Stages Of T-cell Differentiation Research Articles

Excess of body weight is associated with accelerated T-cell senescence in hospitalized COVID-19 patients

BackgroundSeveral risk factors have been involved in the poor clinical progression of coronavirus disease-19 (COVID-19), including ageing, and obesity. SARS-CoV-2 may compromise lung function through cell damage and paracrine inflammation; and obesity has been associated with premature immunosenescence, microbial translocation, and dysfunctional innate immune responses leading to poor immune response against a range of viruses and bacterial infections. Here, we have comprehensively characterized the immunosenescence, microbial translocation, and immune dysregulation established in hospitalized COVID-19 patients with different degrees of body weight.ResultsHospitalised COVID-19 patients with overweight and obesity had similarly higher plasma LPS and sCD14 levels than controls (all p < 0.01). Patients with obesity had higher leptin levels than controls. Obesity and overweight patients had similarly higher expansions of classical monocytes and immature natural killer (NK) cells (CD56+CD16−) than controls. In contrast, reduced proportions of intermediate monocytes, mature NK cells (CD56+CD16+), and NKT were found in both groups of patients than controls. As expected, COVID-19 patients had a robust expansion of plasmablasts, contrasting to lower proportions of major T-cell subsets (CD4 + and CD8+) than controls. Concerning T-cell activation, overweight and obese patients had lower proportions of CD4+CD38+ cells than controls. Contrasting changes were reported in CD25+CD127low/neg regulatory T cells, with increased and decreased proportions found in CD4+ and CD8+ T cells, respectively. There were similar proportions of T cells expressing checkpoint inhibitors across all groups. We also investigated distinct stages of T-cell differentiation (early, intermediate, and late-differentiated – TEMRA). The intermediate-differentiated CD4 + T cells and TEMRA cells (CD4+ and CD8+) were expanded in patients compared to controls. Senescent T cells can also express NK receptors (NKG2A/D), and patients had a robust expansion of CD8+CD57+NKG2A+ cells than controls. Unbiased immune profiling further confirmed the expansions of senescent T cells in COVID-19.ConclusionsThese findings suggest that dysregulated immune cells, microbial translocation, and T-cell senescence may partially explain the increased vulnerability to COVID-19 in subjects with excess of body weight.

Decoding the Immune Landscape of Pathogenic T Cells in Acute Graft-Versus-Host Disease By Single-Cell RNA-Seq

Introduction Graft-versus-host disease (GvHD) is the major cause of non-relapse mortality in allogeneic hematopoietic cell transplantation (alloHCT), and about 40-60% cases would progress into steroid refractory (SR) aGvHD resulting in dismal outcomes. The pathophysiology of aGvHD is complex, and donor derived T cells are identified as the culprits of aGvHD. However, the relationships among distinct T cell subsets and molecular mechanisms underlying aGvHD remain incompletely understood. Methods We employed a single-cell RNA sequencing (scRNA-seq) and T cell receptor (TCR) sequencing (scTCR-seq) to profile 49288 T cells in peripheral blood isolated from alloHCT recipients developed into aGvHD (n=9) or not (as control, n=5). Then, samples from SR-aGvHD patients were further investigated to explore the molecular mechanism of pathogenic T cell subsets in SR-aGvHD. Results A total of thirteen unique T cell subsets with distinct molecular properties were identified, including four clusters of CD8+ T cells (CD8+ TN, CD8+ TCM, CD8+ TEM and CD8+ TEFF), five clusters of CD4+ T cells (CD4+ TN, CD4+ TCM, Th1, Th17 and Treg), NK cell cluster and three clusters of other T cells (IFN-T, γδT and MAIT). The composition shifted towards more-developed CD8+ TCM, CD8+ TEM, CD4+ TCM and Th1 cells in aGvHD group due to rapid cell cycling. The expansion of CD8+ TEM subpopulation was confirmed by multiparameter flow cytometry. The CD8+ TEFF and Th1 were considered to be pathogenic in aGvHD as they exhibited aGvHD-related cytokine/granzyme expression and signaling pathway activation. Upregulated genes in aGvHD-derived-Th1 cells were related to chemotaxis (CCL3, CXCR6), cytokine production (type I interferon, IL-23) and JAK-STAT signaling activation. CD8+ TEFF cells showed activity of chemotaxis (CCL3, CXCR6), granzyme and cytokine expression (GZMB, GZMA, TNF-α, IL-6, IL-10, IL-12) and MAPK/MTOR/NF-kappaB activation. The different composition of cytokine expression and activated signaling pathways suggested distinct functions of each subset. Furthermore, CD8+ TEM represented four traits related to a range of T-cell differentiation stages, spanning rapid cell cycling (transcription factor E2F family), chemotaxis (CCL3, CXCR6), cytotoxicity (GZMA, GZMB, and GZMK) and exhaustion (PDCD1, LAG3, HAVCR2 and TIMD4), suggesting CD8+ TEM as a transient stage which might further develop into CD8+ TEFF for aGvHD pathogenesis (Figure 1). To elucidate the molecular mechanism of SR-aGvHD, we dissected the composition and functional heterogeneity of T-cell subsets between SR- and steroid sensitive (SS)-aGvHD. CD8+ TEM expanded to be the largest subset which resulted in the increase of CD8+ TEFF proportion in SR-aGvHD. CD8+ TEM and CD8+ TEFF showed enrichment of GvHD pathogenic scores and up-regulated expression of inflammation transcription factors STAT1 and STAT3 in SR-aGvHD. Of note, the expression of steroid receptor NR3C1 was downregulated in CD8+ TEM subset, as well as the steroid target gene ANXA1, compared with counterparts. Furthermore, both CD8+ TEM and CD8+ TEFF cells exhibited enrichment of JAK-STAT, MAPK, NF-kappaB, and PI3K-AKT signaling activation (Figure 2). Conclusions The effector T-cell subsets CD8+ TEFF and Th1 were co-activated and undertook distinct expression profiles and signaling pathways, which suggests their different roles in aGvHD pathogenesis. CD8+ TEM, characterized by proliferative, cytotoxicity, chemotaxis and exhaustion capacities, provided functional repertoire for CD8+ TEFF. Reduced NR3C1 expression and activation of by-pass survival pathways of CD8+ TEM might provide persistent differentiation origin of CD8+ TEFF to mediate resistance to steroid therapy. Collectively, our data described the immune landscape of aGvHD and provided novel therapeutic targets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Transcriptome Profiling of Porcine Naïve, Intermediate and Terminally Differentiated CD8+ T Cells.

The pig has the potential to become a leading research model for human diseases, pharmacological and transplantation studies. Since there are many similarities between humans and pigs, especially concerning anatomy, physiology and metabolism, there is necessity for a better understanding of the porcine immune system. In adaptive immunity, cytotoxic T lymphocytes (CTLs) are essential for host defense. However, most data on CTLs come from studies in mice, non-human primates and humans, while detailed information about porcine CD8+ CTLs is still sparse. Aim of this study was to analyze transcriptomes of three subsets of porcine CD8β+ T-cell subsets by using next-generation sequencing technology. Specifically, we described transcriptional profiles of subsets defined by their CD11a/CD27 expression pattern, postulated as naïve (CD8β+CD27+CD11alow), intermediate differentiated (CD8β+CD27dimCD11a+), and terminally differentiated cells (CD8β+CD27-CD11ahigh). Cells were analyzed in ex vivo condition as well as upon in vitro stimulation with concanavalin A (ConA) and PMA/ionomycin. Our analyses show that the highest number of differentially expressed genes was identified between naïve and terminally differentiated CD8+ T-cell subsets, underlining their difference in gene expression signature and respective differentiation stages. Moreover, genes related to early (IL7-R, CCR7, SELL, TCF7, LEF1, BACH2, SATB1, ZEB1 and BCL2) and late (KLRG1, TBX21, PRDM1, CX3CR1, ZEB2, ZNF683, BATF, EZH2 and ID2) stages of CD8+ T-cell differentiation were highly expressed in the naïve and terminally differentiated CD8+ T-cell subsets, respectively. Intermediate differentiated CD8+ T-cell subsets shared a more comparable gene expression profile associated with later stages of T-cell differentiation. Genes associated with cytolytic activity (GNLY, PRF1, GZMB, FASL, IFNG and TNF) were highly expressed in terminally and intermediate differentiated CD8+ T-cell subsets, while naïve CD8+ T cells lacked expression even after in vitro stimulation. Overall, PMA/ionomycin stimulation induced much stronger upregulation of genes compared to stimulation with ConA. Taken together, we provided comprehensive results showing transcriptional profiles of three differentiation stages of porcine CD8+ T-cell subsets. In addition, our study provides a powerful toolbox for the identification of candidate markers to characterize porcine immune cell subsets in more detail.

Artificial thymic organoids reveal an early block in T cell differentiation in patients carrying mutations in IKZF1

Abstract IKZF1 is an essential transcription factor expressed throughout hematopoiesis and involved in both lymphocyte and myeloid differentiation. Heterozygous germline mutations in IKZF1 give rise to distinct clinical phenotypes. IKZF1 haploinsufficiency causes common variable immunodeficiency (CVID) associated with B cell immune deficiency, B-ALL susceptibility and autoimmune manifestations, without clinical T cell defects. Patients carrying dominant negative (DN) IKZF1 mutations have combined immunodeficiency (CID), with an increased proportion of naïve T cells and impaired T cell function. Mouse models of IKZF1 mutations have a distinct phenotype from patients and some models present a more severe defect in T cell development. To address these controversies and gain novel insights into the effects of distinct IKZF1 mutations on human T cell development, we used the artificial thymic organoid (ATO) system, based on the 3D aggregation and culture of a delta-like Notch ligand 4-expressing stromal cell line (MS5-Dll4) with CD34+ cells, to analyze T cell development from CD34+ cells obtained from one patient with IKZF1 haploinsufficiency and one patient with DN IKZF1 mutation. Both patients showed a similar early block in T-cell differentiation at pre-T cell stage. However, the patient with IKZF1 haploinsufficiency showed a more pronounced leakiness, with a residual production of CD3+TCRαβ+ cells, which could account for the milder T-cell phenotype. Interestingly, the DN patient presented an increased accumulation of CD4−CD8β− CD8αα+ cells. These results show an unexpected role for IKZF1 in humans in early stages of T-cell differentiation and indicate IKZF1 as a necessary factor for the induction of CD8β expression in T cells.

Cocaine Use Disorder Is Associated With Changes in Th1/Th2/Th17 Cytokines and Lymphocytes Subsets.

Background: Cocaine is a psychostimulant drug with high addictive proprieties. Evidence suggests that cocaine use leads to critical changes in the immune system, with significant effects on T, B, and natural killer (NK) cells and influencing peripheral levels of cytokines. The presence of abstinence-related symptoms during detoxification treatment is known to influence the prognosis. Here, our aim was to investigate immune profiles in women with cocaine use disorder (CUD) according to withdrawal symptoms severity.Methods: Blood samples and clinical data were collected at onset of detoxification treatment of 50 women with CUD. The patients were stratified according to Cocaine Selective Severity Assessment (CSSA) scores in low withdrawal (L-W) and high withdrawal (H-W) categories. In addition, we also included a control group with 19 healthy women as reference to immune parameters. Peripheral blood was collected and lymphocyte subsets were phenotyped by multi-color flow cytometry (B cells, CD4+ T, CD8+ T, NK cells, and different stages of T-cell differentiation). PBMCs from patients and healthy controls were stimulated in vitro with phytohemagglutinin (1%) for 72 h to assess the production of Th1/Th2/Th17 cytokines.Results: Following stimulation, lymphocytes from women with CUD produced increased levels of Th1/Th2/Th17 cytokines. However, higher levels of IL-2 and IL-17 were observed only in the L-W group, while higher levels of IL-6 were detected in the H-W group compared to controls. H-W group showed lower percentage of early-differentiated Th cells (CD4+CD27+CD28+), elevated percentage of Th cells (CD3+CD4+), intermediate-differentiated Th cells (CD4+CD27−CD28+), and B cells (CD3−CD19+). Both CUD groups showed decreased percentages of naïve T cells (CD3+CD4+CD45RA+ and CD3+CD8+CD45RA+).Conclusion: Our data demonstrated that CUD can lead to increased production of Th1/Th2/Th17 cytokines and lymphocyte changes.

Maladie de Castleman localisée avec prolifération lymphoblastique T indolente

Herein we report the case of a 41-year-old woman who presented with pelvic pain. Magnetic Resonance Imaging exhibited a single pelvic mass, measuring 50mm long axis, alongside the right iliac vessels. Histological examination of the excision specimen showed a lymphoid tumor with features of localized Castleman disease, hyaline vascular type. Moreover we identified multiple interfollicular dark clusters, composed of cells morphologically resembling cortical thymocytes. Their immunophenotype was consistent with an intermediate stage of T-cell differentiation, with the expression of CD3, CD4, CD8, TdT, CD1a, CD99, CD2, CD5, CD7 and CD10, with 40% Ki67. After integration of clinical and molecular data, the retained diagnosis was an indolent T-cell lymphoblastic proliferation associated with hyaline vascular localized Castleman disease. The clinical course confirmed the indolent nature of the proliferation, despite a late local recurrence at 7 years of the initial diagnosis, without histological modification, due to an incomplete initial resection surgery.

ZEB2 and LMO2 Drive Immature T-Cell Lymphoblastic Leukemia Via Distinct Oncogenic Mechanisms

ZEB2, a E-box binding homeobox transcription factor, is able to induce epithelial-to-mesenchymal transition in the context of solid tumors. As such, its expression is correlated with cancer cell invasion, dissemination and metastasis, but also with the acquisition of cancer stem cell properties and therapy resistance.Using conditional knockout mice, we previously have shown that ZEB2 is an essential hematopoietic transcription factors, indispensable for normal embryonic(1) and adult(2) hematopoiesis. In addition, using conditional overexpressing mice we have demonstrated that ZEB2 is a bona fide driver of an immature subtype of T-ALL with increased leukemia-initiating potential(3).Here, we investigated the effects of ZEB2 overexpression on normal T-cell differentiation. Already from young age, ZEB2-overexpressing mice have a smaller thymus, with increased percentages of early T-cell precursors. Detailed flow cytometric analysis indicate a T-cell differentiation delay at the beta-selection checkpoint, with accumulation of DN3 (CD4- CD8- CD44- CD25+) cells. Using in vitro differentiation assays on OP9-DL1 stromal cells, we demonstrate that the perturbed T-cell differentiation is independent of the thymic micro-environment. Lowering the dose of exogenous interleukin-7 levels in the culture media can partially rescue this phenotype, which suggests that ZEB2-overexpressing thymocytes are delayed in T-cell differentiation due to their inability to downregulate the interleukin-7 receptor (IL7R) levels.Both the perturbed T-cell differentiation, and the latency/penetrance of immature subtype of the T-ALL formed in ZEB2-overexpressing mice is similar to what has been described for LMO2-overexpressing mice. Based on this phenocopy, we hypothesized that ZEB2 and LMO2 induce immature T-ALL via converging oncogenic pathway and/or via regulating similar crucial genes for the T-cell differentiation program. However, in contrast to LMO2, ZEB2-overexpresssing pre-leukemic thymocytes do not display aberrant self-renewal potential as demonstrated by transplantation experiments in sublethal irradiated syngeneic hosts. In addition, by using multiple Cre-transgenic lines, allowing the expression of ZEB2 at different stages of T-cell differentiation, we demonstrate that the oncogenic potential of ZEB2 is independent of the perturbed T-cell differentiation observed during the early stage of the disease.Based on these results we conclude that ZEB2 and LMO2 both drive the development of an immature subtype of T-ALL, but via distinct oncogenic mechanisms.(1) Goossens S. et al., Blood 2011 May 26; 117(21):5620-30.(2) Li J. et al., Blood 2017 Jan 26; 129(4):460-472.(3) Goossens S. et al., Nat Commun. 2015 Jan 7;6:5794. DisclosuresNo relevant conflicts of interest to declare.

Pediatric T-ALLs Developing into a Type 2 Relapse Originate from Cells That Carry the Potential of Variable Maturation into Subclones with Distinct Chromatin Landscapes

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that is classified according to surface marker expression. In order to reveal the cells of origin in pediatric T-ALL and to understand mechanisms of relapse we used ATAC-Seq (Assay for Transposase-Accessible Chromatin Sequencing) to compare chromatin accessibility landscapes of healthy T-cell precursors to those of T-ALL cells obtained at initial diagnosis (INI) and relapse (REL).We have FACS sorted 7 differentiation stages of normal T-cell precursors contained in the thymus of infants undergoing cardiac surgery (DN2, DN3, ISP, DPCD3-, DPCD3+, CD4+ and CD8+) and subjected these to ATAC-Seq. Unsupervised learning by principal component analysis (PCA) clustered sorted populations according to the maturation stage, demonstrating that regulatory chromatin signatures of thymocytes are highly stage-specific and re-shaped during T-cell differentiation.We next compared normal T-cell precursors at different stages of maturation to pediatric T-ALLs and found fundamental differences with 30% of open chromatin regions to be more and 28% being less accessible in T-ALL (DESeq2, padj<.05), respectively. We next identified transcription factor (TF) binding sites in open chromatin regions of normal T-cell precursors and T-ALL cells by HOMER analysis. We found 76% of the accessible TF binding sites in double negative (DN2 and DN3) normal precursors to be also accessible in most (20/24) leukemias comparing to 17% of the sites accessible in the more mature (CD4+ and CD8+) stages. These data indicate that T-ALLs originate from cells with an epigenetic profile of early thymic progenitors.We then subjected the ATAC-seq data of all matched leukemia samples obtained at initial disease and at relapse to PCA. INI and REL samples derived from the same patient always clustered in close proximity and were separated according to the T-ALL driving fusion genes. A global analysis of differential accessibility revealed only 0.26% of ATAC-regions to be less- or more-accessible at relapse when compared to the matched initial samples (DESeq2, padj<.05). These data indicate chromatin accessibility to be largely determined by the cell of origin and to generally remain stable during progression from initial diagnosis to relapse. We then considered the 2 types of relapse separately, which we have previously characterized on the basis of subclonal mutation profiles (Kunz et al. Haematologica, 2015). These relapse types are defined by either clonal evolution of cells derived from the predominant clone at primary disease (type 1) or emergence and evolution of a minor initial clone showing a molecular profile that differs from the major initial clone (type 2). We found that the proportion of differentially accessible ATAC-regions between INI and REL is significantly higher in type 2 (1.3%) than in type 1 (0.006%) (DESeq2, padj<.05).Moreover, we trained the deconvolution algorithm CIBERSORT to recognize particular T-cell differentiation stages using ATAC-profiles of the 7 FACS-sorted healthy T-cell populations. We used regulatory chromatin landscape of non-sorted (total) thymus to assess the accuracy of deconvolution. Comparison of predicted fractions in total thymus to FACS measurements revealed highly accurate identification of the maturation stages (r2 = 0.95). CIBERSORT analysis confirmed that the profiles were largely preserved between INI and REL of each sample pair. Notably, however, while in T-ALLs that later developed into a type 1 relapse only one type of early T-cell progenitor dominated the deconvolution profile, T-ALLs that developed into a type 2 relapse showed heterogeneous profiles with contributions of progenitors at different maturation stages.In sum, these epigenomic analyses revealed that the chromatin landscape of normal T-cell precursors evolves in the course of thymic maturation and that the early maturation stages are the likely origin of T-ALL cells. Remarkably, pediatric T-ALLs that later develop a type 2 relapse consist of subclones with a variable profile of chromatin accessibility that define different stages of maturation. These data indicate that T-ALLs with the propensity to develop a type 2 relapse differ from type 1 in that they originate from early precursors that carry the potential of further development into different stages of maturation before the leukemia becomes apparent with a highly subclonal pattern. DisclosuresMuckenthaler:Novartis: Research Funding. Bourquin:Amgen: Other: Travel Support. Kulozik:bluebird bio: Consultancy, Honoraria.

Features of Immunosenescence in Women Newly Diagnosed With Breast Cancer.

Adults exposed to childhood maltreatment have increased stress reactivity. This profile is associated with dysregulation of the immune system, including enhanced inflammatory reactions and accelerated senescence. Subjects exposed to ear stress have increased risk for several age-related diseases, including cardiovascular disease, type II diabetes, and cancer. Although previous studies have reported immune changes in advanced cancer, very little information is available regarding early stage breast cancer. Here, 29 patients with breast cancer were recruited: 15 with history of childhood maltreatment (CM+) and 14 without history (CM−). Twenty-seven healthy women without CM were selected as the control group. Peripheral blood was collected and lymphocyte subsets phenotyped by multi-color flow cytometry (B cells, CD4+ T, CD8+ T, natural killer cells, activated T cells, regulatory T cells, and senescence-associated T cells). Because human cytomegalovirus (CMV) was associated with signatures of early senescence, the CMV serology was determined by ELISA. None of the subjects had IgM reactivity to CMV, excluding acute viral infection. There was a higher proportion of patients with increased CMV IgG levels in the CM+ group as compared to CM− or controls. Different stages of T-cell differentiation can be determined based on the cell-surface expression of the costimulatory molecules CD27 and CD28: ear (CD27+CD28+), intermediate-differentiated (CD27−CD28+), and late-differentiated or senescent T cells (CD27−CD28−). After adjusting for age and education, ear T cells (CD27+CD28+) were found reduced in CM+ and CM− patients (p < 0.0001). In contrast, intermediate-differentiated T cells (CD27−CD28+; p < 0.0001), senescent T cells (CD27−CD28−; p < 0.0001), and exhausted T cells (CD8+CD27−CD28−PD1+; p < 0.0001) were found expanded in both CM+ and CM− groups. Our data suggest that features of immunosenescence are associated with newly diagnosed breast cancer, regardless of the CM history.

Immunophenotypic analysis of adult patients with T-cell lymphoblastic lymphoma treated with hyper-CVAD

ABSTRACTObjectives: Immunophenotype is an important prognostic factor for childhood and adult T-cell acute lymphoblastic leukemia. However, immunophenotypic data from adult patients with T-cell lymphoblastic lymphoma (T-LBL) are scarcely available.Methods: Subjects were unselected adult patients with T-LBL who were treated with intensive chemotherapy. Immunophenotyping of tumor cells was performed according to standard techniques.Results: A total of eight patients with a median age of 31 years were analyzed who received hyper-CVAD treatment for LBL. Immunophenotypic analysis showed that the most common tumor type was cortical T-cell type [early T (n = 2), cortical T (n = 4), and medullary T (n = 2)]. Two patients diagnosed with early T-cell type had early disease progression.Conclusions: Assessment of T-cell differentiation stages in malignant T lymphoblasts would be important in choosing treatment strategies for adult patients with T-LBL.

The T-ALL related gene BCL11B regulates the initial stages of human T-cell differentiation.

The initial stages of T-cell differentiation are characterized by a progressive commitment to the T-cell lineage, a process that involves the loss of alternative (myelo-erythroid, NK, B) lineage potentials. Aberrant differentiation during these stages can result in T-cell acute lymphoblastic leukemia (T-ALL). However, the mechanisms regulating the initial stages of human T-cell differentiation are obscure. Through loss of function studies, we showed BCL11B, a transcription factor recurrently mutated T-ALL, is essential for T-lineage commitment, particularly the repression of NK and myeloid potentials, and the induction of T-lineage genes, during the initial stages of human T-cell differentiation. In gain of function studies, BCL11B inhibited growth of and induced a T-lineage transcriptional program in T-ALL cells. We found previously unknown differentiation stage-specific DNA binding of BCL11B at multiple T-lineage genes; target genes showed BCL11B-dependent expression, suggesting a transcriptional activator role for BCL11B at these genes. Transcriptional analyses revealed differences in the regulatory actions of BCL11B between human and murine thymopoiesis. Our studies show BCL11B is a key regulator of the initial stages of human T-cell differentiation and delineate the BCL11B transcriptional program, enabling the dissection of the underpinnings of normal T-cell differentiation and providing a resource for understanding dysregulations in T-ALL.

Abstract A040: The low antitumor functionality of PD1-positive gp100-specific CD8+ T cell clones isolated from melanoma patients correlates with the presence of CD28 co-stimulatory molecule

Abstract Background: The identification of activation pathways linked to anti-tumor T-cell polyfunctionality in patients responding to immunomodulatory agents is of great relevance in the new era of immunotherapy. An effective anti-tumor immune response is the result of a fine balance between TCR activation and co-stimulatory as well as inhibitory signals. The co-stimulatory molecule CD28 plays an essential role in T-cell activation, and with CD27 characterizes the different T-cell differentiation stages. Terminally differentiated T cells are CD28−CD27−, show an exhausted phenotype, low functionality along with up-regulation of co-inhibitory receptors including PD-1. However, beside its critical role in tumor-induced immune-suppression, PD-1 has recently been described as a marker of highly melanoma-reactive CD8+ T-cells. Accordingly, we have recently reported that Melan-A-specific CD8+ T-cells isolated from melanoma patients treated with combined chemo-immunotherapy [dacarbazine (DTIC) plus Melan-A/gp100 peptide vaccination], showed an enlarged TCR repertoire and high AKT-dependent anti-tumor polyfunctionality sustained by ICOS despite a late differentiated phenotype and high PD-1 expression. We hypothesize that this AKT-dependent anti-tumor polifunctionality may have contributed to protect patients from disease recurrence. Aim of this study is to identify the complex relationship between PD-1 expression and T-cell effector functions, taking advantage from a panel of antigen-specific (Melan-A and gp100) T-cell clones isolated from melanoma patients. Methods: We generated gp100-specific CD8+ T-cell clones from patients treated with peptide-vaccination alone or DTIC plus vaccination. We have analyzed the treatment-induced response in terms of TCR-βsequencing, differentiation phenotype, inhibitory receptor profile, polyfunctionality, cytotoxicity and AKT activation, by flow-cytometric, biochemical and functional analyses. Results: CD8+ gp-100-specific T cells isolated from patients treated with vaccination alone showed an early differentiated phenotype, while those isolated from patients treated with DTIC plus vaccination displayed mostly a late differentiated profile, as defined by the expression of CD28 and/or CD27. These clones possessed an oligoclonal TCR repertoire, irrespective of the treatment received by the patients, differently from results obtained for Melan-A-specific clones. In gp100-specific CD8+ T cells AKT pathway was activated according to their differentiation profile as defined by the expression of CD28 and/or CD27, irrespective of the treatment. High anti-tumor lytic activity and low PD-1 expression were observed in T-cell clones isolated after chemoimmunotherapy, while cells isolated after peptide vaccination alone expressed high level of the inhibitory molecule PD-1, either alone or along with LAG-1 and TIM-3 and were non tumor-reactive. This low anti-tumor polyfunctionality (in terms of TNF-α, IFN-γ and GrB) was increased after anti-PD-1 mAb blockade. Interestingly, differently from Melan A-specific T cells which showed high levels of PD-1 in the absence of CD28, these non-functional gp100-specific T-cell clones expressed PD-1 in the presence of CD28 co-stimulatory molecule. Conclusions: Our results, obtained in a panel of Melan-A and gp100-specific T-cell clones, show that while PD-1-positive-Melan-A specific T-cell clones have a polyfunctional effector profile in the absence of CD28 expression and in the presence of AKT activation sustained by ICOS, non-functional gp-100-specific PD-1-positive T cells express high level of CD28 molecule. The biochemical pathways involved in the control of functionality of PD-1-expressing CD8+ T cells are currently under investigation. Citation Format: Belinda Palermo, Ornella Franzese, Cosmo Di Donna, Mariangela Panetta, Isabella Sperduti, Antonella Soriani, Maria Laura Foddai, Angela Santoni, Paola Nisticò. The low antitumor functionality of PD1-positive gp100-specific CD8+ T cell clones isolated from melanoma patients correlates with the presence of CD28 co-stimulatory molecule [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A040.

Constitutive Expression of MEF2C, LYL1, or LMO2 in CD34+ HSCs Blocks the in Vitro Differentiation before the T-Cell Commitment Point and Could be Oncogenic in Early T-Cell Progenitor ALL

Background:T-cell development in the thymus is a complex process that depends on sequential transcriptional and epigenetic events that induce T-cell lineage commitment and simultaneously suppress alternative cell fates. In T-cell acute lymphoblastic leukemia (T-ALL), aberrantly expressed oncogenes result in the arrest of developing thymocytes, which can lead to the acquisition of secondary mutations, uncontrolled proliferation and disease progression. MEF2C is often expressed as a result of chromosomal rearrangements in immature, early T-cell progenitor ALL (ETP-ALL), but is also expressed in normal thymocyte progenitors before T-cell commitment (in the ETP stage). As the only hematopoietic lineage, thymocytes that have passed the T-cell commitment checkpoint (as well as mature T-cells) do no longer express MEF2C.Aims:We aimed to investigate the effect of constitutive MEF2C expression on early T-cell development. OP9-DL1 co-cultures have been most useful for mimicking in vitro T-cell development starting with hematopoietic stem cells (HSCs) derived from human cord blood or bone marrow. We also aimed to investigate the impact of MEF2C in comparison to LYL1 and LMO2; two T-ALL oncogenes also highly expressed at the ETP stage.Methods:We have utilized the OP9-DL1 in vitro co-culture system to gradually differentiate CD34+ HSCs from umbilical cord blood into the T-cell lineage. HSCs in this co-culture will recapitulate in vivo T-cell development as measured by incremental acquisition of surface markers CD7, CD5, CD1a, and reach the CD4, CD8 double-positive (DP) stage. We generated gene expression profiles of 11 subsequent in vitro stages of differentiation to help us match them to in vivo development stages. We investigated in vitro T-cell differentiation of HSCs after lentiviral transduction with MEF2C or control vectors, as well as with other transcriptional regulators LYL1 and LMO2 that are expressed at the ETP stage.Results:The major change in gene expression of subsequent early T-cell differentiation stages defines two distinct T-cell differentiation clusters that correlate with in vivo pre- and post-T-cell commitment profiles. We found that T-cell commitment occurs in CD7+ CD5+ cells before the acquisition of CD1a surface expression. Expression of control vectors in HSCs does not affect the in vitro T-cell differentiation, but MEF2C expression blocks differentiation into the direction of T-cells as measured by the failure of most cells to acquire CD7 as the first marker. Instead, with increased passage number cells gradually lose CD34 expression and eventually disappear from the co-culture. Similar effects were observed for the expression of LYL1 and LMO2; LYL1 expression arrests the cells at the most immature CD7+ ETP stage and prevents the transition towards CD7+ CD5+ cells, whereas LMO2 expressing cells reach the CD7+ CD5+ stage but fail to acquire CD1a as a marker of T-cell commitment.Summary/Conclusion:The gene expression profiles of 11 human in vitro T-cell differentiation subsets has enabled us to pinpoint T-cell commitment to a stage in which cells have acquired CD7 and CD5, just prior to the acquisition of CD1a. MEF2C, LYL1, and LMO2, expressed in ETP-ALL as well as in normal thymocyte progenitors, do not allow the transition to T-cell commitment when constitutively expressed. These proteins each result in the arrest of in vitro differentiating T-cells at different ETP stages, all before the T-cell commitment as marked by CD1a expression. Constitutive expression of MEF2C, LYL1, or LMO2 in very early thymocyte progenitors is incompatible with development into and beyond the T-cell commitment checkpoint and these proteins could therefore play important roles in the pathogenesis of ETP-ALL. DisclosuresNo relevant conflicts of interest to declare.

Research brief

During chronic infection, pathogen-specific CD8 T cells gradually lose effector functions and upregulate the expression of inhibitory surface receptors. But are “exhausted” T cells completely dysfunctional? A study shows that T cells transferred from mice chronically infected with lymphocytic choriomeningitis virus into mice acutely infected with the virus proliferate and control the infection but retain the exhausted phenotype acquired during chronic infection. Thus, T-cell exhaustion might be a stable stage of T-cell differentiation that limits viral replication during chronic infection without causing overwhelming immunopathology.

Viral infection alters subnuclear localization of PD-1 and CD62L loci in LCMV-specific CD8 T-cells (P6369)

Abstract During chronic viral infection, CD8 T-cell differentiation yields a unique transcriptional profile: T-cell exhaustion. The suboptimal effector function of exhausted cells is partly due to sustained expression of PD-1. Using the LCMV mouse model of infection, we examined pdcd1 (PD-1) and sell (CD62L) gene regulation at different stages of T-cell differentiation. We hypothesized that epigenetic programming and nuclear localization of these genes were coupled to viral persistence and T-cell differentiation. Changes in gene expression are linked to locus positioning in specific compartments such as nuclear lamina (silencing). Using DNA-FISH, we report a significant decrease in pdcd1 alleles associated to nuclear lamina in exhausted cells relative to other CD8 subsets. Dissociation from lamin was consistent with upregulated expression of PD-1 mRNA and protein, and with loss of PD-1 DNA methylation in exhausted cells. In contrast, we observed a significant increase in sell recruitment to lamin with exhaustion, mirroring changes in sell gene expression. CD8 T-cells from Blimp-1-deficient chronically infected mice exhibited an impaired ability to regulate pdcd1 and sell association to lamin, thus implicating Blimp-1 in modulating expression of both genes during chronic infection. Our findings contribute to the understanding of the molecular pathways mediating T-cell memory and exhaustion, and may provide insight into implementing therapeutic strategies to resolve chronic infections.

Long-Term Non-Progression and Broad HIV-1-Specific Proliferative T-Cell Responses.

Complex mechanisms underlying the maintenance of fully functional, proliferative, HIV-1-specific T-cell responses involve processes from early T-cell development through to the final stages of T-cell differentiation and antigen recognition. Virus-specific proliferative CD4 and CD8 T-cell responses, important for the control of infection, are observed in some HIV-1+ patients during early stages of disease, and are maintained in long-term non-progressing subjects. In the vast majority of HIV-1+ patients, full immune functionality is lost when proliferative HIV-1-specific T-cell responses undergo a variable progressive decline throughout the course of chronic infection. This appears irreparable despite administration of potent combination antiretroviral therapy, which to date is non-curative, necessitating life-long administration and the development of effective, novel, therapeutic interventions. While a sterilizing cure, involving clearance of virus from the host, remains a primary aim, a “functional cure” may be a more feasible goal with considerable impact on worldwide HIV-1 infection. Such an approach would enable long-term co-existence of host and virus in the absence of toxic and costly drugs. Effective immune homeostasis coupled with a balanced response appropriately targeting conserved viral antigens, in a manner that avoids hyperactivation and exhaustion, may prove to be the strongest correlate of durable viral control. This review describes novel concepts underlying full immune functionality in the context of HIV-1 infection, which may be utilized in future strategies designed to improve upon existing therapy. The aim will be to induce long-term non-progressor or elite controller status in every infected host, through immune-mediated control of viremia and reduction of viral reservoirs, leading to lower HIV-1 transmission rates.

Targeting antiapoptotic A1/Bfl-1 by in vivo RNAi reveals multiple roles in leukocyte development in mice

AbstractGene-targeting studies in mice have identified the essential roles of most prosurvival Bcl-2 family members in normal physiology and under conditions of stress. The function of one member, Bcl2a1/Bfl-1/A1, is only poorly understood because of quadruplication of its gene locus in mice, hindering conventional knockout studies. To overcome this problem, we generated mouse models allowing traceable constitutive or reversible ablation of A1 in the hematopoietic system by RNA interference. Knockdown of A1 impaired early stages of T-cell differentiation, B-cell homeostasis, and sensitized transitional as well as follicular B cells to apoptosis induced by ligation of the B-cell receptor. As a consequence, B-cell proliferation in response to mitogens was severely impaired, whereas that of T cells appeared unaffected. Furthermore, depending on the extent of A1 knockdown, granulocytes showed increased spontaneous death in culture or failed to accumulate in significant numbers in vivo. These models highlight the critical role of A1 in leukocyte development and homeostasis, constituting valuable tools for investigating presumed roles of this Bcl-2 family member in immunity, tumorigenesis, and drug resistance.

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.

Differentiation-dependent functional and epigenetic landscapes for cytokine genes in virus-specific CD8+T cells

Although the simultaneous engagement of multiple effector mechanisms is thought to characterize optimal CD8(+) T-cell immunity and facilitate pathogen clearance, the differentiation pathways leading to the acquisition and maintenance of such polyfunctional activity are not well understood. Division-dependent profiles of effector molecule expression for virus-specific T cells are analyzed here by using a combination of carboxyfluorescein succinimidyl ester dilution and intracellular cytokine staining subsequent to T-cell receptor ligation. The experiments show that, although the majority of naive CD8(+) T-cell precursors are preprogrammed to produce TNF-α soon after stimulation and a proportion make both TNF-α and IL-2, the progressive acquisition of IFN-γ expression depends on continued lymphocyte proliferation. Furthermore, the extensive division characteristic of differentiation to peak effector activity is associated with the progressive dominance of IFN-γ and the concomitant loss of polyfunctional cytokine production, although this is not apparent for long-term CD8(+) T-cell memory. Such proliferation-dependent variation in cytokine production appears tied to the epigenetic signatures within the ifnG and tnfA proximal promoters. Specifically, those cytokine gene loci that are rapidly expressed following antigen stimulation at different stages of T-cell differentiation can be shown (by ChIP) to have permissive epigenetic and RNA polymerase II docking signatures. Thus, the dynamic changes in cytokine profiles for naive, effector, and memory T cells are underpinned by specific epigenetic landscapes that regulate responsiveness following T-cell receptor ligation.

Phenotype and TCR-gamma gene rearrangements in a Malaysian cohort of T-cell leukaemia/lymphoma cases

T cells undergo a series of complex phenotypic changes before achieving maturation. Discrete stages of T-cell differentiation are simplified to four stages (pro-, pre-, cortical and mature-T cell) and used in the classification of T-cell leukaemia. HLA-DR has been reported to be expressed in immature T-cell acute lymphoblastic leukemia (ALL) and also confer a poorer treatment outcome. Simultaneously, the genotype goes through distinct pattern changes due to rearrangement of T-cell receptor (TCR) genes. TCR gene rearrangement is important in the diagnosis of clonality and used as markers to detect minimal residual disease in lymphoproliferative disorders. We identified a subset within Pro-T and Pre-T cell cases distinguished by the expression of HLA-DR. These subgroups appeared to be more immature as rearrangement of the TCR-gamma gene was either at germline or involved only the first constant region (C1) unlike a more rearranged pattern in the HLA-DR-subgroups. We also observed a higher incidence of mediastinal mass (67%) in the HLA-DR-subgroup in the Pre-T stage. These characteristics may be useful as markers to further refine staging of T-cell ALL and determine prognosis.