Thymic Microenvironment Research Articles

MiR-155 promotes T reg cell development by safeguarding medullary thymic epithelial cell maturation.

During thymocyte development, medullary thymic epithelial cells (mTECs) provide appropriate instructive cues in the thymic microenvironment for not only negative selection but also the generation of regulatory T (T reg) cells. Here, we identify that miR-155, a microRNA whose expression in T reg cells has previously been shown to be crucial for their development and homeostasis, also contributes to thymic T reg (tT reg) cell differentiation by promoting mTEC maturation. Mechanistically, we show that RANKL stimulation induces expression of miR-155 to safeguard the thymic medulla through targeting multiple known and previously uncharacterized molecules within the TGFβ signaling pathway, which is recognized for its role in restricting the maturation and expansion of mTECs. Our work uncovers a miR-155-TGFβ axis in the thymic medulla to determine mTEC maturity and, consequently, the quantity of tT reg cells and suggests that miR-155 ensures proper tT reg cell development in both cell-intrinsic and -extrinsic manners.

The Responsiveness of Thymic Stromal Cells to semaphorin-3A

ABSTRACT Background The thymus is responsible for thymocyte differentiation into immunocompetent T lymphocytes. Different cell types in the thymic microenvironment actively cooperate in this process, interacting with the developing thymocytes through soluble factors, extracellular matrix (ECM) molecules, and receptors. In addition, this microenvironment can be influenced by several factors, such as semaphorin-3A (Sema3A), which is a multifunctional protein involved in cell migration. We evaluated the Sema3A effects on the cellular parameters and functional features of thymic stromal cells. Methods Thymic stromal cells were obtained by enzymatic digestion of the murine thymus. These cells were treated with Sema3A and evaluated as follows: cell morphology by scanning electron microscope, F-actin cytoskeleton and deposition of ECM molecules by fluorescence microscopy, and adhesion assays with freshly obtained thymocytes. Results The obtained thymic stroma was composed of 67% of thymic epithelial cells (TECs), and 90% of the TECs were positive for the Sema3A receptor neuropilin-1. These cells secreted CXCL12, IL-7 and extended thymocyte survival. Sema3A changed the morphology of thymic stromal cells and promoted F-actin reorganization. In addition, the fibronectin fibers were reoriented, and the laminin production was increased in Sema3A-treated thymic stromal cells. In the adhesion assays, there was an increase in the number of adhered thymocytes when thymic stromal cells were pretreated with Sema3A. Conclusion Our data strongly suggest the active participation of Sema3A in thymic physiology, highlighting its role as an immunomodulatory molecule. This may provide important knowledge for understanding the interactions of thymic cells.

Tumor-associated myeloid cells provide critical support for T-ALL

AbstractDespite harboring mutations in oncogenes and tumor suppressors that promote cancer growth, T-cell acute lymphoblastic leukemia (T-ALL) cells require exogenous cells or signals to survive in culture. We previously reported that myeloid cells, particularly dendritic cells, from the thymic tumor microenvironment support the survival and proliferation of primary mouse T-ALL cells in vitro. Thus, we hypothesized that tumor-associated myeloid cells would support T-ALL in vivo. Consistent with this possibility, in vivo depletion of myeloid cells results in a significant reduction in leukemia burden in multiple organs in 2 distinct mouse models of T-ALL and prolongs survival. The impact of the myeloid compartment on T-ALL growth is not dependent on suppression of antitumor T-cell responses. Instead, myeloid cells provide signals that directly support T-ALL cells. Transcriptional profiling, functional assays, and acute in vivo myeloid-depletion experiments identify activation of IGF1R as a critical component of myeloid-mediated T-ALL growth and survival. We identify several myeloid subsets that have the capacity to directly support survival of T-ALL cells. Consistent with mouse models, myeloid cells derived from human peripheral blood monocytes activate IGF1R and directly support survival of primary patient T-ALL cells in vitro. Furthermore, enriched macrophage gene signatures in published clinical samples correlate with inferior outcomes for pediatric T-ALL patients. Collectively, these data reveal that tumor-associated myeloid cells provide signals critical for T-ALL growth in multiple organs in vivo and implicate tumor-associated myeloid cells and associated signals as potential therapeutic targets.

Type II Cytokines Fine-Tune Thymic T Cell Selection to Offset Murine Central Nervous System Autoimmunity.

Early thymic progenitors (ETPs) are bone marrow-derived hematopoietic stem cells that remain multipotent and give rise to a variety of lineage-specific cells. Recently, we discovered a subset of murine ETPs that expresses the IL-4Rα/IL-13Rα1 heteroreceptor (HR) and commits only to the myeloid lineage. This is because IL-4/IL-13 signaling through the HR inhibits their T cell potential and enacts commitment of HR+ETPs to thymic resident CD11c+CD8α+ dendritic cells (DCs). In this study, we discovered that HR+-ETP-derived DCs function as APCs in the thymus and promote deletion of myelin-reactive T cells. Furthermore, this negative T cell selection function of HR+-ETP-derived DCs sustains protection against experimental allergic encephalomyelitis, a mouse model for human multiple sclerosis. These findings, while shedding light on the intricacies underlying ETP lineage commitment, reveal a novel, to our knowledge, function by which IL-4 and IL-13 cytokines condition thymic microenvironment to rheostat T cell selection and fine-tune central tolerance.

Selective expression of claudin-5 in thymic endothelial cells regulates the blood-thymus barrier and T-cell export.

T-cell development depends on the thymic microenvironment, in which endothelial cells (ECs) play a vital role. Interestingly, vascular permeability of the thymic cortex is lower than in other organs, suggesting the existence of a blood-thymus barrier (BTB). On the other hand, blood-borne molecules and dendritic cells bearing self-antigens are accessible to the medulla, facilitating central tolerance induction, and continuous T-precursor immigration and mature thymocyte egress occur through the vessels at the cortico-medullary junction (CMJ). We found that claudin-5 (Cld5), a membrane protein of tight junctions, was expressed in essentially all ECs of the cortical vasculatures, whereas approximately half of the ECs of the medulla and CMJ lacked Cld5 expression. An intravenously (i.v.) injected biotin tracer hardly penetrated cortical Cld5+ vessels, but it leaked into the medullary parenchyma through Cld5- vessels. Cld5 expression in an EC cell line caused a remarkable increase in trans-endothelial resistance in vitro, and the biotin tracer leaked from the cortical vasculatures in Cldn5-/- mice. Furthermore, i.v.-injected sphingosine-1 phosphate distributed selectively into the medulla through the Cld5- vessels, probably ensuring the egress of CD3high mature thymocytes from Cld5- vessels at the CMJ. These results suggest that distinct Cld5 expression profiles in the cortex and medulla may control the BTB and the T-cell gateway to blood circulation, respectively.

S1584 Autoimmune Exocrine Pancreatic Insufficiency: A Novel Paraneoplastic Mechanism for Diarrhea in Patients With Thymoma

INTRODUCTION: Paraneoplastic syndromes are an exceedingly rare cause of chronic diarrhea. Thymomas have been associated with two known paraneoplastic diarrheal syndromes, namely in the context of hypogammaglobulinemia (Good’s syndrome) or a graft-versus-host disease (GVHD)-like condition. However, exocrine pancreatic insufficiency has yet to be reported as a cause of diarrhea in patients with thymoma. CASE DESCRIPTION/METHODS: A 36-year-old woman with no known medical morbidities presented with postprandial watery diarrhea, emesis, and 100-lb unintentional weight loss over two years. Labs were notable for lymphocytosis up to 8,100/mL with normal flow cytometry and no abnormalities on blood smear. Celiac disease serologies and stool infectious studies were negative. Immunoglobulin G levels were elevated at 3,010 mg/dL, and stool pancreatic elastase levels were low (< 15 mg/g). Additional labs were consistent with fat soluble vitamin deficiency: vitamin A < 5 mcg/dL, 25-hydroxyvitamin D < 4 ng/dL, vitamin E 7 mg/L, vitamin K 69 pg/mL. Computed tomography (CT) of the chest revealed an 11-cm anterior mediastinal mass with pleural and nodal metastases, and biopsy was suggestive of malignant thymoma (Figure 1). CT of the abdomen revealed a normal-appearing pancreas. Subsequent endoscopy and colonoscopy showed healthy-appearing mucosa with no abnormalities (Figure 2). However, colonic biopsies were notable for mild diffuse inflammatory changes including scattered intraepithelial lymphocytes and neutrophils, increased apoptotic bodies, and crypt architectural distortion. Her diarrhea ultimately improved after four cycles of induction chemotherapy. DISCUSSION: GVHD-like colitis in patients with malignant thymoma has been reported in several instances. This has been attributed to alterations in the thymic microenvironment, which lead to dysregulation and loss of anergy. Autoreactive T-cells can mount an immune response that impacts a number of organs, especially in the gastrointestinal tract. Although our patient had histopathological changes reflective of colitis secondary to GVHD, the severity of her diarrhea was out-of-proportion to these findings. The postprandial nature of her symptoms and markedly low stool pancreatic elastase were suggestive of possible pancreatic insufficiency, likely secondary to an autoimmune phenomenon impacting the exocrine glands of the pancreas. This case proposes a possible novel paraneoplastic mechanism for diarrhea in patients with thymoma.Figure 1.: Cross-section image of anterior mediastinal mass.Figure 2.: Normal-appearing duodenal mucosa.

Resveratrol ameliorates thymus senescence changes in D-galactose induced mice.

The thymic microenvironment plays an important role in the development of T cells. A decrease of thymic epithelial cells is the main cause of age-related thymic atrophy or degeneration. Resveratrol (RSV), a phytoalexin produced from plants, has been shown to inhibit the adverse effects of dietary obesity on the structure and function of the thymus. D-Galactose (D-gal) can induce accelerated aging in mice. In the present study, young mice (2 months old) were injected with D-gal (120 mg/kg/day) for 8 consecutive weeks to construct an accelerated aging model. Compared with normal control mice, the thymus epithelium of the D-gal treated mice had structural changes, the number of senescent cells increased, the number of CD4+ T cells decreased, and CD8+ T cells increased. After RSV administration by gavage for 6 weeks, it was found that RSV improved the surface phenotypes of D-gal treated mice, and recovered thymus function by maintaining the ratio of CD4+ to CD8+ cells. It also indicated that RSV enhanced the cell proliferation and inhibited cell senescence. Increased autoimmune regulator (Aire) expression was present in the RSV treated mice. The lymphotoxin-beta receptor (LTβR) expression also increased. These findings suggested that RSV intake could restore the alterations caused by D-gal treatment in the thymus via stimulation of Aire expression.

Thymic Engraftment by in vitro-Derived Progenitor T Cells in Young and Aged Mice.

T cells play a critical role in mediating antigen-specific and long-term immunity against viral and bacterial pathogens, and their development relies on the highly specialized thymic microenvironment. T cell immunodeficiency can be acquired in the form of inborn errors, or can result from perturbations to the thymus due to aging or irradiation/chemotherapy required for cancer treatment. Hematopoietic stem cell transplant (HSCT) from compatible donors is a cornerstone for the treatment of hematological malignancies and immunodeficiency. Although it can restore a functional immune system, profound impairments exist in recovery of the T cell compartment. T cells remain absent or low in number for many months after HSCT, depending on a variety of factors including the age of the recipient. While younger patients have a shorter refractory period, the prolonged T cell recovery observed in older patients can lead to a higher risk of opportunistic infections and increased predisposition to relapse. Thus, strategies for enhancing T cell recovery in aged individuals are needed to counter thymic damage induced by radiation and chemotherapy toxicities, in addition to naturally occurring age-related thymic involution. Preclinical results have shown that robust and rapid long-term thymic reconstitution can be achieved when progenitor T cells, generated in vitro from HSCs, are co-administered during HSCT. Progenitor T cells appear to rely on lymphostromal crosstalk via receptor activator of NF-κB (RANK) and RANK-ligand (RANKL) interactions, creating chemokine-rich niches within the cortex and medulla that likely favor the recruitment of bone marrow-derived thymus seeding progenitors. Here, we employed preclinical mouse models to demonstrate that in vitro-generated progenitor T cells can effectively engraft involuted aged thymuses, which could potentially improve T cell recovery. The utility of progenitor T cells for aged recipients positions them as a promising cellular therapy for immune recovery and intrathymic repair following irradiation and chemotherapy, even in a post-involution thymus.

Immunohistochemical mapping of thymic microenvironment in sterlet (Acipenser ruthenus)

In this study, we describe the immunohistochemical characterisation of the thymus, the main lymphoid organ, in sturgeon. The wide range cytokeratin, vimentin, S-100 protein, LCA (CD45) and CD3 were selected as the immunohistochemical markers to map the thymus in juvenile sterlet (Acipenser ruthenus). The epithelial cells and Hassall’s corpuscles were labelled with a wide range cytokeratin. The fibroblasts and connective tissue within the thin fibrous capsule on the thymic surface expressed vimentin positivity. The stromal reticular cells were S-100 protein positive. The Leukocyte Common Antigen LCA (CD45) was negative on the thymic lymphocytes. The CD3 was negative on the thymic lymphocytes with cross-reactivity on the non-targeted structures. In conclusion, the commercially available antibodies against the wide range cytokeratin, vimentin and S-100 protein can be used to differentiate components of the sturgeon thymus, while the LCA (CD45) and CD3 application failed. We suggest that further studies are needed to generate fish specific antibodies.

English

Bowdichia virgilioides Kunth (Fabaceae) is a tree species used in folk medicine against inflammatory disorders, and it is still unclear whether consumption of the B. virgilioides stem bark extract has any effect on immune cells. Thus, this study aimed to evaluate the effects of B. virgilioides stem bark extract (AEBv) on the functions of the thymus and B lymphocytes. The AEBv was obtained by decoction and administrated orally once a day in male mice for 7 days. After treatment, thymi were obtained for morphology analysis, relative weight, cellularity and deposition of laminin. Thymocytes obtained from control and treated animals were submitted to adhesion and migration assays followed by immunophenotyping. Also, mesenteric lymph nodes were removed, and the lymphoid cells were obtained to measure B cell proliferation and cytokine production. Mice treated with AEBv and their controls were also immunized with sheep red blood cells (SRBC), and 8 days post immunization the sera were collected to perform antibody titration using hemagglutination assays. AEBv administration decreased thymus weight and cellularity and increased the laminin deposition without any significant effect on the expression of the laminin receptor VLA-6 in the thymus microenvironment. Additionally, thymocytes of AEBv-treated mice showed reduced adhesion and migration on laminin substrates, and the B lymphocytes showed reduced proliferation, enhanced IL-10 secretion, decreased TNF-&alpha; production, and attenuated anti-SRBC antibody production. These findings showed that AEBv intake might cause modulatory effects on the functions of thymocytes and B-lymphocytes, contributing to the treatment of immune diseases. Key words: Thymocytes, cell migration, cell adhesion, immunosuppression, B cell. &nbsp;

Off the beaten track: defining the developmental path of T cells through the human thymus

The thymus is a primary lymphoid organ in which T cells are developed and T cell receptor repertoire is formed. T cell development starts when T cell precursors migrate from the bone marrow or the fetal liver to the thymus.1 In the thymic microenvironment, these precursor cells undergo a series of differentiation and selection events in different regions of the thymus, resulting in a diverse repertoire of mature T cells to establish immune protection against foreign antigens and immune tolerance toward self-antigens.

Factors that influence the thymic selection of CD8αα intraepithelial lymphocytes.

Thymocytes bearing αβ T cell receptors (TCRαβ) with high affinity for self-peptide-MHC complexes undergo negative selection or are diverted to alternate T cell lineages, a process termed agonist selection. Among thymocytes bearing TCRs restricted to MHC class I, agonist selection can lead to the development of precursors that can home to the gut and give rise to CD8αα-expressing intraepithelial lymphocytes (CD8αα IELs). The factors that influence the choice between negative selection versus CD8αα IEL development remain largely unknown. Using a synchronized thymic tissue slice model that supports both negative selection and CD8αα IEL development, we show that the affinity threshold for CD8αα IEL development is higher than for negative selection. We also investigate the impact of peptide presenting cells and cytokines, and the migration patterns associated with these alternative cell fates. Our data highlight the roles of TCR affinity and the thymic microenvironments on T cell fate.

Generation and Regeneration of Thymic Epithelial Cells.

The thymus is unique in its ability to support the maturation of phenotypically and functionally distinct T cell sub-lineages. Through its combined production of MHC-restricted conventional CD4+ and CD8+, and Foxp3+ regulatory T cells, as well as non-conventional CD1d-restricted iNKT cells and invariant γδT cells, the thymus represents an important orchestrator of immune system development and control. It is now clear that thymus function is largely determined by the availability of stromal microenvironments. These specialized areas emerge during thymus organogenesis and are maintained throughout life. They are formed from both epithelial and mesenchymal components, and collectively they support a stepwise program of thymocyte development. Of these stromal cells, cortical, and medullary thymic epithelial cells represent functional components of thymic microenvironments in both the cortex and medulla. Importantly, a key feature of thymus function is that levels of T cell production are not constant throughout life. Here, multiple physiological factors including aging, stress and pregnancy can have either short- or long-term detrimental impact on rates of thymus function. Here, we summarize our current understanding of the development and function of thymic epithelial cells, and relate this to strategies to protect and/or restore thymic epithelial cell function for therapeutic benefit.

PD-1/PD-L1 signaling regulates the thymic niche size of agonistly selected T cells

Abstract Effective peripheral T cell responses rely upon a diverse T cell repertoire, generated during thymic selection. Signaling through the TCR establishes thresholds for thymocyte selection in order to create this repertoire. Because co-stimulation modifies TCR signaling, it may contribute to the regulation of T cell development by determining signal strength thresholds that direct thymocytes into specific lineages. Programmed death-1 (PD-1), a member of the B7/CD28 family of costimulatory/inhibitory receptors, has been implicated in the development of thymocytes because it is expressed in CD4− CD8− double-negative thymocytes during TCRb rearrangement. Further, the ligands for PD-1, PD-L1 and PD-L2, are expressed on thymic epithelial cells. PD-L2 is restricted to the medulla whereas PD-L1 is expressed throughout the thymus. Here, we show that PD-1 modulates agonist selection, a process in which some high-affinity thymocytes will mature into unconventional T cell lineages (e.g. regulatory T cells, NKT cells). Transgenic mice lacking either PD-L1 or PD-1 have increased numbers of agonistly selected T cells compared to wildtype animals. Interestingly, loss of PD-L1 increases the selection of conventional and agonistly selected T cells whereas loss of PD-1 limits the phenotype to agonist lineages. These data demonstrate that PD-1, PD-L1, and possibly PD-L2 interactions, potentially within the thymic microenvironment in which they are expressed, play a role in determining T cell fate and shaping the final T cell repertoire.

Thymic epithelial miR-155 promotes regulatory T cell development through safeguarding medullary thymic epithelial cell maturation

Abstract During thymocyte development, medullary thymic epithelial cells (mTECs) provide appropriate instructive cues in the thymic microenvironment for not only negative selection but also the generation of regulatory T (Treg) cells. Here, we identify that miR-155, a microRNA whose expression in Treg cells has previously been shown to be crucial for their development and homeostasis, also contributes to thymic Treg (tTreg) cell differentiation by promoting mTEC maturation. Mechanistically, we show that RANKL stimulation induces expression of miR-155 to safeguard the thymic medulla through targeting multiple known and previously uncharacterized molecules within the TGFβ signaling pathway, which is recognized for its role in restricting the maturation and expansion of mTECs. Our work uncovers a miR-155-TGFβ axis in the thymic medulla to determine mTEC maturity and, consequently, the quantity of tTreg cells and suggests that miR-155 ensures proper tTreg cell development in both cell-intrinsic and -extrinsic manners.

Redox regulation of autophagy in thymic stromal cell function

Abstract T lymphocytes develop in the thymus, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells (TSCs) directs the progenitors along a well-characterized program of differentiation. However, the biology of the TSCs comprising the lymphopoietic thymic microenvironment remains relatively under-characterized because stromal cells are rare and difficult to isolate. Using a deconvolution technique to study gene expression essentially in situ, we previously identified a deficiency in the H202 quenching enzyme catalase (CAT) in TSCs, and found that CAT deficiency results in high H202 levels in this population, eventually leading to thymic atrophy. Our current studies address the possibility that high H202 levels serve physiological functions in TSCs in the young, steady state thymus. TSCs exhibit high basal levels of autophagy at the steady state, which is critical for self-antigen presentation and T cell selection. The mechanisms governing high basal autophagy in TSCs are unknown, however autophagy is induced by many stressors, including high H202 levels. Our data indicate that catalase overexpression targeted to mitochondria in transgenic mice (mCAT Tg) results in diminished autophagy in TSCs and causes diminished negative selection in the thymus, eventually leading to autoimmunity. The effects on negative selection in mCAT Tg mice are rescued by increased basal autophagy on the beclin 1 knock-in (Becn1F121A/F121A) background. Our results suggest that the high basal autophagy level required in TSCs for T cell negative selection is promoted by physiologically low levels of catalase expression in the steady state thymus.

Alteration in Three-Dimensional Microenvironment of Themis-deficient Murine Thymus Using Image Cytometry Approach

Abstract Themis is a T-cell intrinsic protein involved in selection during thymocyte development in the thymus. Thus, Themis-deficient mice have gravely reduced numbers of T-cells. Despite extensive research on thymocyte signalling, the exact role of Themis remains a mystery. As the thymic 3-dimensional (3D) microenvironment is a critical element in supporting thymocyte development and vice versa, we were interested to investigate the effect of Themis deficiency on thymic components and processes. In this study, we paired 3D imaging with conventional flow cytometry to understand thymic development in normal and Themis-deficient condition. Current conventional 2-dimensional (2D) histology and 3D reconstruction from 2D sections are unsatisfactory for studying thymus morphology. We thus established a novel approach for whole thymus 3D imaging to visualize developing thymocytes and supporting stroma, such as epithelial cells, myeloid cells and vasculature. 3D imaging coupled with quantitative analysis of images using image cytometry allows for objective comparison of the thymic microenvironment between sample groups. Here, we show an altered medullary and vasculature organization in Themis−/− thymus, suggesting a suboptimal 3D niche supporting thymocyte development. Correspondingly, mature single-positive thymocytes which are dependent on the medullary environment are altered phenotypically. In addition, we detected higher levels of apoptosis in Themis−/− cortex and an increase in clonally deleted double-positive thymocytes. Our findings demonstrate how Themis-deficiency affects developing thymocytes and its supporting stromal components.

Gelatin-Based 3D Microgels for In Vitro T Lineage Cell Generation.

T cells are predominantly produced by the thymus and play a significant role in maintaining our adaptive immune system. Physiological involution of the thymus occurs gradually with age, compromising naive T cell output, which can have severe clinical complications. Also, T cells are utilized as therapeutic agents in cancer immunotherapies. Therefore, there is an increasing need for strategies aimed at generating naive T cells. The majority of in vitro T cell generation studies are performed in two-dimensional (2D) cultures, which ignore the physiological thymic microenvironment and are not scalable; therefore, we applied a new three-dimensional (3D) approach. Here, we use a gelatin-based 3D microgel system for T lineage induction by co-culturing OP9-DL4 cells and mouse fetal-liver-derived hematopoietic stem cells (HSCs). Flow cytometric analysis revealed that microgel co-cultures supported T lineage induction similar to 2D cultures while providing a 3D environment. We also encapsulated mouse embryonic thymic epithelial cells (TECs) within the microgels to provide a defined 3D culture platform. The microgel system supported TEC maintenance and retained their phenotype. Together, these data show that our microgel system has the capacity for TEC maintenance and induction of in vitro T lineage differentiation with potential for scalability.

Biomodulina T May Restore Immunity in Older Adults.

Worldwide, there has been a progressive demographic shift over the past 50 years resulting in a larger proportion of older adults in the general population. Aging itself is a complex biological phenomenon characterized in part by changes in the immune system known as "immunosenescence", which makes older adults more susceptible to infectious, cardiovascular and autoimmune diseases, as well as cancers. Several strategies have been proposed in an attempt to reverse immunosenescence, including use of hormones, cytokines and thymic factors. A promising drug in the search to restore the thymic microenvironment (which plays an important role in the regulation and maintenance of the immune system) in older adults is Biomodulina T, a Cuban product registered for use in patients with recurrent respiratory infections. The administration of Biomodulina T increases the number of naïve T-lymphocyte, CD4-positive cells that have recently migrated from the thymus gland (recent thymic emigrants) and memory CD8-positive T lymphocytes, which have characteristics akin to stem cells (stem cell-like memory). Furthermore, the expression of programmed cell death 1 protein in CD4-positive T lymphocytes and CD4-positive T lymphocytes decreases, and the proliferative capacity of CD4-positive T lymphocytes increases, without changes in the frequency of regulatory T lymphocytes. These results suggest that the administration of Biomodulina T could be used to restore immunity in older adults and in other immunocompromised individuals, improve response to other immunotherapies in cancer patients, and increase the effi cacy of vaccinations in older adults. Its use has been approved in Cuba for immune system restoration.KEYWORDS Immunosenescence, aging, immunotherapy, immuno-modulation, antineoplastic protocols, Cuba.

Testing the Efficiency and Kinetics of Negative Selection Using Thymic Slices.

Central tolerance is an efficient barrier to autoimmunity and negative selection of self-reactive thymocytes is one of its major manifestations. Because of its importance, negative selection has been studied extensively through numerous in vitro and in vivo approaches that have tremendously increased our understanding of the process. Recently, in situ experimental systems using thymus slices have been developed that combine some of the advantages of in vitro assays such as ease of manipulation and high throughput with the existence of three dimensional mature thymus microenvironment. These approaches offer unprecedented opportunity to study negative selection. Here, we describe how thymic slices can be used to measure the kinetics and magnitude of negative selection. Taking the OT-1/Ova system as an example, we provide detailed guidance on cutting thymic slices, labeling and overlaying thymocytes on them and reading out the extent of negative selection by flow cytometry. The system can easily be adapted to evaluate the effects of various mutations or treatments on negative selection or to study the behavior of different cells in the thymus through time-lapse imaging.