Thymic CD4 Research Articles

Precocious Interleukin 21 Expression in Naive Mice Identifies a Natural Helper Cell Population in Autoimmune Disease

Interleukin 21 (IL-21) plays key roles in humoral immunity and autoimmune diseases. It is known to function in mature CD4+ T follicular B cell helper (TFH) cells, but its potential involvement in early Tcell ontogeny is unclear. Here, we find that a significant population of newly activated thymic and peripheral CD4+ Tcells functionally expresses IL-21 soon after birth. This naturally occurring population, termed natural (n)TH21 cells, exhibits considerable similarity to mature TFH cells. nTH21 cells originating and activated in the thymus are strictly dependent on autoimmune regulator (AIRE) and express high levels of NUR77, consistent with a bias toward self-reactivity. Their activation/expansion in the periphery requires gut microbiota and is held in check by FoxP3+ TREG cells. nTH21 cells are the major thymic and peripheral populations of IL-21+ cells to expand in an IL-21-dependent humoral autoimmune disease. These studies link IL-21 to Tcell ontogeny, self-reactivity, and humoral autoimmunity.

Thymic Output and CD4 T-Cell Reconstitution in HIV-Infected Children on Early and Interrupted Antiretroviral Treatment: Evidence from the Children with HIV Early Antiretroviral Therapy Trial.

Early treatment of HIV-infected children and adults is important for optimal immune reconstitution. Infants' immune systems are more plastic and dynamic than older children's or adults', and deserve particular attention. This study aimed to understand the response of the HIV-infected infant immune system to early antiretroviral therapy (ART) and planned ART interruption and restart. Data from HIV-infected children enrolled the CHER trial, starting ART aged between 6 and 12 weeks, were used to explore the effect of ART on immune reconstitution. We used linear and non-linear regression and mixed-effects models to describe children's CD4 trajectories and to identify predictors of CD4 count during early and interrupted ART. Early treatment arrested the decline in CD4 count but did not fully restore it to the levels observed in HIV-uninfected children. Treatment interruption at 40 or 96 weeks resulted in a rapid decline in CD4 T-cells, which on retreatment returned to levels observed before interruption. Naïve CD4 T-cell count was an important determinant of overall CD4 levels. A strong correlation was observed between thymic output and the stable CD4 count both before and after treatment interruption. Early identification and treatment of HIV-infected infants is important to stabilize CD4 counts at the highest levels possible. Once stabilized, children's CD4 counts appear resilient, with good potential for recovery following treatment interruption. The naïve T-cell pool and thymic production of naive cells are key determinants of children's CD4 levels.

Ghrelin Protects the Thymic Epithelium From Conditioning-Regimen-Induced Damage and Promotes the Restoration of CD4+ T Cells in Mice After Bone Marrow Transplantation.

The delay in immune reconstitution after hematopoietic stem cell transplantation (HSCT), especially a delay in central immune reconstitution, leads to opportunistic infections and disease relapse after transplantation and affects the long-term outcome of HSCT. This delay is mainly attributable to thymic damage after myeloablative chemotherapy and radiotherapy. We established a model of allogeneic bone marrow transplantation (BMT) in mice and administered ghrelin (GRL) 7 days before the conditioning regimen or the day after BMT to explore the effect of GRL on thymus. All the GRL-treated mice, especially those administered GRL before the conditioning regimen, exhibited more intact thymic architecture and a more rapid restoration of CD4 T lymphocytes after BMT than those of the corresponding control mice. Moreover, the levels of T cell receptor excision circles were significantly higher in the mice treated with GRL before the conditioning regimen than in the control mice at 28 days after BMT. Our findings suggest that GRL may be a novel potential therapeutic approach to protecting the thymic epithelium from conditioning regimen-induced damage and promoting rapid and durable thymic and peripheral CD4 T cell recovery after HSCT.

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.

Central immune tolerance establishment under thymic involution (Supported by NIH/NIAID 1R01AI121147 to D-M Su)

Abstract The establishment of central immune tolerance in the thymus is occurs via thymocyte negative selection to deplete self-reactive T clones and thymic CD4+ regulatory T cell (tTreg) generation. Due to limited published data, it is largely unclear whether these two mechanisms are both defective in the involuted thymus, as a result of aging or other insults. We used mouse models with thymic epithelial cell (TEC) conditional defect (FoxN1 conditional knockout) and naturally aged defect to study mechanistic changes in central tolerance establishment. We identified a defect in thymocyte negative selection but not in tTreg generation. In comparison to tTreg with thymic conventional CD4+ T cells (tTcon) in the same thymus, the ratios were increased with severity of thymic involution, suggesting that activity of tTreg generation is increased in the involuted condition. We found that these changes were due to alterations in signaling strength received by T cell receptors, characterized by a decline of Nur77 and CD5 in CD4+ single positive thymocytes and an enhancement of Zap-70 in newly-generated tTregs. This is due to weakened signaling provided by MHC/self-peptide complex ligand from defective TECs, but not due to changes in tTreg development-required factors (TGFb, IL-2, IL-15, and NF-kB). Our results suggest that the involuted thymus attempts to balance the defective negative selection by enhancing tTreg generation to maintain central immune tolerance. If this balance is broken, age-related inflammatory diseases could take place.

Natural ThPOK− CD4 cytotoxic T lymphocytes : Thymic development of a new distinct self-specific CD4 T cell population.

Abstract The lineage commitment to either CD4 or CD8 T cells is made in the thymus, where thymocytes with specificity for MHC-II molecules differentiates into the CD4 “helper” linage whereas ones specific for MHC-I molecules commit to the CD8 “cytotoxic” lineage. The molecular regulation is controlled by the action of key transcription factors. Notably ThPOK, which promotes the CD4 fate and suppress the cytotoxic fate of CD4 T cells. Recently, we made a major breakthrough and showed that these lineages is not fixed and the mature peripheral CD4 T cells have the plasticity to lose ThPOK expression and reprogram to ThPOKneg CD4 CTL (cytotoxic T lymphocytes) marked by the expression of CD8a, Granzyme B and potent killer capacity. Investigating the origin of CD4 CTL, we observed in ThPOK fate maping mouse a strong proportion of CD4 CTL that never expressed ThPOK, suggesting that some CD4 CTL are natural cytotoxic cells that develop in the thymus. Based on a recent works allowing the analysis of mature CD4 cells in the thymus, we observed a substantial number of mature CD4 T cells that do not express ThPOK and display an active-like phenotype. Moreover, once injected, thymic ThPOKneg mature CD4 T cells remain ThPOKneg in the periphery and start to acquire CD8a expression demonstrating that natural CD4 CTL arise from the thymus. We also demonstrated that this population requires a strong TCR signal during their development, suggesting that natural CD4 CTL have a repertoire specific for self-antigens. We have identified a new distinct population of mature CD4 T cells that develop in the thymus: the natural cytotoxic CD4 T lymphocytes. We also established mechanism underlying these process by demonstrating that CD4 CTL needs a strong TCR signal for their differentiation.

Increased generation of regulatory T cells in the presence of reduced T cell receptor signaling capacity correlates with a reduction in positive selection of thymocytes

Abstract The T cell antigen receptor (TCR) is the primary signal transducing structure expressed on thymocytes and mature T cells, transmitting signals necessary for maturation and activation. The TCRα and β chains are rearranged from germline gene segments to generate a highly variable repertoire of antigen receptors. Thymocytes undergo a selection process in the thymus to ensure presence of a functional, but not highly autoreactive, T cell receptor on their surface. These processes, and T cell function, depend on the invariant CD3 molecules to transduce intracellular signals from the surface TCRαβ chains. We have created a knock-in mouse with Cre-recombinase-inducible tyrosine (6Y)-to-phenylalanine (6F) mutations in the 6 Immunoreceptor Tyrosine Activation Motif (ITAM) tyrosines of the 2 CD3ζ chains of the TCR/CD3 complex, resulting in a theoretical 60% decrease in TCR signaling potential. We found that pre-selection, but not post-selection, switching of wild-type (6Y) CD3ζ to non-signaling (6F) CD3ζ results in a significant increase in the percentage of thymic and peripheral regulatory CD4+ T cells (Tregs) relative to control germline 6Y CD3ζ mice. Mice with a pre-selection 6Y to 6F CD3ζ switch also have increased CD4+ CD25+ Foxp3− Treg precursors. This increase in Tregs and thymic Treg precursors is cell autonomous, and correlates with a reduction in positive, but not negative, selection of CD4SP thymocytes. Tregs with reduced signaling capacity do not, however, have suppressive capacity equivalent to wild-type Tregs, despite expressing normal levels of transcription factors and surface proteins characteristic of Tregs. Collectively, the data support an instructive, TCR-signal-based model of Treg lineage commitment in the thymus.

Tcf1 at the crossroads of CD4+ and CD8+ T cell identity

Transcription factors and DNA/histone modification enzymes work in concert to establish and maintain cell identity. CD4+ and CD8+ T cells are key players in cellular immunity with distinct functions. Recent studies offer novel insights into how their identities are established in the thymus and maintained in the periphery during immune responses. During thymic maturation, Thpok, HDAC1 and HDAC2 guard CD4+ T cells from activation of CD8+ cytotoxic genes, and Tcf1 and Lef1 utilize their intrinsic HDAC activity to shut down CD4+ lineage-associated genes in CD8+ T cells. In activated CD4+ T cells, Tcf1 and Lef1 act upstream of the Bcl6-Blimp1 axis to direct differentiation of follicular helper T (Tfh) cells, and prevent diversion of Tfh to IL-17-producing cells. In parallel, T-bet, together with Eomes or Blimp1, ensures proper induction of the cytotoxic program in CD8+ effectors elicited by acute infection, and prevents generation of pathogenic, IL-17-producing CD8+ effector T cells. Antigen persistence due to chronic viral infection leads to CD8+ T cell exhaustion. A portion of exhausted CD8+ T cells has the capacity to activate the Tfh program in a Tcf1-dependent manner. Those Tfh-like CD8+ Tcells exhibit enhanced proliferative capacity in response to PD-1 blockage therapy and are more effective in curtailing viral replication. Thus, dissecting the molecular aspects of T cell identity, during development and immune responses, may lead to new therapies for treating autoimmunity, tumors, and persistent infections.

Thymic Function Failure Is Associated With Human Immunodeficiency Virus Disease Progression.

Thymic function has been mainly analyzed with surrogate peripheral markers affected by peripheral T-cell expansion, making it difficult to assess the role of thymic failure in human immunodeficiency virus (HIV) disease progression. The assay of signal-joint/DβJβ T-cell rearrangement excision circles (sj/β-TREC ratio) overcomes this limitation but has only been assayed in small cohorts. Thus, the aim of this study was to determine the role of thymic function, measured by the sj/β-TREC ratio, on CD4 T-cell maintenance in prospective HIV cohorts that include patients with a wide age range and different immunological phenotypes. Seven hundred seventy-four patients including typical progressors, long-term nonprogressors (LTNPs), and vertically HIV-infected subjects were analyzed. Thymic function was quantified in peripheral blood samples using the sj/β-TREC ratio. Associations between thymic function and CD4 T-cell dynamics and combination antiretroviral therapy (cART) onset were analyzed using linear, logistic, and Cox proportional hazard models. Thymic function failure (sj/β-TREC ratio <10) was independently associated with HIV progression. In agreement, patients with distinctive high CD4 T-cell levels and low progression rates (vertically HIV-infected patients and LTNPs, including HIV controllers) had significantly higher thymic function levels whereas patients with thymic function failure had lower CD4 T-cell levels, lower nadir, and faster CD4 T-cell decay. This work establishes the relevance of thymic function, measured by sj/β-TREC ratio, in HIV disease progression by analyzing a large number of patients in 3 cohorts with different HIV disease progression phenotypes. These results support and help to understand the mechanisms underlying the rationale of early cART onset.

Survival of mature T cells depends on signaling through HOIP.

T cell development in the thymus is controlled by a multistep process. The NF-κB pathway regulates T cell development as well as T cell activation at multiple differentiation stages. The linear ubiquitin chain assembly complex (LUBAC) is composed of Sharpin, HOIL-1L and HOIP, and it is crucial for regulating the NF-κB and cell death pathways. However, little is known about the roles of LUBAC in T-cell development and activation. Here, we show that in T-HOIPΔlinear mice lacking the ubiquitin ligase activity of LUBAC, thymic CD4+ or CD8+ T cell numbers were markedly reduced with severe defects in NKT cell development. HOIPΔlinear CD4+ T cells failed to phosphorylate IκBα and JNK through T cell receptor-mediated stimulation. Mature CD4+ and CD8+ T cells in T-HOIPΔlinear mice underwent apoptosis more rapidly than control T cells, and it was accompanied by lower CD127 expression on CD4+CD24low and CD8+CD24low T cells in the thymus. The enforced expression of CD127 in T-HOIPΔlinear thymocytes rescued the development of mature CD8+ T cells. Collectively, our results showed that LUBAC ligase activity is key for the survival of mature T cells, and suggest multiple roles of the NF-κB and cell death pathways in activating or maintaining T cell-mediated adaptive immune responses.

Thymic CD4 T cell selection requires attenuation of March8-mediated MHCII turnover in cortical epithelial cells through CD83.

Deficiency of CD83 in thymic epithelial cells (TECs) dramatically impairs thymic CD4 T cell selection. CD83 can exert cell-intrinsic and -extrinsic functions through discrete protein domains, but it remains unclear how CD83's capacity to operate through these alternative functional modules relates to its crucial role in TECs. In this study, using viral reconstitution of gene function in TECs, we found that CD83's transmembrane domain is necessary and sufficient for thymic CD4 T cell selection. Moreover, a ubiquitination-resistant MHCII variant restored CD4 T cell selection in Cd83(-/-) mice. Although during dendritic cell maturation CD83 is known to stabilize MHCII through opposing the ubiquitin ligase March1, regulation of March1 did not account for CD83's TEC-intrinsic role. Instead, we provide evidence that MHCII in cortical TECs (cTECs) is targeted by March8, an E3 ligase of as yet unknown physiological substrate specificity. Ablating March8 in Cd83(-/-) mice restored CD4 T cell development. Our results identify CD83-mediated MHCII stabilization through antagonism of March8 as a novel functional adaptation of cTECs for T cell selection. Furthermore, these findings suggest an intriguing division of labor between March1 and March8 in controlling inducible versus constitutive MHCII expression in hematopoietic antigen-presenting cells versus TECs.

Ubiquitin ligase MARCH 8 cooperates with CD83 to control surface MHC II expression in thymic epithelium and CD4 T cell selection.

Major histocompatibility complex class II (MHC II) expression is tightly regulated, being subjected to cell type-specific mechanisms that closely control its levels at the cell surface. Ubiquitination by the E3 ubiquitin ligase MARCH 1 regulates MHC II expression in dendritic cells and B cells. In this study, we demonstrate that the related ligase MARCH 8 is responsible for regulating surface MHC II in thymic epithelial cells (TECs). March8(-/-) mice have elevated MHC II at the surface of cortical TECs and autoimmune regulator (AIRE)(-) medullary TECs (mTECs), but not AIRE(+) mTECs. Despite this, thymic and splenic CD4(+) T cell numbers and repertoires remained unaltered in March8(-/-) mice. Notably, the ubiquitination of MHC II by MARCH 8 is controlled by CD83. Mice expressing a mutated form of CD83 (Cd83(anu/anu) mice) have impaired CD4(+) T cell selection, but deleting March8 in Cd83(anu/anu) mice restored CD4(+) T cell selection to normal levels. Therefore, orchestrated regulation of MHC II surface expression in TECs by MARCH 8 and CD83 plays a major role in CD4(+) T cell selection. Our results also highlight the specialized use of ubiquitinating machinery in distinct antigen-presenting cell types, with important functional consequences and implications for therapeutic manipulation.

Distinct recirculation potential of CD69+CD103- and CD103+ thymic memory CD8+ T cells.

Tissue-resident memory T (TRM) cells occupy peripheral and lymphoid tissues where they confer protection against local infection. While epithelial CD8+ TRM cells coexpress CD69 and CD103, CD103- memory cells have been described in various organs and are often presumed non-recirculating based on their expression of CD69. We found that both CD69+CD103+ and CD69+CD103- memory cells populated the thymus upon transfer of CD8+ effector T cells into uninfected recipients. Transcriptionally and phenotypically, CD103+ thymic cells resembled non-lymphoid TRM cells, whereas CD69+CD103- cells displayed a profile that was more closely related to recirculating cells. Although CD69 was required for optimal CD103+ TRM formation, its expression alone did not identify permanently resident cells, as CD69+CD103- cells disappeared from the thymus following antibody-mediated depletion of recirculating cells. Our findings highlight a distinct migration potential of phenotypically divergent thymic CD8+ memory T cells and emphasise the inadequacy of CD69 as a marker of tissue residency.

The spleen dictates platelet destruction, anti-platelet antibody production, and lymphocyte distribution patterns in a murine model of immune thrombocytopenia.

For many years, splenectomy has been used to treat immune thrombocytopenia (ITP), and this procedure benefits approximately two-thirds of the treated patients. Although splenectomy may raise platelet counts, antibody-coated platelets and cytotoxic T lymphocytes appear to persist or can change over time. To better understand how the spleen may affect anti-platelet immune responses, we used a murine model of ITP demonstrating both antibody-mediated and T lymphocyte-mediated thrombocytopenia. Mice with severe combined immunodeficiency (SCID) were either splenectomized or not and transfused with splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61(+) platelets. Platelet counts and anti-platelet antibody levels were performed weekly. After 4weeks, the mice were sacrificed, and lymphoid organs were harvested and examined by flow cytometry to quantify CD4(+)CD25(+)FoxP3(+) Tregs and conventional cross-presenting XCR1(+) and tolerizing SIRPα+ dendritic cells. The results indicate that compared with control non-splenectomized mice, thrombocytopenia was improved and anti-platelet antibody production was significantly diminished in all splenectomized mice that received immune splenocytes. Splenectomized SCID mice also had a marked reduction in Tregs in the thymus together with an increased proportion of both thymic dendritic cell subsets that correlated with increased platelet counts. Of interest, although splenectomy diminished anti-platelet antibody production and raised platelet counts, marrow megakaryocyte densities were still significantly reduced in mice that received immune splenocytes. These results suggest that the spleen in murine ITP not only is the primary site responsible for platelet destruction, but it also controls, to a significant extent, the antibody response against platelets and the migration patterns of lymphocyte subsets.

Transcriptome analysis indicated that Salmonella lipopolysaccharide-induced thymocyte death and thymic atrophy were related to TLR4-FOS/JUN pathway in chicks.

BackgroundThymus is the crucial site for T cell development and once believed to be immune privileged. Recently, thymus has gained special attention as it is commonly targeted by infectious agents which may cause pathogenic tolerance and subsequent immunosuppression.ResultsWe analyzed thymic responses to the challenge with Salmonella typhimurium (STm) or lipopolysaccharide (LPS) derived from STm in chicks. Newly hatched chicks were injected intraperitoneally with 5 × 104 CFU/mL STm or 50 mg/kg LPS. After LPS treatment, maximum thymocyte death (3 ~ 5-fold change) compared to controls was found at 12 h, and maximum loss of thymic weight (35 %) and reduced thymic index (20 %) were found at 36 h. After STm infection, maximum thymocyte death and thymic atrophy occurred at 36 and 72 h, respectively. No significant changes of thymic structure, chT1+ and CD4+/CD8+ T cell ratio were observed in thymus or spleen tissues after LPS treatment. Furthermore, transcriptome analysis revealed important roles for the TLR4-FOS/JUN signaling pathway in thymic injury. Thus, the major process of thymic atrophy in this study first involved activation of transcriptional factors FOS/JUN upon LPS binding to TLR4 that caused release of inflammatory factors, thereby inducing inflammatory responses and DNA damage and ultimately cell cycle arrest and thymic injury.ConclusionsSTm and Salmonella LPS could induce acute chick thymic injury. LPS treatment acted faster than STm. TLR4-FOS/JUN pathway may play an important role in LPS induced chick thymic injury.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2674-6) contains supplementary material, which is available to authorized users.

Thymic hyperplasia after chemotherapy in adults with mature B cell lymphoma and its influence on thymic output and CD4+ T cells repopulation

ABSTRACTTo investigate the thymic regenerative potential in adults accepting chemotherapy for lymphoma. The dynamics of thymic activity in 54 adults from baseline to 12 mo post-chemotherapy was analyzed by assessing thymic structural changes with serial computed tomography (CT) scans, and correlating these with measurements of thymic output by concurrent analysis of single-joint (sj) T-cell receptor excision circles (sjTREC) and CD31+ recent thymic emigrants (RTE) in peripheral blood. Furthermore, the consequence of thymic renewal on peripheral CD4+ T cell recovery after chemotherapy was evaluated. Time-dependent changes of thymic size and thymic output assessed by both sjTREC levels and CD31+ RTE counts in peripheral blood were observed during and after chemotherapy. Enlargement of thymus over baseline following chemotherapy regarded as rebound thymic hyperplasia (TH) was identified in 20 patients aged 18−53 y (median 33 y). By general linear models repeated measure analysis, it was found that, patients with TH (n = 20) had a faster recovery of sjTREC levels and CD31+ RTE counts after chemotherapy than patients with comparable age, gender, diagnosis, disease stage, thymic volume and output function at baseline but without TH (n = 18) (p = 0.035, 0.047); besides, patients with TH had a faster repopulation of both naïve CD4+ T cell and natural regulatory CD4+ T cell subsets than those without TH (p = 0.042, 0.038). These data suggested that adult thymus retains the capacity of regeneration after chemotherapy, especially in young adults. The presence of TH could contribute to the renewal of thymopoiesis and the replenishment of peripheral CD4+ T cell pool following chemotherapy in adults.

Treatment of Post- HSCT Immunodeficiency By Infusion of Ex Vivo- Generated T Cell Precursors from Adult and Cord Blood Hematopoietic Stem and Progenitor Cells

Non-HLA identical hematopoietic stem cell transplantation (HSCT) provides a corrective therapy for most life-threatening primary immunodeficiencies (PID) and some malignant hemopathies. Despite advances made, severe complications following the treatment such as the prolonged persistence of T cell immunodeficiency still limit the use of this partially incompatible HSCT. After HSCT, the reconstitution of a functional T cell compartment relies on the availability of T cell precursors to rapidly seed the thymus and differentiate into mature T cells.We have previously demonstrated that an in vitro culture system based on the use of a modified Delta-like-4 (DLL4) Notch ligand and T cell cytokines allows for the effective generation of human T cell precursors from cord blood within 7 days. Moreover, once injected into NOD/SCID/gcko mice, T cell precursors generated in this system were able to colonize the thymus and generate a diversified and functional T-cell compartment. Here, we aimed at testing the capacity of adult HSPCs in this reconstitution system. We found that, like their CB- derived counterparts, T cell precursors generated from adult HPSCs phenotypically resembled thymic CD34+CD7+ cells with high in vitro T-cell differentiation potential. Interestingly, the peak of T cell progenitors for adult HSPCs occurred around day 3, compared to day 7 in CB. At this timepoint, T cell precursors derived from adult HSPC already expressed all critical genes for T cell lineage development, as well as the major chemokine receptors implicated in thymus homing. The introduction of retronectin further improved differentiation and proliferation of T cell progenitors from both HPSC sources in our in vitro system. Comparative molecular analysis of adult- and CB- derived progenitors suggested, that differential requirements for Notch receptor/ligand interactions may explain the differences in kinetics observed during the culture of the two types of HSPC. It remains to be further evaluated, whether targeted modifications of the Notch signaling pathway can improve the outcome of this in vitro T cell differentiation system for adult HPSCs.Overall our results suggest that adult HSPCs, like their CB- derived counterparts, provide an effective source of in vitro cultured T cell progenitors harboring all the necessary requirements for the in vivo -reconstitution of a functional T cell compartment. This is particularly important in the context of future clinical applications in HSCT where adult HSPCs are more available and more frequently used than CB HSPCs. Based on our results, we propose that upon injection into a patient, DLL4- cultured T cell precursors from both HSPC sources could significantly accelerate the reconstitution of the adaptive immune system after a partially HLA-incompatible HSCT. Currently, we are translating these results into a phase I clinical trial including adult and pediatric patients transplanted for malignant hemopathies or PIDs requiring an allogeneic HSCT from a HLA-partially mismatched donors. DisclosuresNo relevant conflicts of interest to declare.

Thymic B cells promote thymus-derived regulatory T cell development and proliferation

Thymic CD4+ FoxP3+ regulatory T (Treg) cells are critical for the development of immunological tolerance and immune homeostasis and requires contributions of both thymic dendritic and epithelial cells. Although B cells have been reported to be present within the thymus, there has not hitherto been a definition of their role in immune cell development and, in particular, whether or how they contribute to the Treg cellular thymic compartment. Herein, using both phenotypic and functional approaches, we demonstrate that thymic B cells contribute to the maintenance of thymic Treg cells and, using an in vitro culture system, demonstrate that thymic B cells contribute to the size of the thymic Treg compartment via cell–cell MHC II contact and the involvement of two independent co-stimulatory pathways that include interactions between the CD40/CD80/CD86 co-stimulatory molecules. Our data also suggest that thymic B cells promote the generation of thymic Treg cell precursors (pre-Treg cells), but not the conversion of FoxP3+ Treg cells from pre-Treg cells. In addition, thymic B cells directly promote the proliferation of thymic Treg cells that is MHC II contact dependent with a minimal if any role for co-stimulatory molecules including CD40/CD80/CD86. Both pathways are independent of TGFβ. In conclusion, we rigorously define the critical role of thymic B cells in the development of thymic Treg cells from non-Treg to precursor stage and in the proliferation of mature thymic Treg cells.

Selective deletion of estrogen receptor alpha in FoxP3+ CD4+ T cells influences peripheral T cell homeostasis (BA11P.139)

Abstract Being female confers a greater risk of developing an autoimmune disease than any other known genetic or environmental risk factor. Given that a loss of regulatory T cell (Treg) function contributes to autoimmunity and estrogen receptor alpha (ERα) promotes Tregs and suppresses experimental autoimmune encephalomyelitis (EAE), ERα-induced Tregs offers a new strategy to treat autoimmune disorders. The objective of these studies was to determine whether selective ablation of ERα in FoxP3+ Tregs dysregulates peripheral T cell homeostasis. To the end, mice harboring a floxed Esr1 gene (ERαf/f) were bred to mice expressing a YFP-Cre fusion protein driven by an IRES following foxp3 to obtain FoxP3+ Treg restricted deletion of Ers1. Thymic and peripheral CD4+ and CD8+ T cell frequency appeared unaffected with Esr1 ablation except for a sex-biased reduction in the total number of thymic and peripheral FoxP3+ Tregs and an increase in total number of splenocytes in ERαf/f FoxP3Cre mice compared to ERα+/+ FoxP3Cre mice. Spotaneous chronic inflammation and autoimmunity was undetected in ERαf/f FoxP3Cre mice. In a CD4+ T cell transfer model of experiment colitis, selective ablation of the ERα in FoxP3+ Tregs resulted in exacerbated inflammation compared to mice harboring ERα-sufficient Treg cells. Overall, these preliminary results suggest that FoxP3+ Treg-specific ERα is essential for the function of peripheral Tregs and immune homeostasis in a sex-biased manner.

Runx1 is required for T cell maturation (HEM2P.246)

Abstract T cell development depends on coordinated gene expression changes in order to progress through developmental checkpoints. The loss of transcription factor Runx1 in CD4-cre Runx1 conditional knockout (cKO) mice leads to decreased numbers of CD4 single positive (SP) thymocytes, CD4 peripheral T cells and lower expression of IL-7Rα, a key player of T cell homeostasis, by CD4+ Runx1 deficient T cells . Low IL-7Rα is thought to cause decreased CD4 T cell survival. However, we show that constitutive expression of an IL-7Rα transgene in CD4-cre Runx1 cKO mice does not restore thymic or peripheral CD4 cellularity. We also see similar expression of Bcl-2 and Bcl-xL in WT and Runx1-deficient CD4 T cells, indicating that a Runx1-mediated IL-7Rα-independent mechanism might be responsible. As IL-7Rα expression increases over the course of T cell maturation, we asked if Runx1 is needed for maturation. During thymic maturation, post-positive selection CD4 SP thymocytes migrate from cortex to medulla, changing from CCR7lowCD24high to CCR7highCD24high and finally CCR7highCD24low before thymic egress. We saw a block at the CCR7lowCD24high stage in CD4-cre Runx1 cKO CD4 SP thymocytes. Additionally, other maturation markers including CD55 and Qa2 were also downregulated. Thus, Runx1 is required for thymic maturation of CD4 SP thymocytes.