Single-positive CD4 Research Articles

Long-Term Human Immune Reconstitution, T-Cell Development, and Immune Reactivity in Mice Lacking the Murine Major Histocompatibility Complex: Validation with Cellular and Gene Expression Profiles.

Humanized mice transplanted with CD34+ hematopoietic cells (HPCs) are broadly used to study human immune responses and infections in vivo and for testing therapies pre-clinically. However, until now, it was not clear whether interactions between the mouse major histocompatibility complexes (MHCs) and/or the human leukocyte antigens (HLAs) were necessary for human T-cell development and immune reactivity. We evaluated the long-term (20-week) human hematopoiesis and human T-cell development in NOD Scid Gamma (NSG) mice lacking the expression of MHC class I and II (NSG-DKO). Triplicate experiments were performed with HPCs obtained from three donors, and humanization was confirmed in the reference strain NOD Rag Gamma (NRG). Further, we tested whether humanized NSG-DKO mice would respond to a lentiviral vector (LV) systemic delivery of HLA-A*02:01, HLA-DRB1*04:01, human GM-CSF/IFN-α, and the human cytomegalovirus gB antigen. Human immune reconstitution was detectable in peripheral blood from 8 to 20 weeks after the transplantation of NSG-DKO. Human single positive CD4+ and CD8+ T-cells were detectable in lymphatic tissues (thymus, bone marrow, and spleen). LV delivery harnessed the detection of lymphocyte subsets in bone marrow (αβ and γδ T-cells and NK cells) and the expression of HLA-DR. Furthermore, RNA sequencing showed that LV delivery increased the expression of different human reactome pathways, such as defense responses to other organisms and viruses. Human T-cell development and reactivity are independent of the expression of murine MHCs in humanized mice. Therefore, humanized NSG-DKO is a promising new model for studying human immune responses, as it abrogates the xenograft mouse MHC interference.

Abstract We059: Age-associated Alterations in T cell Output and Functions

The elderly have a higher risk for a wide range of diseases, including infection, tumor, and cardiovascular disease. This could be, at least partially, attributed to T lymphocyte changes during aging. The goal of this project was to characterize age-related changes in T cell output and functions. In this study, young (3-6 months old) and aged (18-24 months old) male and female C57BL/6J mice were utilized. Our data showed that compared to young mice, aged mice demonstrated significantly smaller thymus and fewer developing thymocytes, including double negative CD4 - CD8 - , double positive CD4 + CD8 + , as well as single positive CD4 + and CD8 + thymocytes, which implies that aged mice generate less T cells. Accordingly, circulating T cell count was significantly lower in aged mice than young controls. To investigate how aging affects T cell functions, we isolated splenic T cells and stimulated them with PMA (100 nM) + ionomycin (1 µM) (P+I) or vehicle for 4 hours. At the steady state, aged T cells expressed higher levels of Ifng , Tnfα , granzyme b , and perforin than young T cells, suggesting that aged T cells show a senescence-associated secretory phenotype (SASP). After stimulation with P+I, both young and aged T cells expressed dramatically higher levels of Ifng , Tnfα , Il2 , Il4 , Il17 , and granzyme b , indicative of T cell activation. Interestingly, aged T cells had higher expression of Ifng, Il17, and granzyme b, but lower Tnfα and Il2 levels. Moreover, aged T cells expressed higher levels of exhaustion markers, such as Pd1 , Ctla4 , Lag3 , and Tigit , indicating that aged T cells exhibit an exhaustion phenotype. In conclusion, our study demonstrates that aged mice show smaller thymus and fewer thymocytes and blood T cells than young mice. In addition, aged T cells exhibit a SASP and exhaustion phenotype. Upon activation, aged T cells might be more cytotoxic by secreting higher levels of IFN-γ, IL-17, and granzyme B.

Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells

Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells

Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells

Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells

Abstract 3979: PD-1 signals regulate NKG7 that is required for CD8+ T cells to sustain cytotoxicity and durable antitumor memory responses

Abstract Cytotoxic CD8+ T cells play a key role in response to anti-PD-1/PD-L1 immune checkpoint inhibitors (ICI) therapy. However, the exact effector molecules that are regulated by PD-1 signals in cytotoxic CD8+ T cells have not been completely defined. Lack of this knowledge is a crucial problem in addressing how to overcome the low clinical response rate to ICI therapy. When exploring why certain patients not responding to ICI therapy, we found non-responders did not have an elevated NKG7 expression in CD8+ T cells as responders to ICI therapy. The difference in NKG7 expression in CD8+ T cells between non-responders and responders to anti-PD-1 therapy prompted us to investigate the potential role of PD-1 signaling in regulating NKG7 expression. To that end, we generated a CD8+ T cell-specific Pdcd1 conditional knockout mice (CD8 E8ICre-Pdcd1fl/fl, also known as CD8-PD-1 cKO) where Pdcd1 is deleted after thymic selection of single positive CD8+ T cells. We observed a delayed tumor growth and prolonged survival in CD8-PD-1 cKO mice compared to control mice after B16-OVA melanoma tumor challenge. On Day 12, when the CD8-PD-1 cKO mice demonstrated their ability to control tumor growth, we conducted single cell RNA sequencing on isolated tumor infiltrating CD8+ T cells, revealing a significant increase of Nkg7 expression in effector CD8+ T cells in the Pdcd1 deficient CD8+ T cells compared to the control wild type T cells. Pdcd1-deficient effector CD8+ T cells also had increased expression of genes linked to NFAT/Calcineurin signaling pathway including Nfatc1, S100a4, Tbet and Tox, suggesting Nkg7 could be regulated by Pdcd1 via the NFAT pathway. To determine the impact of increased NKG7 expression in CD8+ T cells, we used OVA protein as a surrogate tumor antigen in vaccination. We found a complete B16-OVA tumor rejection in CD8-PD-1 cKO mice compared to control mice when tumor cells were injected on day 7 after vaccination with OVA protein and poly (I:C) (as an adjuvant), but only a partial rejection on day 21. The CD8-PD-1 cKO mice that were tumor-free for 30-40 days rejected 2nd tumor challenge with B16-OVA tumors. This long-term protection is tumor antigen (OVA) specific because these mice did not reject B16F10 tumor cells that do not express OVA antigen. We also found effector memory CD8+ T cells with high cytotoxicity increased in the spleen of CD8-PD-1 cKO mice after OVA antigen immunization compared to control. In conclusion, we found NKG7 is one of the effector molecules in cytotoxic CD8+ T cells, which can be regulated by PD-1 signals through NFAT signaling. Targeting the PD-1-NFAT pathway may be a new option to overcome low responses of T cells to ICI therapy in treatment of refractory cancer. Citation Format: Zhiming Mao, Joanina Gicobi, Jacob Hirdler, Xin Liu, Michelle Hsu, Emilia Dellacecca, Wenjing Zhang, Fabrice Lucien-Matteoni, Hai dong. PD-1 signals regulate NKG7 that is required for CD8+ T cells to sustain cytotoxicity and durable antitumor memory responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3979.

Genetically corrected RAG2-SCID human hematopoietic stem cells restore V(D)J-recombinase and rescue lymphoid deficiency

AbstractRecombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development and function by initiating the variable (V), diversity (D), and joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCIDs), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phase of the cell cycle in the early stages of T- and B-cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based “universal gene-correction” approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and RAG2-SCID patient. V(D)J-recombinase activity was restored after gene correction of RAG2-SCID-derived HSPCs, resulting in the development of T-cell receptor (TCR) αβ and γδ CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of CDR3 length and preserved usage of the distal TRAV genes. We confirmed the in vivo rescue of B-cell development with normal immunoglobulin M surface expression and a significant decrease in CD56bright natural killer cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients.

In Vivo Modeling of T-Cell Acute Lymphoblastic Leukemia Reveals Synergistic Oncogenic Pathways and Bcl11b Haploinsufficiency As a Potential Therapeutic Vulnerability

For decades, T-ALL models were based on retroviral overexpression of NOTCH1 in murine hematopoietic stem cells (HSCs). This induces aggressive T-ALL but does not reflect the genetic and clinical heterogeneity of the disease. Large-scale genomic analyses have mapped the detailed landscape of T-ALL driver events over recent years, but how these diverse oncogenic events interact in T-ALL pathogenesis and which vulnerabilities this creates remains incompletely explored. This is in part due to the lack of appropriate mouse models that recapitulate the complex disease biology and are suitable to identify and study potential therapeutic targets and interventions in a more realistic context. We modeled the genetic heterogeneity of T-ALL through in vivo multiplexing of gain-of-function and loss-of-function oncogenic events known to drive the disease. We developed a novel mouse strain that expresses Cre-recombinase and an inducible Cas9 only in the thymus ( LSL.Cas9 x Lck-cre) along with a lentiviral vector system into which T-ALL specific oncogenes are cloned in antisense flanked by LOX66/71 sites. Gain-of-function events, such as the overexpression of transcriptional regulators ( Tlx1, Tal1, or Lmo2) and signal transducers ( NRAS G12D, PIK3CD E1021K) were induced in developing thymocytes by Cre-mediated inversion together CRISPR-Cas9 editing of recurrent T-ALL loss-of-function drivers. This combinatorial approach allowed to probe more than 2000 possible oncogenic mutational combinations in vivo. Transplantation of HSCs from LSL.Cas9 x Lck-cre mice transduced with our vector system into sublethally irradiated mice led to T-ALL development with different morphology and disease phenotypes. Developing leukemia harbored reproducible phenotypes, covering very immature CD4-CD8-CD25+CD44+ T-ALL in the Tlx1_NRAS subgroup, in contrast to the classical cortical CD4+CD8+CD3- phenotype (Tlx1_PIK3CD subgroup), as well as mature single-positive CD4+CD3+ or CD8+CD3+ blasts (Tal1_Lmo2 subgroup). We identified Tlx1, Tal1, Lmo2, NRAS G12Dand PIK3CD E1021Kas the main driver of disease phenotype and gene expression profile, whereas the loss-of-function mutations contributed to and accelerated the onset of the disease. Tumor-derived cell lines could be easily established and kept their phenotypic characteristics and mutation patterns. The resulting T-ALL tumors contained up to eight different disease-relevant genetic alterations, thus recapitulating the genetic complexity in humans and overcoming the long latency for spontaneous mutations in less complex mouse models. Among these, co-evolution of insertions and deletions in Notch1, Cdkn2a, Bcl11b, and Pten were among the most frequently observed mutations in definitive T-ALL, whereas others, e.g., in Dnm2, Phf6, Etv6, and Lef1, occurred more randomly. While gene editing by CRISPR-Cas9 mostly resulted in frame-shift mutations in both alleles, Bcl11b exhibited a strong haploinsufficient phenotype with exactly one knock-out and one intact allele in each case examined. Following this observation, further suppression of Bcl11b by shRNAs in Bcl11b +/- cell lines derived from our tumor models demonstrated rapid induction of cell death, which was more intense in T-ALL cells compared to non-malignant thymocytes. In addition, re-analysis of CRISPR-based loss-of-function screens of human hematopoietic cell lines retrieved from the Cancer Dependency Map confirmed BCLL11B as a strong selective dependency in human T-ALL. In summary, we model subgroup-specific T-ALL transformation through conditional and multiplexed in vivo gene editing and oncogene overexpression, thus creating a resource of novel mouse models that recapitulate the genetic and biological heterogeneity of the disease. Using our resource, we identified Bcl11b as a strong and selective context-specific dependency in T-ALL. Although Bcl11b is a transcription factor, our results could serve as an starting point for novel therapies that interfere with Bcl11b function, such as molecular glue degraders or protein interaction inhibitors.

Ligustilide prevents thymic immune senescence by regulating Thymosin β15-dependent spatial distribution of thymic epithelial cells

BackgroundThymus is the most crucial organ connecting immunity and aging. The progressive senescence of thymic epithelial cells (TECs) leads to the involution of thymus under aging, chronic stress and other factors. Ligustilide (LIG) is a major active component of the anti-aging Chinese herbal medicine Angelica sinensis (Oliv.) Diels, but its role in preventing TEC-based thymic aging remains elusive. PurposeThis study explored the protective role of Ligustilide in alleviating ADM (adriamycin) -induced thymic immune senescence and its underlying molecular mechanisms. MethodThe protective effect of Ligustilide on ADM-induced thymic atrophy was examined by mouse and organotypic models, and conformed by SA-β-gal staining in TECs. The abnormal spatial distribution of TECs in the senescent thymus was analyzed using H&E, immunofluorescence and flow cytometry. The possible mechanisms of Ligustilide in ADM-induced thymic aging were elucidated by qPCR, fluorescence labeling and Western blot. The mechanism of Ligustilide was subsequently validated through actin polymerization inhibitor, genetic engineering to regulate Thymosin β15 (Tβ15) and Tβ4 expression, molecular docking and β Thymosin-G-actin cross-linking assay. ResultsAt a 5 mg/kg dose, Ligustilide markedly ameliorated ADM-induced weight loss and limb grip weakness in mice. It also reversed thymic damage and restored positive selection impaired by ADM. In vitro, ADM disrupted thymic structure, reduced TECs number and hindered double negative (DN) T cell differentiation. Ligustilide counteracted these effects, promoted TEC proliferation and reticular differentiation, leading to an increase in CD4+ single positive (CD4SP) T cell proportion. Mechanistically, ADM diminished the microfilament quantity in immortalized TECs (iTECs), and lowered the expression of cytoskeletal marker proteins. Molecular docking and cross-linking assay revealed that Ligustilide inhibited the protein binding between G-actin and Tβ15 by inhibiting the formation of the Tβ15-G-actin complex, thus enhancing the microfilament assembly capacity in TECs. ConclusionThis study, for the first time, reveals that Ligustilide can attenuate actin depolymerization, protects TECs from ADM-induced acute aging by inhibiting the binding of Tβ15 to G-actin, thereby improving thymic immune function. Moreover, it underscores the interesting role of Ligustilide in maintaining cytoskeletal assembly and network structure of TECs, offering a novel perspective for deeper understanding of anti thymic aging.

IPSC-Derived CD4 T Cell Generation and Investigation of CD4/CD8 T Cell Lineage Choice

iPSC-derived functionally rejuvenated antigen-specific cytotoxic T lymphocytes generated from exhausted CTLs show promising antitumor effect towards refractory tumors. Currently, we are preparing for clinical trials using iPSC-derived CTLs for EBV-associated lymphomas. These iPSC-derived T cells (iPSC-Ts) all generated into CD8 T cells, and stable generation of CD4 single positive (SP) T cells from iPSCs has never been accomplished without enforced CD4 gene transduction even though many research groups have challenged to do so. Even when an iPSC is established from a CD4 T cell clone, differentiating this iPSC does not generate into CD4 T cells, but in fact it generates into CD8 T cells. Therefore, we aimed to investigate why iPSC-Ts only differentiate into CD8T cells, and not into CD4T cells. We focused on adult T cell leukemia (ATL), because HTLV-1 infected CD4 T cells clonally expand. We reprogrammed CD4T cells obtained from acute-type ATL patients into iPSCs (ATL-iPSCs), then differentiated them into T cells using our established method. Interestingly, ATL-iPSCs from different donors all successfully differentiated into CD4 SP T cells (ATL-iPSC-Ts). Upon characterization of ATL-iPSC-Ts we confirmed that they showed FOXP3 positive naive regulatory T cell (Treg) phenotype. To investigate the function of these cells, we performed 51Cr-release assay. The cytotoxicity of HTLV-1 Tax-specific CTLs (Tax-CTLs) with or without ATL-iPSC-Ts against primary ATL cells was measured. The percentages of ATL cells lysed by Tax-CTLs was 34 ± 3.2%, whereas by the addition of ATL-iPSC-Ts, the cytotoxicity decreased to 23.4 ± 1.0% revealing the functionally suppressive effect of ATL-iPSC-Ts. In order to further understand the mechanism of CD4/CD8 T cell lineage choice, and discover the key regulators needed for generation of CD4 SP T cells from iPSCs, we performed single cell RNA-sequencing (scRNA-seq) on ATL-iPSC-Ts. As a control we used healthy donor (HD) CD4 SP T cell clone-derived iPSCs (HD-iPSCs), which differentiated into CD8 T cells (HD-iPSC-Ts [CD8 +]). We identified several genes expressed at a significantly higher level on HD-iPSC-Ts (CD8 +) than on ATL-iPSC-Ts (CD4 +). We presumed these genes to be essential for CD8 T cell differentiation. Therefore, we used CRISPR/Cas9 technology to knock out the respective gene in HD-iPSCs and confirmed if these iPSCs could differentiate into CD4 SP T cells, instead of CD8 T cells. This resulted in successful generation of CD4 SP T cells. We confirmed that these CD4 SP T cells were FOXP3 negative and were not Tregs. On the other hand, overexpression of this gene on ATL-iPSCs differentiated into CD8 SP T cells, instead of CD4 T cells. In conclusion, our work represents the first successful generation of natural CD4 SP T cells from iPSCs. The gene we identified appears to be a pivotal regulator of CD4/CD8 T cell lineage choice in our iPSC-T differentiation system. While more investigation is required, our method shows promise towards facilitating stable generation of CD4 T cells for T cell therapy.

FRI616 Combined Elf5a And Notch Inhibition Reprograms The Effector T Cell Fate Decisions Towards Treg Phenotype Without Any Off Target Effect

Abstract Disclosure: S. Rafiqi: None. A. Aldasouqi: None. S. faisal: None. S. Qureshi: None. A. Mahmood: None. J.C. Jaume: None. S. Imam: None. We explored immune resetting and plasticity of T effector cells towards T regulatory cells (CD4+CD25+FOXP3+) by elF5A and Notch inhibition. We validated our approach for plasticizing human T effector cells into Treg cells conducted on recent onset latent autoimmune diabetes of adult (LADA) patients (n=3), and nondiabetic patients (n=4) with simultaneous use of GC7 (100 μM) and anti-DLL4 (10 μg/ml). We also investigated the functional stability of plasticized Tregs and compared it with freshly isolated naïve Tregs from the same patient. Tregs were co-cultured with T effector cells and suppression/proliferation was accessed after 5 days. To create an ex-vivo Treg deficient environment, we sorted out CD4+ cells from the PBMCs. CD4+ deficient PBMCs and sorted single positive CD4+CD25- cells were treated with anti-DLL4+GC7+rhGAD65 with and without IL-2 supplementation and were compared with conventional anti-(CD3+CD28) stimulation and absolute control (20%FCS+RPMI). The presence of Treg cells was confirmed by flow cytometry. 30.5±5.1% of CD4 cells were plasticized into Tregs in the treatment group supplemented with IL-2 while only 18.8±4.6 % were plasticized in treatment group without IL-2 supplementation. Also, 11.2±3.1% plasticized to Tregs in treatment group in presence of CD3/CD28 microbeads compared to 6.19±2.4% in absolute control group. Similarly, 13±4.3% of CD8 cells had plasticized into Tregs in treatment group supplemented with IL-2 while only 11.5±6.7% plasticized in treatment group without IL-2 supplementation. 14.3±5.8% plasticized to Tregs in treatment group in presence of CD3/CD28 microbeads compared to 12.4±6.3% in absolute control group. Interestingly, the plasticized Tregs from LADA patients were more robust than those of non-diabetic patients at a 1:2 ratio (Treg:Tresp). Furthermore, the plasticized Tregs had comparable suppressive capacity over Tresp (CD4+CD25-) cells as that of freshly isolated naïve human Tregs. To investigate any off-target effects of (GC7 and anti-DLL4), live, dead, and apoptotic cell count at 24, 48, 96 hours, and 7 days post-treatment using dead cell apoptosis kit (Invitrogen). In our study, there was no significant difference between treatment and control groups in terms of live, dead, and apoptotic cells at different time intervals. On the 7th day of culture 64.06±8.66% and 68.88±6.15% of cells were live in treatment and absolute control groups respectively while 7.24±1.59% and 10.0±2.02% cells were found apoptotic respectively. Therefore, we define the reprogramming of T effector cells towards Treg cells with functional suppressive capacity using GC7 and anti-DLL4 without any off-target effect (toxicity). The present study expands the concept of T cell plasticity and establishes the functional stability of plasticized T cells that can accelerate pre-clinical trials for therapeutic use in T1D and as an adjunct to immunotherapies. Presentation: Friday, June 16, 2023

CD1b glycoprotein, a crucial marker of thymocyte development during T cell maturation in cynomolgus monkeys

Phenotypic markers that denote different developmental stages of thymocytes are important for understanding T cell development in the thymus. Here, we show that CD1b is a critical discriminator of thymocyte maturation stage in cynomolgus monkeys. CD1b was expressed by immature thymocytes prior to β-selection, and its expression decreased as cells became fully mature in the thymus. MHC-I expression was lowest at the CD3loCD1b+ immature double-positive (DP) stage, while the ratio of CD1d:MHC-I expression was significantly higher at this stage than at other developmental stages. PLZF was expressed by < 0.2% of thymocytes; most PLZF+ thymocytes were CD3-/loCD1b+ immature DP thymocytes with the potential to produce IL-4. EOMES+ thymocytes, which accounted for > 2% of total thymocytes, were mostly CD3+CD1b- mature thymocytes and predominantly of the CD8 single-positive (SP) lineage. An unconventional CD8+ T cell subset expressing the NKG2AC+CXCR3+ innate-like T cell marker was identified within the EOMES+ CD8 SP lineage; these cells exhibited a memory phenotype. Taken together, these findings show that CD1b is a valuable discriminatory marker of thymocyte development. The data presented herein can be used to characterize the features of PLZF- and EOMES-associated unconventional T cells in the thymus.

Abstract P3058: Myocardial Infarction Impairs T Lymphopoiesis In Mice

T lymphocytes, especially CD4 + T cells, play beneficial roles in regulating cardiac repair after acute myocardial infarction (MI). Some patients with MI show lymphopenia, a reduction in blood lymphocyte count. More importantly, lymphopenia negatively correlates with patient outcome. We have previously shown that circulating lymphocyte trafficking to the bone marrow contributes to lymphopenia post-MI. However, whether MI affects T cell production remains uninvestigated. The aim of this study was to investigate whether MI impairs T lymphopoiesis, the process of T cell development. Three to six months old male C57BL/6J mice underwent MI surgery by permanent ligation of the left anterior descending coronary artery. Sham controls were subject to the same procedure with the exception of artery ligation. At 7 days post-MI, we quantified thymic mass, cellularity, developmental thymocytes, and hematopoietic stem/progenitor cells in the bone marrow and thymus using multiparameter flow cytometry. Thymocyte apoptosis was evaluated using an Annexin V assay kit. Compared to sham controls, MI mice at day 7 post-MI exhibited smaller thymus, lower cellularity, and less thymocytes at different developmental stages, including double negative CD4 - CD8 - , double positive CD4 + CD8 + , and single positive CD4 + thymocytes. Early thymic progenitors (ETP) that generate thymocytes in the thymus were also lower in MI mice than sham counterparts. Interestingly, common lymphoid progenitors (CLP) in the bone marrow, the ancestors of ETP, were not affected by MI. To determine the reason that MI mice show less thymocytes, we assessed thymocyte apoptosis. The data showed that thymocytes in MI mice exhibited higher apoptotic rate than sham controls. In conclusion, our study, for the first time, demonstrates that MI impairs T lymphopoiesis, and this impairment mainly occurs in the thymus, but not in the bone marrow. The underlying mechanisms are related to increased thymocyte apoptosis.

T Cell Development and Function.

T cells play critical roles in the immune system, including in responses to cancer, autoimmunity, and tissue regeneration. T cells arise from common lymphoid progenitors (CLPs) that differentiate from hematopoietic stem cells in the bone marrow. CLPs then traffic to the thymus, where they undergo thymopoiesis through a number of selection steps, resulting in mature single positive naive CD4 helper or CD8 cytotoxic T cells. Naive T cells are home to secondary lymphoid organs like lymph nodes and are primed by antigen-presenting cells, which scavenge for both foreign and self-antigens. Effector T cell function is multifaceted, including direct target cell lysis and secretion of cytokines, which regulate the functions of other immune cells (refer to "Graphical Abstract"). This review will discuss T cell development and function, from the development of lymphoid progenitors in the bone marrow to principles that govern T cell effector function and dysfunction, specifically within the context of cancer.

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

Inflammatory CD4/CD8 double-positive human T cells arise from reactive CD8 T cells and are sufficient to mediate GVHD pathology.

An important paradigm in allogeneic hematopoietic cell transplantations (allo-HCTs) is the prevention of graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) activity of donor T cells. From an observational clinical study of adult allo-HCT recipients, we identified a CD4+/CD8+ double-positive T cell (DPT) population, not present in starting grafts, whose presence was predictive of ≥ grade 2 GVHD. Using an established xenogeneic transplant model, we reveal that the DPT population develops from antigen-stimulated CD8 T cells, which become transcriptionally, metabolically, and phenotypically distinct from single-positive CD4 and CD8 T cells. Isolated DPTs were sufficient to mediate xeno-GVHD pathology when retransplanted into naïve mice but provided no survival benefit when mice were challenged with a human B-ALL cell line. Overall, this study reveals human DPTs as a T cell population directly involved with GVHD pathology.

Distinct populations of eosinophils in the human thymus with capacity to modulate thymocyte maturation.

Local differentiation of eosinophil precursors occurs in the human thymus. Thymic eosinophils are often positioned in the corticomedullary junction between the CD4+ CD8+ double-positive (DP) thymocytes and the CD4+ or CD8+ single-positive (SP) thymocytes. The aims of this study were to (1) determine if there are distinct thymic eosinophil populations that differ from the blood eosinophil populations and (2) evaluate the capacity of thymic eosinophils to promote the development of SP thymocytes from DP thymocytes. Thymic and blood eosinophils from thymectomized infants (n=7) were compared regarding the expression of 34 molecules using cytometry by time-of-flight (CyTOF). In addition, FACS-sorted thymic eosinophils were co-cultured with autologous CD3/CD28-stimulated DP, CD4 SP, and CD8 SP thymocytes and analysed by flow cytometry and CyTOF. X-shift clustering analysis and viSNE dimensionality reduction were performed. Seven eosinophil populations were identified within the blood and thymus, respectively, five of which were specific for either tissue. Whereas the blood eosinophil populations varied between individuals, the thymic eosinophil populations were more uniform. The eosinophil-thymocyte co-cultures resulted in (1) an increase in CD4 SP thymocytes when eosinophils were cultured with DP thymocytes, (2) decreased frequency of CD8 SP thymocytes when these were cultured with eosinophils, and (3) a more mature thymic phenotype when eosinophils were cultured with CD4 SP thymocytes. Thymic eosinophils are a specialized population of eosinophils with a distinct phenotype that separates them from their blood counterparts, and in vitro they appear to favour CD4 SP thymocyte development to the detriment of CD8 SP thymocytes.

IGF-1 increases survival of CD4+ lineage in a 2D model of thymocyte/thymic stromal cell co-culture.

Insulin-like growth factor-1 (IGF-1), in addition to its classic effects on cell proliferation and organism growth, has pleiotropic actions on the immune system, particularly on the thymus. Thus, the objective of this study was to evaluate the influence of IGF-1 on molecules involved in the survival of thymocytes in vitro using a co-culture system with thymic stromal cells obtained from C57BL/6 mice. The obtained thymic stroma has contained thymic epithelial cells, macrophages, dendritic cells, fibroblasts, and preserved the expression of the major histocompatibility complex (MHC) molecules. Fresh thymocytes were added to these cultures and the co-culture were treated daily with IGF-1 (100ng/mL) for 3days. In this scheme, the viability of the thymocytes was about 70%, either in the control (non-treated cells) or in the IGF-1-treated cultures. It was found that IGF-1 was able to increase the percentage of thymocytes from the CD4+ single-positive (SP) subset. This result was accompanied by an increase in the MHC II expression on thymic stromal cells and an augment on the interleukin-7 receptor (CD127) on the surface of the CD4 SP thymocytes after treatment with IGF-1. Finally, IGF-1 treatment increased the expression of the ThPOK encoding gene Zbtb7b, which is involved in the differentiation of CD4+ SP thymocytes. Our study demonstrates the participation of IGF-1 in the thymocyte/thymic stroma interactions, especially in the extended survival of the CD4+ lineage in the thymus.

Tracking Regulatory T Cell Development in the Thymus Using Single-Cell RNA Sequencing/TCR Sequencing.

Recent studies have demonstrated that regulatory T cells (Tregs) develop in the thymus via two pathways involving distinct Treg progenitors (TregP): CD25+FOXP3- (CD25+ TregP) and CD25-FOXP3lo (FOXP3lo TregP) Treg progenitors. To examine this process in more detail, we carried out single-cell RNA sequencing (scRNA-Seq) and TCR-Seq on sorted murine CD4+CD8+ double-positive (DP) thymocytes, CD4+ single-positive (CD4SP) thymocytes, CD25+FOXP3-CD73- TregP, CD25-FOXP3loCD73- TregP, newly generated mature CD25+FOXP3+CD73- Tregs, and FOXP3+CD73+ recirculating/long-term resident Tregs (RT-Tregs). Sorted populations were individually hashtagged and then combined into one scRNA-Seq/TCR-Seq library before sequencing and subsequent analysis. We found that both CD25+ TregP and FOXP3lo TregP arise via an initial agonist-activated state that gives rise to a second transitional stage before differentiating into mature Tregs Using both scRNA-Seq and bulk RNA-Seq on sorted thymocyte subsets, we demonstrate that CD25+ TregP are significantly enriched for Il2 production, suggesting that they are the major source of IL-2 needed to convert TregP into mature Tregs Using TCR-Seq, we found that several TCRs were clearly biased in favor of the conventional or Treg lineages, but that a large fraction of TCRs were found in both these lineages. Finally, we found that RT-Tregs in the thymus are not monomorphic but are composed of multiple distinct subsets and that these RT-Tregs express the most diverse TCR repertoire of all CD4SP thymocytes. Thus, our studies define multiple stages of Treg differentiation within the murine thymus and serve as a resource for future studies on CD4+ thymocyte development and Treg differentiation.

The circadian clock sets a spatial-temporal window for recent thymic emigrants.

The diurnal timing system regulates multiple functions of lymphocytes in peripheral lymphoid organs. Whether T-cell development in the thymus and T-cell egress from the thymus are affected by the circadian clock is not clear. Herein, we used flow cytometry to examine the cell number and percentage of total thymocytes and various thymocyte subsets from Zeitgeber time (ZT) 1 to ZT21. CD4 and CD8 single-positive (SP) thymocytes, in particular, the mature CD4 SP4 thymocyte subset with emigration capability and P-phycoerythrin+ CD4 SP thymocytes in the perivascular space of the thymus, exhibited robust circadian oscillations. The diurnal expression of sphingosine-1-phosphate receptor-1 (S1PR1) and CCR2 on SP thymocytes and the rhythmic sphingosine-1-phosphate (S1P) and CCL2 gradient formed between peripheral blood and thymus likely promoted SP thymocyte egress in a circadian pattern. Switching the daylight cycle disturbed the rhythm of S1PR1 and CCR2 expression and subsequent thymocyte output. We further demonstrated that the core clock molecule BMAL1 had rhythmic binding of the promoters of Klf2, S1pr1 and Sphk2. Together, we elucidated the circadian dynamic characteristics of mature thymocyte egress, which coordinated with the diurnal changes in T-cell homing to the lymph nodes. The core rhythmic molecule BMAL1 likely promoted thymocyte emigration through transcriptional regulation of emigration-related molecules.

TET proteins regulate T cell and iNKT cell lineage specification in a TET2 catalytic dependent manner.

TET proteins mediate DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and other oxidative derivatives. We have previously demonstrated a dynamic enrichment of 5hmC during T and invariant natural killer T cell lineage specification. Here, we investigate shared signatures in gene expression of Tet2/3 DKO CD4 single positive (SP) and iNKT cells in the thymus. We discover that TET proteins exert a fundamental role in regulating the expression of the lineage specifying factor Th-POK, which is encoded by Zbtb7b. We demonstrate that TET proteins mediate DNA demethylation - surrounding a proximal enhancer, critical for the intensity of Th-POK expression. In addition, TET proteins drive the DNA demethylation of site A at the Zbtb7b locus to facilitate GATA3 binding. GATA3 induces Th-POK expression in CD4 SP cells. Finally, by introducing a novel mouse model that lacks TET3 and expresses full length, catalytically inactive TET2, we establish a causal link between TET2 catalytic activity and lineage specification of both conventional and unconventional T cells.