice during disease had similar percentages of MOG-specific T cells, but an underrepresentation of TH17 and T H1-polarized CD4 T cells compared to the periphery. These findings demonstrate that mir-181ab1 also plays a role in modulating the differentiation and function of self-reactive T cells. Together, data from our study illustrate the diverse roles of mir-181ab1 in selection and autoimmunity.

Subcellular distribution of Lck during CD4 T-cell maturation in the thymic medulla regulates the T-cell activation threshold

Mature peripheral T cells respond to foreign but not to self-antigens. During development in the thymus, deletion of high-affinity self-reactive immature thymocytes contributes to tolerance of mature T cells. However, double-positive thymocytes are positively selected to survive if they respond to self-peptide-MHC complexes; thus, there must be mechanisms to prevent overt reactivity to those same complexes in the periphery. "Developmental tuning" is the active process through which T-cell receptor (TCR)-associated signaling pathways of single-positive (SP) thymocytes are attenuated to respond appropriately to self-peptide-MHC complexes in the periphery. We previously showed that MHC class II expression in the thymic medulla was necessary to tune CD4(+) SP (CD4 SP) thymocytes. CD4 SP thymocytes from mice lacking medullary MHC class II expression had inappropriately enhanced proximal TCR signaling to low-affinity self-ligands that was associated with altered cellular distribution of the tyrosine kinase Lck. Now, we report that activation of both tuned and untuned CD4 SP thymocytes is Lck-dependent. Untuned CD4 SP cells contain a pool of Lck with increased basal phosphorylation that is not associated with the CD4 coreceptor. Phosphorylation of this pool of Lck decreases with tuning. Immunogold transmission electron microscopy of membrane sheets permitted direct visualization of Lck. In the absence of tuning, a significant proportion of Lck and the TCR subunit CD3ζ are expressed on the same protein island; this close association of Lck and the TCR probably explains the enhanced activation of untuned CD4 SP cells. Thus, changes in membrane topography during thymic maturation determine the set point for TCR responsiveness.

The effects of leflunomide on CD4+CD25+Foxp3+ T regulatory cells in mice receiving allogeneic bone marrow transplantation

Leflunomide (LEF) is effective not only in different animal models of autoimmune diseases and the therapy of patients with rheumatoid arthritisbut also in graft rejection. The effect of LEF on CD4(+)CD25(+)T regulatory cells (Treg) was determined in a mouse model of allogeneic bone marrow transplantation. BALB/c and C57BL/6 mice were used as donors and recipients, respectively. C57BL/6 mice were given 2 Gy total-body irradiation, followed by an intravenous injection of 2 × 10(7) BALB/c bone marrow cells (BMCs). Mice were treated with LEF daily at a dose of 30 mg/kg/day for 2 weeks. In naïve mice, LEF significantly decreased the percentage of CD4(+)CD25(+) Treg cells in spleens (P < 0.05), but not in lymph nodes, though LEF enhanced the percentages of CD4(+)CD25(+) Treg cells in CD4 single positive cells in the thymocytes and blood (P < 0.05). Furthermore, LEF significantly decreased the percentage of CD4(+)CD25(+) Treg cells in the spleens of mice that received allogeneic BMCs. LEF decreases peripheral CD4(+)CD25(+) Treg cells in un-immunizing and immunizing recipients, indicating that LEF might not be an ideal candidate for the treatment of autoimmune diseases or graft rejection with respect to induction of immune tolerance.

Pre-TCR Signaling and CD8 Gene Bivalent Chromatin Resolution during Thymocyte Development

The CD8 gene is silent in CD4(-)CD8(-) double-negative thymocytes, expressed in CD4(+)CD8(+) double-positive cells, and silenced in cells committing to the CD4(+) single-positive (SP) lineage, remaining active in the CD8(+) SP lineage. In this study, we show that the chromatin of the CD8 locus is remodeled in C57BL/6 and B6/J Rag1(-/-) MOM double-negative thymocytes as indicated by DNaseI hypersensitivity and widespread bivalent chromatin marks. Pre-TCR signaling coincides with chromatin bivalency resolution into monovalent activating modifications in double-positive and CD8 SP cells. Shortly after commitment to CD4 SP cell lineage, monovalent repressive characteristics and chromatin inaccessibility are established. Differential binding of Ikaros, NuRD, and heterochromatin protein 1α on the locus during these processes may participate in the complex regulation of CD8.

LKB1 regulates TCR-mediated PLCγ1 activation and thymocyte positive selection

The serine/threonine kinase LKB1 is a tumour suppressor that regulates cell growth, polarity, and proliferation in many different cell types. We previously demonstrated that LKB1 controls thymocyte survival via regulation of AMPK activation. In this study, we show that LKB1 was also involved in thymocyte positive selection through regulation of T cell receptor (TCR) signalling. Both Lck-Cre- and CD4-Cre-mediated deletion of LKB1 impaired the generation of mature CD4 and CD8 single positive (SP) thymocytes that might have resulted from the attenuated tyrosine phosphorylation of phospholipase C-γ 1 (PLCγ1) in the absence of LKB1. We found that LKB1 was directly phosphorylated by Lck at tyrosine residues 36, 261, and 365 and predominately interacted with LAT and PLCγ1 following TCR stimulation. Loss of LKB1 led to impaired recruitment of PLCγ1 to the LAT signalosome. Correlatively, LKB1-deficient thymocytes failed to upregulate lineage-specifying factors, and to differentiate into SP thymocytes even if their impaired survival was rescued. These observations indicated that LKB1 is a critical component involved in TCR signalling, and our studies provide novel insights into the mechanisms of LKB1-mediated thymocyte development.

RUNX Transcription Factor-Mediated Association of Cd4 and Cd8 Enables Coordinate Gene Regulation

SummaryT cell fate is associated with mutually exclusive expression of CD4 or CD8 in helper and cytotoxic T cells, respectively. How expression of one locus is temporally coordinated with repression of the other has been a long-standing enigma, though we know RUNX transcription factors activate the Cd8 locus, silence the Cd4 locus, and repress the Zbtb7b locus (encoding the transcription factor ThPOK), which is required for CD4 expression. Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart. Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8. Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells.

Interplay of transcription factors in T-cell differentiation and function: the role of Runx

Over the past years, increasing numbers of distinct subsets have been discovered and identified for a T lymphocytes' entity. Differentiation and function of each T cell subset are controlled by a specific master transcription factor. Importantly, Runt-related transcription factors, particularly Runx1 and Runx3, interplay with these master regulators in various aspects of T cells' immunity. In this review article, we first explain roles of Th-Pok and Runx3 in differentiation of CD4 versus CD8 single positive cells, and later focus on cross-regulation of Th-Pok and Runx3 and their relationship with other factors such as TCR strength. Next, we provide evidences for the direct interplay of Runx1/3 with T-bet and GATA3 during Th1 versus Th2 commitment to activate or silence transcription of signature cytokine genes, IFNγ and IL4. Lastly, we explain feed-forward relationship between Runx1 and Foxp3 and discuss roles of Runx1 in regulatory T cells' suppressive activity. This review highlights an essential importance of Runx molecules in controlling various T cell subsets' differentiation and functions through molecular interplay with the master transcription factors in terms of protein-protein interaction as well as regulation of gene expression.