Thymic Activity Research Articles

Advanced three-dimensional X-ray imaging unravels structural development of the human thymus compartments

BackgroundThe thymus, responsible for T cell-mediated adaptive immune system, has a structural and functional complexity that is not yet fully understood. Until now, thymic anatomy has been studied using histological thin sections or confocal microscopy 3D reconstruction, necessarily for limited volumes.MethodsWe used Phase Contrast X-Ray Computed Tomography to address the lack of whole-organ volumetric information on the microarchitecture of its structural components. We scanned 15 human thymi (9 foetal and 6 postnatal) with synchrotron radiation, and repeated scans using a conventional laboratory x-ray system. We used histology, immunofluorescence and flow cytometry to validate the x-ray findings.ResultsApplication to human thymi at pre- and post-natal stages allowed reliable tracking and quantification of the evolution of parameters such as size and distribution of Hassall’s Bodies and medulla-to-cortex ratio, whose changes reflect adaptation of thymic activity. We show that Hassall’s bodies can occupy 25% of the medulla volume, indicating they should be considered a third thymic compartment with possible implications on their role. Moreover, we demonstrate compatible results can be obtained with standard laboratory-based x-ray equipment, making this research tool accessible to a wider community.ConclusionsOur study allows overcoming the resolution and/or volumetric limitations of existing approaches for the study of thymic disfunction in congenital and acquired disorders affecting the adaptive immune system.

Awakening the thymus to cure SARS-CoV-2 infection: a matter of genes

Establishing an appropriate immune response is essential to recover from viral diseases such as COVID-19.The thymus, an organ of the immune system, plays a vital role in producing new T-lymphocytes that help eliminate the virus that causes COVID-19. We recently discovered that a genetic trait determines the effectiveness of thymic activity in fighting this virus, and thus the clinical outcome.

Active thymus in adult with lung cancer: preliminary results from the Adult Thymic Project.

Thymus is considered a non-functional remnant in adults, but some evidence suggest that it may harbor residual activity. Lung cancer patients represent the ideal model to study thymic residual activity, as their thymus can be easily harvested during surgery. This study was designed to confirm the presence of residual thymic activity both in adult mice (step 1) and in humans (step 2). In step 1, lung cancer was induced by activating k-ras mutation in a cohort of 20 young and adult mice. After killing, thymus and lungs were analyzed. Thymus was considered active when medullary was evident, cortico-medullary ratio was 50:50 or higher and adipose involution was present. In step 2, a cohort of 20 patients, undergoing surgery for lung cancer, had biopsy of pericardial fat pad, site of ectopic thymus. Thymus was considered present if Hassall's bodies were detected. In mice, active thymus was detected in a high proportion of cases, without significant difference between adult and young (70% vs 44.4% respectively). Two cases without evidence of lung tumor had a fully functional thymus. In humans, ectopic thymus was detected in the pericardial fat pad in 2 cases (10.5%), confirmed by immunohistochemistry. Signs of previous thymic activity were detected in 8 additional patients. Results confirmed thymus activity in animal models and humans with lung cancer, providing the rationale for future systematic mediastinal thymic biopsy. The comprehension of interactions between thymus, lymphocytes and tumor may open a new potentially targetable perspective in lung cancer.

Genetically determined thymic function affects strength and duration of immune response in COVID patients with pneumonia.

Thymic activation improves the outcome of COVID-19 patients with severe pneumonia. The rs2204985 genetic polymorphism within the TCRA-TCRD locus, which affects thymic output in healthy individuals, was found here to modify SARS-CoV-2-specific immunity and disease severity in COVID-19 patients with severe pneumonia. Forty patients with severe COVID-19 pneumonia were investigated. The GG genotype at the rs2204985 locus was associated, independently of age and sex, with stronger and long-lasting anti-SARS-CoV-2 helper and cytotoxic T cell responses 6 months after recovery. The GG genotype was also associated with less severe lung involvement, higher thymic production, and higher counts of blood naïve T lymphocytes, including recent thymic emigrants, and a larger population of activated stem cell memory CD4+ T cells. Overall, GG patients developed a more robust and sustained immunity to SARS-CoV-2. Polymorphism at rs2204985 locus should be considered as an additional predictive marker of anti-SARS-CoV-2 immune response.

B cell subsets in thymus from infants with Down syndrome

B lymphocytes are still poorly characterized in the human thymus, and due to the immunological peculiarities of Down syndrome, a detailed investigation of thymic B lymphocytes and their subsets was performed in <2-year-old infants. This is a period of intense thymic activity, and here we compared thymuses from infants with Down syndrome (DG) and age-matched control group (CG) with heart disease as the only described malformation, no clinical signs of immunodeficiency and normal blood lymphocyte numbers. MethodsThis study, approved by the institutional ethics committee, included 10 thymic samples from DG infants, and 20 from CG infants. Depending on thymus size and the type of surgical approach, babies were submitted to total thymectomy or only the removal of a small thymic fragment (mainly in the DG), always during cardiac surgery in order to correct congenital defects. Flow cytometry was used for immunophenotyping of cell suspension obtained from thymic tissue; and the B lymphocyte topographic localization was investigated by immunohistochemistry, and a quantitative evaluation of B-cells in each region was carried out using methods of digitized images analysis. ResultsFlow cytometry revealed that CD20+ cells represent around 1% of total lymphocytes in both groups, with higher naive and lower unswitched memory B lymphocyte frequencies in DG when compared to the CG, while B1 cells were present in much higher frequency in DG than in CG. All the other B-cell subpopulations (transitional, switched memory, plasmablasts and plasmocytes) were statistically equivalent between groups. Immunohistochemistry showed higher frequencies of CD20+ cells in medullary region compared to cortex, without differences between the groups (DG = 16.8% and CG = 17.1% of total cell number), while the cortex region had few B cells in both groups, with significantly lower frequency in DG group, reaching 0.25%, compared with the CG, which showed 0.6%. Conclusion: Flow cytometry revealed a different distribution of B-cell subtypes in DG thymus compared to CG and, thus, data obtained from such rare material could be relevant in the characterization and further analysis and understanding of the immunological mechanisms that involve the participation of B lymphocyte subpopulations in the thymus. FAPESP (grant 2014/50489-9).

SARS-CoV-2 infection of thymus induces loss of function that correlates with disease severity

Lymphopenia, particularly when restricted to the T-cell compartment, has been described as one of the major clinical hallmarks in patients with coronavirus disease 2019 (COVID-19) and proposed as an indicator of disease severity. Although several mechanisms fostering COVID-19-related lymphopenia have been described, including cell apoptosis and tissue homing, the underlying causes of the decline in T-cell count and function are still not completely understood. Given that viral infections can directly target thymic microenvironment and impair the process of T-cell generation, we sought to investigate the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on thymic function. We performed molecular quantification of T-cell receptor excision circles and κ-deleting recombination excision circles to assess, respectively, T- and B-cell neogenesis in SARS-CoV-2-infected patients. We developed a system for invitro culture of primary human thymic epithelial cells (TECs) to mechanistically investigate the impact of SARS-CoV-2 on TEC function. We showed that patients with COVID-19 had reduced thymic function that was inversely associated with the severity of the disease. We found that angiotensin-converting enzyme 2, through which SARS-CoV-2 enters the host cells, was expressed by thymic epithelium, and in particular by medullary TECs. We also demonstrated that SARS-CoV-2 can target TECs and downregulate critical genes and pathways associated with epithelial cell adhesion and survival. Our data demonstrate that the human thymus is a target of SARS-CoV-2 and thymic function is altered following infection. These findings expand our current knowledge of the effects of SARS-CoV-2 infection on T-cell homeostasis and suggest that monitoring thymic activity may be a useful marker to predict disease severity and progression.

Ageing Affects Thymopoiesis and Experimental Autoimmune Encephalomyelitis Development in a Strain-Dependent Manner

Introduction: Considering significance of mechanisms of central tolerance for development of autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), and suppressive influence of circulating proinflammatory cytokines and alterations in brain-thymus communication, characteristic for the central nervous system (CNS) autoimmune diseases, on thymopoiesis, the study interogated putative strain-based thymus-related specificities relevant for the opposite effects of ageing on susceptibility of Dark Agouti (DA) and Albino Oxford (AO) rats to EAE. Methods: Quantitative and qualitative changes in thymopoiesis including underlying mechanisms were examined using flow cytometry and RT-qPCR quantification of mRNAs for molecules relevant for integrity of stroma and T-cell development, respectively. Results: With ageing, differently from DA rats, in AO rats the surface density of CD90, a negative regulator of selection threshold, on thymocytes undergoing lineage commitment was upregulated (consistent with TGF-β expression downregulation), whereas the generation of natural CD4+CD25+Foxp3+ regulatory T cells (nTregs) was impaired reflecting differences in thymic expression of cytokines supporting their development. Additionally, specifically in old AO rats, in whom EAE development depends on IL-17-producing CD8+ T cells, their thymic differentiation was augmented, reflecting augmented thymic IL-4 expression. In turn, differently from old DA rats developing self-limiting EAE, in age-matched AO rats developing EAE of prolonged duration, EAE development led to impaired generation of nTregs and accumulation of proinflammatory, cytotoxic CD28–CD4+ T cells in the periphery. Discussion: The study indicates that strain differences in age-related changes in the efficacy of central tolerance, in addition to enhanced thymic generation of CD8+ T cells prone to differentiate into IL-17-producing cells, could partly explain the opposite effect of ageing on DA and AO rat susceptibility to EAE induction. Additionally, it suggested that EAE development leading to a less efficient thymic output of CD4+ cells and nTregs in old AO rats than their DA counterparts could contribute to prolonged EAE duration in AO compared with DA rats. Conclusion: The study warns to caution when designing therapeutic interventions to enhance thymic activity in genetically diverse populations, e.g., humans, and interpreting their outcomes. Furthermore, it indicates that CNS autoimmune pathology may additionally worsen thymic involution and age-related immune changes.

Characterization of immune checkpoint inhibitor-induced cardiotoxicity reveals interleukin-17A as a driver of cardiac dysfunction after anti-PD-1 treatment.

Immune checkpoint inhibitors (ICI), such as anti-PD-1 monoclonal antibodies, have revolutionized cancer therapy by enhancing the cytotoxic effects of T-cells against tumours. However, enhanced T-cell activity also may cause myocarditis and cardiotoxicity. Our understanding of the mechanisms of ICI-induced cardiotoxicity is limited. Here, we aimed to investigate the effect of PD-1 inhibition on cardiac function and explore the molecular mechanisms of ICI-induced cardiotoxicity. C57BL6/J and BALB/c mice were treated with isotype control or anti-PD-1 antibody. Echocardiography was used to assess cardiac function. Cardiac transcriptomic changes were investigated by bulk RNA sequencing. Inflammatory changes were assessed by qRT-PCR and immunohistochemistry in heart, thymus, and spleen of the animals. In follow-up experiments, anti-CD4 and anti-IL-17A antibodies were used along with PD-1 blockade in C57BL/6J mice. Anti-PD-1 treatment led to cardiac dysfunction and left ventricular dilation in C57BL/6J mice, with increased nitrosative stress. Only mild inflammation was observed in the heart. However, PD-1 inhibition resulted in enhanced thymic inflammatory signalling, where Il17a increased most prominently. In BALB/c mice, cardiac dysfunction was not evident, and thymic inflammatory activation was more balanced. Inhibition of IL-17A prevented anti-PD-1-induced cardiac dysfunction in C57BL6/J mice. Comparing myocardial transcriptomic changes in C57BL/6J and BALB/c mice, differentially regulated genes (Dmd, Ass1, Chrm2, Nfkbia, Stat3, Gsk3b, Cxcl9, Fxyd2, and Ldb3) were revealed, related to cardiac structure, signalling, and inflammation. PD-1 blockade induces cardiac dysfunction in mice with increased IL-17 signalling in the thymus. Pharmacological inhibition of IL-17A treatment prevents ICI-induced cardiac dysfunction.

Extracellular vesicles from IFN-γ-primed mesenchymal stem cells repress atopic dermatitis in mice

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by immune dysregulation, pruritus, and abnormal epidermal barrier function. Compared with conventional mesenchymal stem cell (MSC), induced pluripotent stem cell (iPSC)-derived mesenchymal stem cell (iMSC) is recognized as a unique source for producing extracellular vesicles (EVs) because it can be obtained in a scalable manner with an enhanced homogeneity. Stimulation of iMSCs with inflammatory cytokines can improve the immune-regulatory, anti-inflammatory, and tissue-repairing potential of iMSC-derived EVs. Proteome analysis showed that IFN-γ-iMSC-EVs are enriched with protein sets that are involved in regulating interferon responses and inflammatory pathways. In AD mice, expression of interleukin receptors for Th2 cytokines (IL-4Rα/13Rα1/31Rα) and activation of their corresponding intracellular signaling molecules was reduced. IFN-γ-iMSC-EVs decreased itching, which was supported by reduced inflammatory cell infiltration and mast cells in AD mouse skin; reduced IgE receptor expression and thymic stromal lymphopoietin and NF-kB activation; and recovered impaired skin barrier, as evidenced by upregulation of key genes of epidermal differentiation and lipid synthesis. IFN-γ-iMSC-EVs inhibit Th2-induced immune responses, suppress inflammation, and facilitate skin barrier restoration, contributing to AD improvement.

A Comparison for Type 2 Cytokines and Lesional Inflammatory Infiltrations in Bullous Pemphigoid and Atopic Dermatitis.

Bullous pemphigoid (BP) and atopic dermatitis (AD) are both type 2 inflammatory skin diseases with similar clinical features. Thymic stromal lymphopoietin (TSLP) is an epithelial-derived cytokine which is upregulated in AD. However, the expression of TSLP in BP and the correlation between TSLP and inflammatory infiltrations have not been fully studied. To characterize the serum Th2 cytokines level and Th2 inflammatory cell infiltrations in BP and AD. To study TSLP levels in serum, blister fluids and expression in lesional skin in patients with BP and AD. TSLP level in serum and blister fluids was measured by enzyme-linked immunosorbent assay (ELISA). Inflammatory cells (CD4+ T cells, CD8+ T cells, CD1a+ cells, eosinophils and mast cells) were stained immunohistochemically and quantified by image analysis. TSLP level was significantly increased in blister fluids of BP and was highly expressed in lesional skin of BP and AD. Serum levels of IL-6, IL-4, IL-22, IFN-γ and thymic activation regulates chemokines (TARC) were significantly higher in patients with BP and AD than in healthy controls. CD4+ T cells, CD8+ T cells and CD1a+ cells were significantly more in upper dermis of BP and AD lesions. Eosinophils were found more in BP lesions while mast cells were found more in AD lesions than in healthy controls. A distinct correlation was found between TSLP levels and the intensities of CD4+ T cells, CD1a+ cells infiltrations. TSLP was significantly higher in blister fluids and skin lesions of BP, suggesting that it might contribute to the pathogenesis of BP. BP exhibited a similar type 2 immune response and a slight difference in cells infiltrations with AD.

Two types of human TCR differentially regulate reactivity to self and non-self antigens

SummaryBased on analyses of TCR sequences from over 1,000 individuals, we report that the TCR repertoire is composed of two ontogenically and functionally distinct types of TCRs. Their production is regulated by variations in thymic output and terminal deoxynucleotidyl transferase (TDT) activity. Neonatal TCRs derived from TDT-negative progenitors persist throughout life, are highly shared among subjects, and are reported as disease-associated. Thus, 10%–30% of most frequent cord blood TCRs are associated with common pathogens and autoantigens. TDT-dependent TCRs present distinct structural features and are less shared among subjects. TDT-dependent TCRs are produced in maximal numbers during infancy when thymic output and TDT activity reach a summit, are more abundant in subjects with AIRE mutations, and seem to play a dominant role in graft-versus-host disease. Factors decreasing thymic output (age, male sex) negatively impact TCR diversity. Males compensate for their lower repertoire diversity via hyperexpansion of selected TCR clonotypes.

Towards a unified model of naive T cell dynamics across the lifespan.

Naive CD4 and CD8 T cells are cornerstones of adaptive immunity, but the dynamics of their establishment early in life and how their kinetics change as they mature following release from the thymus are poorly understood. Further, due to the diverse signals implicated in naive T cell survival, it has been a long-held and conceptually attractive view that they are sustained by active homeostatic control as thymic activity wanes. Here we use multiple modelling and experimental approaches to identify a unified model of naive CD4 and CD8 T cell population dynamics in mice, across their lifespan. We infer that both subsets divide rarely, and progressively increase their survival capacity with cell age. Strikingly, this simple model is able to describe naive CD4 T cell dynamics throughout life. In contrast, we find that newly generated naive CD8 T cells are lost more rapidly during the first 3-4 weeks of life, likely due to increased recruitment into memory. We find no evidence for elevated division rates in neonates, or for feedback regulation of naive T cell numbers at any age. We show how confronting mathematical models with diverse datasets can reveal a quantitative and remarkably simple picture of naive T cell dynamics in mice from birth into old age.

Evaluation of physiological Waldeyer's ring, mediastinal blood pool, thymic, bone marrow, splenic and hepatic activity with 18F-FDG PET/CT: exploration of normal range among pediatric patients.

While 18F-FDG PET/CT pediatrics applications have increased in number and indications, few studies have addressed normal maximum standardized uptake values (SUVmax) of referral organs in children. The purpose of this study is to assess these in a cohort of pediatric patients. 285 18F-FDG PET/CT scans in 229 patients were reviewed. SUVmax were assessed for mediastinal blood pool (MBP), thymus (T), liver (L), spleen (S), bone marrow (BM) and Waldeyer's Ring (Wald). L/MBP and S/L ratios were calculated. Same day complete blood counts (CBC) were available for 132 studies and compared to BM and S. Means, standard deviations and correlation coefficients with age, weight and body surface area (BSA) were calculated. Weak correlation with age, weight or BSA was found for Wald. Strong correlations with weight/BSA more than with age were demonstrated for MBP, L and BM and moderate for S and T. After initial decrease between age 0 and 2, thymic activity peaked at age 11years then involuted. No correlation was found between CBC ad BM or S. In 28 studies, L was less or equal to MBP. In 74 S was superior to L. Referral organs 18F-FDG uptake varies in children more in relation with weight and BSA than with age for key referral organs, such as L, S and MBP. In a significant number of studies, L activity may impede evaluation of treatment response in comparison with MBP or inflammation/infection evaluation in comparison with S.

Cellular and molecular mediators of thymic DC homeostasis and activation

Abstract Central tolerance in the thymus ensures that autoreactive thymocytes undergo negative selection or diversion to the regulatory T cell (Treg) lineage. Thymic antigen presenting cells (APCs) such as medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present myriad self-peptides to thymocytes to induce central tolerance. We and others have previously shown that thymic cDCs can be subdivided into activated CCR7+MHC-IIhi and resting CCR7− MHC-IIlo cDC1 and cDC2 subsets with distinct functional properties, suggesting the thymic DC compartment may be more heterogeneous than previously described. Here, we performed single-cell RNA sequencing (scRNAseq) of hematopoietic APCs isolated from adult mouse thymi to comprehensively identify thymic DC subsets with distinct gene expression profiles. Our data reveal 11 transcriptionally distinct cDC subsets. We identify 6 cDC1 subsets, 3 cDC2 subsets, 2 activated CCR7+ “cDC3” subsets distinguished by expression of cDC1 and cDC2 markers, and 4 plasmacytoid DC clusters. Through functional assays, we find that the XCR1+ cDC3s are the most efficient at presenting mTEC-derived peptides on MHC-I and MHC-II, while 2 distinct cDC2 subsets are the most efficient at generating Tregs in vitro. Additional scRNA-seq and flow cytometry analyses of thymic DCs from mice in which thymocyte maturation was blocked at different developmental stages reveal candidate cellular and molecular crosstalk signals that regulate homeostasis and/or activation of pDC and cDC subsets. Our findings reveal hitherto unknown functional heterogeneity within the thymic DC compartment and identify potential interactions that support DC homeostasis and activation which in turn could impact central tolerance. Supported by NIH P01 AI139449-02

Correlation between circulating innate lymphoid cell precursors and thymic function

SummaryThe thymus has a high capacity to support the differentiation of ILCs, especially when E protein transcription factors are ablated. Whether it contributes to the homeostasis of ILC pools in tissues is not clear. Single-cell RNA sequencing analysis shows a substantial amount of ILC precursors in wild type but not athymic nude blood. The precursors express CD3 intracellularly (ic) but not on the surface. The abundance of Lin−CD127+CD62L+icCD3ε+ precursors varies with age, peaking at 2–3 months. These cells can differentiate into various ILC subsets on OP9-DL1 stroma in vitro. In the lung, small intestine, and epidermis, icCD3ε+ cells differentiate into diverse ILC subsets in different tissue environments in steady state. Helminth infection promotes their differentiation toward functional ILC2s. Thus, the thymus appears to play a role in replenishing ILC pools in different peripheral tissues. Because thymic activity is age-dependent, this finding may help explain age-related differences in immune responses.

RNA Metabolism in T Lymphocytes.

RNA metabolism plays a central role in regulating of T cell-mediated immunity. RNA processing, modifications, and regulations of RNA decay influence the tight and rapid regulation of gene expression during T cell phase transition. Thymic selection, quiescence maintenance, activation, differentiation, and effector functions of T cells are dependent on selective RNA modulations. Recent technical improvements have unveiled the complex crosstalk between RNAs and T cells. Moreover, resting T cells contain large amounts of untranslated mRNAs, implying that the regulation of RNA metabolism might be a key step in controlling gene expression. Considering the immunological significance of T cells for disease treatment, an understanding of RNA metabolism in T cells could provide new directions in harnessing T cells for therapeutic implications.

Interruption of Thymic Activity in Adult Mice Improves Responses to Tumor Immunotherapy.

The thymus produces precursors of both conventional T cells (Tconv; also known as effector T cells) and regulatory T cells (Treg) whose interactions prevent autoimmunity while allowing efficient protective immune responses. Tumors express a composite of self-antigens and tumor-specific Ags and engage both Tconv and Treg. Along the aging process, the thymus involutes, and tumor prevalence increases, a correlation proposed previously to result from effector cell decline. In this work, we directly tested whether interruption of thymic activity in adult mice affects Foxp3-expressing Treg composition and function and alters tumor immune surveillance. Young adult mice, on two different genetic backgrounds, were surgically thymectomized (TxT) and analyzed or challenged 2 mo later. Cellular analysis revealed a 10-fold decrease in both Tconv and Treg numbers and a bias for activated cells. The persisting Treg displayed reduced stability of Foxp3 expression and, as a population, showed a compromised return to homeostasis upon induced perturbations. We next tested the growth of three tumor models from different tissue origins and/or presenting distinct degrees of spontaneous immunogenicity. In none of these conditions, adult TxT facilitated tumor growth. Rather, TxT enhanced the efficacy of antitumor immunotherapies targeting Treg and/or the immune checkpoint CTLA4, as evidenced by the increased frequency of responder mice and decreased intratumoral Treg to CD8+IFN-γ+ cell ratio. Together, our findings point to a scenario in which abrogation of thymic activities affects preferentially the regulatory over the ridding arm of the immune activities elicited by tumors and argues that higher prevalence of tumors with age cannot be solely attributed to thymic output decline.

Bipotent Lymphoid Progenitors Independently Maintain Long-Term Genetically Engineered T and NK Cell Production in Humans

Historically, survival and activity of individual human hematopoietic progenitor subtypes have been studied exclusively via transplantation in permissive mouse models. Despite being a relevant investigational tool, such animal models do not recapitulate the human hematopoietic milieu. Specifically, the production of human T and NK cells in these mice is severely impaired therefore, as of today, it has been impossible to measure with enough confidence the in vivo dynamics of human T/NK lymphoid progenitors. Hematopoietic stem cell gene therapy (GT) using integrating viral vectors has opened a unique opportunity to trace the fate of transplanted cells for the first time directly in vivo in humans by means of integration sites (IS) clonal tracking. Our mathematical modelling of IS data in clinical GT supported the existence of a population of long-term lymphoid progenitors (LtLP) capable of surviving independently from hematopoietic stem cells (HSC). However, to date, no experimental setting has been available to validate such statistical prediction neither in the mouse nor in humans. We here report the first formal evidence, directly in vivo in humans, that a population of bipotent lymphoid progenitors is capable of surviving and maintaining de novo T/NK production for at least 15 years after loss of transplanted HSC. In 5 SCID-X1 patients treated with gammaretroviral vector HSC-GT we observed that genetically engineered myeloid and B cells have stayed constantly below detection limits starting from 6 months after GT. On the contrary T and NK cells remained vector positive for up to 19 years (average vector copies/cell equal 2.1 and 2.3 respectively). We initially thought that such data would be consistent with permanent loss of engineered HSC and survival of vector-marked long-living circulating mature lymphocytes. However, strikingly and unexpectedly, by a comprehensive immunophenotyping of T cells overtime, we detected vector-positive short-living CD45RA+CD62L+CD95- naïve T cells (Tn) in the circulation of these patients. We first obtained functional validation of the identity of such Tn cells by IFN-y productionassays and we confirmed positive thymic activity by measurement of TREC content. High throughput sequencing of 1,193 T-cell receptor (TCR) rearrangements in FACS-sorted T cell subtypes confirmed that these engineered Tn cells had normal TCR diversity and that new rearrangements were detectable overtime. Similarly, Vbeta repertoire measured by spectratyping displayed normal profiles in all patients. These results suggested that a de novo T-cells production is maintained in these patients by a putative LtLP population in absence of supply by gene-corrected HSC. Using Tn data as surrogate markers of LtLP clonal dynamics and composition, we collected and analysed 12,756 unique IS from 5 populations including Tn cells in a window of 10.1 to 14.9 years after the observed loss of transplanted HSC. Clonal diversity was stable in all T subtypes and IS sharing across subpopulations and timepoints was high and consistent with active in vivo output by LtLP and differentiation of Tn into memory/effector T cells. Analyzing nature and recapture probability of IS we observed that IS in LtLP are significantly enriched in genes involved in lymphocyte survival/activation and we could estimate that T cell production is currently sustained by 2,092-6,056 individual engineered LtLP clones. Moreover, by studying 651 IS collected from CD3-CD56+ purified NK cells we observed that up to 41.2% of them were shared with independently analyzed Tn suggesting that the LtLP active in these patients have a dual T/NK restricted potential. Lastly, we detected and tracked overtime 52 clones bearing IS in MECOM, CCND2 and LMO2 including the IS originally associated with vector-induced leukemia in one patient. Our data show absence of any sustained clonal expansions within the engineered LtLP population for up 19 years after GT, the longest follow up available to date in clinical HSC-GT. In conclusion, our data provide the first formal evidence in vivo in human that a de novo production of genetically engineered T and NK cells can be physiologically maintained by a population of LtLP surviving up to 15 years after loss of transplanted HSC (manuscript submitted). Identification and exploitation of such human LtLP population might be crucial for the development of next generation GT and cancer immunotherapy approaches. Disclosures Baricordi: AVROBIO Inc: Current equity holder in publicly-traded company, Other: Trainee. Thrasher:Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Factors associated with immunosenescence during early adulthood in HIV-infected patients after durable efficient combination antiretroviral therapy

Perinatally HIV-infected patients face the consequences of both chronic infection effects per se and long-term combination antiretroviral therapy (cART) on immunosenescence. Aims of our study were to evaluate which factors independently contribute to immunosenescence in HIV-infected young adults with a very different HIV infection duration (perinatally HIV-infected young individuals -pHIVy- and age-matched non perinatally HIV-infected youths –npHIVy), after durable efficient cART. We considered low thymic and bone marrow output, respectively evaluated by quantifying T-cell receptor excision circles (TRECs), K-deleting recombination excision circles (KRECs), and shorter telomeres lenght (TL) as surrogate biomarkers of immunosenescence. Twenty-one pHIVy and 19 npHIVy (with a mean HIV duration of 3–8 years) were included; mean age was 27 years for both groups. Immunosenescence biomarkers were comparable between pHIVy and npHIVy (despite longer HIV-infection, higher frequency of AIDS events, past cART-free periods and concomitant chronic viral infections in pHIVy). At the multivariate analysis, CD4+ was the only variable independently associated with TRECs and TL. Our data suggest that a good level of thymic activity can compensate the deleterious effects of past periods without cART, if HIV replication is suppressed for a sufficient time.

Developmental exposure to low doses of dichlorodiphenyltrichloroethane impairs proliferative response of thymic lymphocytes to Concanavalin A in rats

The aim of the research was to study formation of thymic lymphocytes proliferative response to T cell mitogen Concanavalin A in 7, 42, and 70 days-old male Wistar rats developmentally exposed to low doses of endocrine disruptor dichlorodiphenyltrichloroethane (2.90 ± 0.13 μg/kg body weight). The thymus of the exposed rats did not show morphological abnormalities. Exposure to the endocrine disrupter was found to alter age-dependent changes of thymic lymphocyte proliferative activity and attenuate proliferative response to Concanavalin A in puberty and adulthood. Insufficient response to mitogen was mediated by higher content of actively proliferating Ki-67-positive lymphoblasts compared to the control values. Insufficient proliferative response to mitogen in developmentally exposed to the endocrine disruptor rats may provide higher risk of impaired cellular immune reactions.