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Thymic output and homeostatic mature cell proliferation both influence T cell repopulation following depletional induction, though the relative contribution of each and their association with recipient age have not been well studied. We investigated the repopulating T cell kinetics in kidney transplant recipients who underwent alemtuzumab induction followed by belatacept/rapamycin-based immunosuppression over 36-month posttransplantation. We focused specifically on the correlation between repopulating T cell subsets and the age of patients. Substantial homeostatic Ki67-expressing T cell proliferation was seen posttransplantation. A repertoire enriched for naive T (TNaive ) cells emerged posttransplantation. Analysis by generalized estimating equation linear models revealed a strong negative linear association between reconstituting TNaive cells and advancing age. A relationship between age and persistence of effector memory cells was shown. We assessed thymic output and found an increase in the frequency of recent thymic emigrants (RTEs, CD4+ CD31+ ) at 12-month posttransplantation. Patients under 30 years of age showed significantly higher levels of CD4+ CD31+ cells than patients over 55 years of age pre- and posttransplantation. IL-7 and autologous mature dendritic cells (mDCs) induced CD57- cell proliferation. In contrast, mDCs, but not IL-7, induced CD57+ cell proliferation. This study establishes the relationship between age and thymic output during T cell homeostatic repopulation after alemtuzumab induction. Trial Registration: ClinicalTrials.gov - NCT00565773.

Immunosuppressive drugs such as cyclosporine A (CSA) can disrupt thymic structure and functions, ultimately inducing syngeneic/autologous graft-versus-host disease together with involuted medullas. To elucidate the effects of CSA on the thymus more precisely, we analyzed the effects of CSA on the thymus and T cell system using rats. In addition to confirming the phenomena already reported, we newly found that the proportion of recent thymic emigrants also greatly decreased, suggesting impaired supply. Immunohistologically, the medullary thymic epithelial cells (mTECs) presented with a relative decrease in the subset with a competent phenotype and downregulation of class II major histocompatibility complex molecules. In control rats, thymic dendritic cells (DCs) comprised two subsets, XCR1+SIRP1α−CD4− and XCR1−SIRP1α+CD4+. The former had a tendency to selectively localize in the previously-reported epithelium-containing areas of the rat medullas, and the number was significantly reduced by CSA treatment. The epithelium-free areas, another unique domains in the rat medullas, contained significantly more Foxp3+ thymic Tregs. With CSA treatment, the epithelium-free areas presented strong involution, and the number and distribution of Tregs in the medulla were greatly reduced. These results suggest that CSA inhibits the production of single-positive thymocytes, including Tregs, and disturbs the microenvironment of the thymic medulla, with a decrease of the competent mTECs and disorganization of epithelium-free areas and DC subsets, leading to a generation of autoreactive T cells with selective medullary involution.

Background Chemotherapy can cause thymic atrophy and reduce T-cell output in cancer patients. However, the thymus in young adult patients has regenerative potential after chemotherapy, manifesting as thymic hyperplasia which can be easily mistaken as residual disease or recurrence in patients suffering lymphoma. Case presentation This study reports a case of lymphoma in a young female adult who was initially diagnosed with an anterior mediastinal mass, and was found to have soft tissue occupying the anterior mediastinum repeatedly after chemotherapy, suggesting a lymphoma residue or disease progression. From discussions by a multi-disciplinary team (MDT), the anterior mediastinal mass of the patient was considered unknown and might be thymus tissue or tumor tissue, and it was eventually identified as thymus tissue via histopathology. Conclusions The anterior mediastinal mass appearing after chemotherapy in patients with lymphoma can be considered as enlarged thymus, and such phenomenon is frequent in young adult patients who undergo chemotherapy or autologous hematopoietic stem cell transplantation. Additionally, detection of thymic output cells in peripheral blood might be a feasible approach to differentiate thymic hyperplasia from lymphoma.

Background The relationship between circulating regulatory T-cell (Tregs) subset distribution and hypertension severity in children with primary hypertension is not known. We aimed to find out if target organ damage (TOD) in children with primary hypertension is related to defects in Tregs distribution reflected by their phenotype characteristics. Methods The study constituted 33 nontreated hypertensive children and 35 sex-matched and age-matched controls. Using multicolor flow cytometry technique, we assessed a distribution of the total Tregs (CD4CD25CD127) and their subsets (CD45RA-naive Tregs, CD45RA memory/activated Tregs, CD45RACD31 recent thymic emigrants Tregs and mature naive CD45RACD31 Tregs) in the whole blood. Results Hypertensive children showed decreased percentage of the total Tregs, the CD45RA-naive Tregs, the total CD31 Tregs and the recent thymic emigrants Tregs but elevation of the CD45RA memory/activated Treg and mature naive CD45RACD31 Tregs. Decreased frequency of the total Tregs, naive Tregs and CD31-bearing Treg cell subsets (CD31 total Tregs, CD45RACD31 recent thymic emigrants Tregs) negatively correlated to TOD markers, arterial stiffness and blood pressure elevation. In contrast, increased percentage of memory Tregs and CD31 Tregs subsets positively correlated to organ damage markers, arterial stiffness and blood pressure values. These changes were independent of BMI, age, sex and hsCRP. Conclusion Both diagnosis of hypertension, TOD and arterial stiffness in hypertensive children were associated with decreased population of total CD4 Tregs, limited output of recent thymic emigrants Tregs, and increased pool of activated/memory Tregs. Hypertension was an independent predictor of the circulating Treg subsets distribution irrespective of hsCRP.

Supplemental Digital Content is available in the text, Objective: Around 20–30% of HIV-infected individuals (HIV+) on successful antiretroviral therapy (ART) fail to normalize their CD4+ T-cell counts. Various factors could contribute to the lack of immune reconstitution, one of them being thymic insufficiency. We aimed to explore associations between recent thymic emigrants (RTEs) and CD4+ T-cell recovery. Design: ART-naive HIV+ individuals who started ART with advanced AIDS were selected. Good versus poor immune reconstitution was defined by CD4+ gains above or below 100 CD4+ T cells/μl. The follow-up period was 6 months. Methods: Peripheral blood mononuclear cells were isolated and flow cytometry was used to characterize RTEs as the fraction of naive CD4+ T cells expressing CD31+, the platelet endothelial cell adhesion molecule. Markers of cellular activation, senescence, exhaustion and cycling were also assessed. Results: After 6 months on ART, HIV+ individuals with good immune reconstitution had higher absolute numbers of RTEs, compared with those with poor immune reconstitution, and these strongly correlated with CD4+ gains in those individuals with good immune reconstitution but not with poor immune reconstitution. We also found that CD8+ T-cell immune activation decreased as early as 2 months post-ART initiation in individuals with good immune reconstitution, but only at month 6 post-ART in individuals with poor immune reconstitution. Levels of immune activation were inversely correlated with the absolute numbers of RTEs in both groups, but more strongly so in individuals with poor immune reconstitution. Conclusion: We show that RTEs are linked to CD4+ T-cell recovery and that the degree of immune reconstitution is not directly linked to persistent immune activation.

Secondary lymphoid organs (SLO; including the spleen and lymph nodes) are critical both for the maintenance of naïve T (TN) lymphocytes and for the initiation and coordination of immune responses. How they age, including the exact timing, extent, physiological relevance, and the nature of age-related changes, remains incompletely understood. We used time-stamping to indelibly mark cohorts of newly generated naïve T cells (a.k.a. recent thymic emigrants - RTE) in mice, and followed their presence, phenotype and retention in SLO. We found that SLO involute asynchronously. Skin-draining lymph nodes (LN) atrophied early (6-9 months) in life and deeper tissue-draining LN and the spleen late (18-20 months), as measured by the loss of both TN numbers and the fibroblastic reticular cell (FRC) network. Time-stamped RTE cohorts of all ages entered SLO and successfully completed post-thymic differentiation. However, in older mice, these cells were poorly retained, and those found in SLO exhibited an emigration phenotype (CCR7loS1P1hi). Transfers of adult RTE into recipients of different ages formally demonstrated that the defect segregates with the age of the SLO microenvironment and not with the age of T cells. Finally, upon intradermal immunization, RTE generated in mice as early as 6-7 months of age barely participated in de novo immune responses and failed to produce well-armed effector cells. These results highlight changes in structure and function of superficial secondary lymphoid organs in laboratory mice that are earlier than expected and are consistent with the long-appreciated and pronounced reduction of cutaneous immunity with aging.

Prolonged lymphopenia represents a major clinical problem after cytoreductive therapies such as chemotherapy and the conditioning required for hematopoietic stem cell transplant (HCT), contributing toward the risk of infections and malignant relapse. Restoration of T cell immunity is dependent on tissue regeneration in the thymus, the primary site of T cell development; although the capacity of the thymus to repair itself diminishes over lifespan. However, although boosting thymic function and T cell reconstitution is of considerable clinical importance, there are currently no approved therapies for treating lymphopenia. Here we found that Zinc (Zn), is critically important for both normal T cell development as well as repair after acute damage. Accumulated Zn in thymocytes during development was released into the extracellular milieu after HCT conditioning, where it triggered regeneration by stimulating endothelial cell-production of BMP4 via the cell surface receptor GPR39. Dietary supplementation of Zn was sufficient to promote thymic function in a mouse model of allogeneic HCT, including enhancing the number of recent thymic emigrants in circulation; although direct targeting of GPR39 with a small molecule agonist enhanced thymic function without the need for prior Zn accumulation in thymocytes. Together, these findings not only define an important pathway underlying tissue regeneration, but also offer an innovative preclinical approach to treat lymphopenia in HCT recipients.KEY POINTSThymocytes release zinc after HCT conditioning is sensed by GPR39 and promotes epithelial repairPharmacologic stimulation of GPR39 promotes T cell reconstitution after HCT

BackgroundEnd-stage renal disease is associated with premature ageing of the T cell immune system but inter-individual variation is substantial. The hypothesis was tested that advanced immunological T cell ageing assessed by peripheral T cell differentiation increases the long-term mortality risk after renal transplantation.ResultsCirculating T cells of 211 recipients of a kidney from a living donor were analyzed before and in the first year after transplantation. The number of CD31-positive naive T cells (as a marker for recent thymic emigrants) and the differentiation status of the memory T cells was assessed. Thirty recipients died during follow-up of at least 5 years. Absolute numbers of naive CD4+(living:258 cells/μl vs. deceased:101 cells/μl,p +T cells (living:97 cells/μl vs. deceased:37 cells/μl, p +T cells remained associated with all-cause mortality (HR 0.98, CI 0.98–0.99,p +naive T cells) indicating a lowered thymus function. In addition, the physiological age-related compensatory increase in CD31−naïve T cells was not observed. Within the first year after transplantation, the number and characteristics of naive T cells remained stable.ConclusionsA severe reduction in circulating naïve T cells because of a decrease in recent thymic emigrants is highly associated with all-cause mortality after renal transplantation.

Introduction: Common variable immunodeficiency (CVID) is characterized by recurrent infections, autoimmunity, lymphoproliferation, hypogammaglobulinemia, and defective antibody production. In CVID, B-cell abnormalities were described to predict end organ involvement and prognosis. Pediatric-onset CVID is much rarer than adult CVID, and lymphocyte subset abnormalities have not been thoroughly evaluated. Objective: We sought to determine lymphocyte subset abnormalities and their association with end organ involvement in pediatric-onset CVID patients. Methods: The clinical manifestations and laboratory findings including absolute numbers and percentages of B-, T-, and NK cell populations were assessed in pediatric-onset CVID patients and compared to age-matched healthy controls. The patients were divided into 2 groups according to age at assessment (pediatric CVID patients: 10–16 years, n = 9; and adult CVID patients: >16 years, n = 13). The comparisons between lymphocyte subsets and organ involvement were also evaluated. Results: Mean age at symptom onset was 18 (3–204) months. All CVID patients with pediatric onset had decreased levels of total and memory B cells, CD4+ T cells, CD4+CD45RA+ naive T cells, and recent thymic emigrant (RTE) cells. On the other hand, they had increases in CD8+CD45RO+ memory T cells. Interestingly, adult CVID patients demonstrated high frequencies of activated and double-negative T cells, which were unique only for this group of patients. Specific cellular abnormalities associated with the reduction in B and NK cells and increase in CD8+ T cells were found in patients with bronchiectasis. Moreover, in pediatric CVID patients, low serum IgA levels and decreased numbers of naive T and RTE cells were determined as risk factors for chronic diarrhea. Conclusions: Specific abnormalities in B- and T-lymphocyte compartments were identified in pediatric-onset CVID patients and appear to be associated with end organ manifestations.

Thymic output was studied prospectively in 52 children who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). Thymic activity was assessed by quantification of recent thymic emigrants (RTE) discriminated from the rest of naive T cells by immunophenotype CD3+/CD4+/CD31+/CD45RA+. Thymic output was analyzed in correlation with the kinetics of immune recovery and in relation to other potential risk factors that may influence thymopoiesis: underlying disease, type of HSCT, source of stem cells, age of recipient and donor, type of conditioning, implemented graft versus host disease (GvHD) prophylaxis, viral reactivations (herpes viruses cytomegalovirus – CMV, Epstein-Barr virus – EBV, adenovirus – ADV, BK virus – BKV), occurrence and grade of both acute and chronic graft versus host disease (aGvHD, cGvHD) and number of transplanted CD34 cells/kg. The absolute count of RTE in peripheral blood was evaluated at 6 time points: before the conditioning and on days +15, +30, +60 , +90 and +180 after HSCT. Occurrence of grade II-IV aGvHD was the most important factor associated with low RTE counts after HSCT. History of malignant disease, and transplantation from matched unrelated donor were risk factors for lower thymic output. We found a weak inverse correlation between the age of the recipient and thymic output on post-HSCT day +180. Source of stem cells, type of conditioning, viral reactivations, occurrence of chronic GvHD, age of the donor and the number of transplanted CD34 cells/kg did not affect thymopoiesis in our study group. These preliminary findings and identification of risk factors for deterioration of thymic activity may in the future help in selecting candidates for thymus rejuvenation strategies.

Most of primary immunodeficiencies with hypogammaglobulinemia are associated with reduced memory B cells. T cell development may be interesting as well, but increased recent thymic emigrants are rarely reported in these patients. We report the case of a family (mother and her two sons) diagnosed with common variable immunodeficiency 10 due to a mutation in the NFKB2 gene. Laboratory findings showed that all three patients presented hypogammaglobulinemia, reduced memory B cells and elevated naïve T lymphocytes and recent thymic emigrants. This feature, in the absence of glucocorticoid deficiency, may suggest a primary thymic dysfunction. Interestingly, the mother presented the worst immune phenotype, as regards both antibody production and NK function, indicating that immune function may deteriorate in the course of time. We conclude that close monitoring of immune functions may widen the knowledge on the CVID10 and improve the patients’ care.

In our previous study, we found that low thymic output and short telomere length were associated with a higher risk of tumor in elderly cancer patients. Here, we aimed to examine in depth the impact of immunological and biological senescence and immune activation on disease outcome in elderly patients with colorectal cancer (CRC).Peripheral blood samples from 81 CRC patients were studied for immune activation, immune senescence and recent thymic emigrant(RTE) CD4 and CD8 cells by flow cytometry. T-cell receptor rearrangement excision circle (TREC) levels and telomere lengths were measured by real-time PCR. Plasma levels of microbial translocation markers, LPS and sCD14, were quantified by ELISA. While TREC levels and telomere length were not prognostic of disease outcome, high percentages of immune senescent and immune activated CD8 cells were associated with a higher risk of a negative event (relapse, progression, or death) in all studied patients and disease relapse in I-III staged patients. Levels of sCD14 and LPS were higher in patients who will experience a negative event than in patients who will not. In conclusion, in elderly CRC patients higher immunological senescence and immune activation negatively impact the disease outcome; how these characteristics influence the antineoplastic treatments remains to be investigated.

Regulatory T cells (Tregs) are indispensable for the control of immune homeostasis and have clinical potential as a cell therapy for treating autoimmunity. Tregs can lose expression of the lineage-defining Foxp3 transcription factor and acquire effector T cell (Teff) characteristics, a process referred to as Treg plasticity. The extent and reversibility of such plasticity during immune responses remain unknown. Here, using a murine genetic fate-mapping system, we show that Treg stability is maintained even during exposure to a complex microbial/antigenic environment. Furthermore, we demonstrate that the observed plasticity of Tregs after adoptive transfer into a lymphopenic environment is a property limited to only a subset of the Treg population, with the nonconverting majority of Tregs being resistant to plasticity upon secondary stability challenge. The unstable Treg fraction is a complex mixture of phenotypically distinct Tregs, enriched for naïve and neuropilin-1-negative Tregs, and includes peripherally induced Tregs and recent thymic emigrant Tregs These results suggest that a "purging" process can be used to purify stable Tregs that are capable of robust fate retention, with potential implications for improving cell transfer therapy. ispartof: SCIENCE IMMUNOLOGY vol:6 issue:61 ispartof: location:United States status: published

Most patients with primary immune deficiency suffer from recurrent infections, however, paradoxical autoimmune phenomena can also manifest. The aim of this study was to identify immunological markers of autoimmune phenomena associated with common variable immunodeficiency (CVID). The study included 33 adults with CVID divided into two groups: (1) those with noninfectious autoimmune complications (CVID-C (n = 24)) and (2) those with only infectious symptoms (CVID-OI (n = 9)). Flow cytometry of peripheral blood was performed and compared with systemic lupus erythematosus (SLE) patients (n = 17) and healthy controls (n = 20). We found that all lymphocytes were lower in CVID-C and SLE. NK cells were lowest in CVID-C. Th17 cells were significantly reduced in CVID-C and SLE. Tregs were significantly lower in CVID-C and SLE. Bregs did not significantly differ between any groups. Class-switched memory B cells were significantly lower in CVID-C and CVID-OI. Lastly, plasmablasts were significantly higher in SLE. Among the T cell subsets, CVID-C patients had lower naive and recent thymic emigrant CD4+ T cells. In conclusion, reduced Treg, Th17, and NK cells are features of CVID with autoimmune complications, and class-switched memory B cells can help distinguish patients with different causes of autoimmunity. Future studies are needed to confirm whether reductions of Treg, Th17, and NK cells might be a biomarker of more complicated CVID cases.

Age‐related thymic atrophy results in reduced output of naïve conventional T (Tcon) cells. However, its impact on regulatory T (Treg) cells is insufficiently understood. Given evidence that thymic Treg (tTreg) cell generation is enhanced in the aged, atrophy thymus and that the aged periphery accumulates peripheral Treg (pTreg) cells, we asked why these Treg cells are unable to effectively attenuate increased autoreactivity‐induced chronic inflammation in the elderly. We designed a mock‐self‐antigen chimera mouse model, in which membrane‐bound ovalbumin (mOVA) transgenic mice, bearing a FoxN1‐floxed gene for induction of conditional thymic atrophy, received OVA‐specific (OT‐II) T‐cell receptor (TCR) transgenic progenitor cells. The chimeric mice with thymic atrophy exhibited a significant decrease in OVA‐specific tTreg and pTreg cells but not polyclonal (pan)‐Treg cells. These OVA‐specific pTreg cells were significantly less able to suppress OVA‐specific stimulation‐induced proliferation in vitro and exhibited lower FoxP3 expression. Additionally, we conducted preliminary TCR repertoire diversity sequencing for Treg cells among recent thymic emigrants (RTEs) from Rag(GFP)‐FoxP3(RFP) dual‐reporter mice and observed a trend for decreased diversity in mice with thymic atrophy compared to littermates with normal thymus. These data indicate that although the effects of age‐related thymic atrophy do not affect pan‐Treg generation, certain tissue‐specific Treg clones may experience abnormal agonist selection. This, combined with enhanced pan‐pTreg cells, may greatly contribute to age‐related chronic inflammation, even in the absence of acute autoimmune disease in the elderly.

As the thymus represents the primary site of T-cell development, optimal thymic function is of paramount importance for the successful reconstitution of the adaptive immunity after allogeneic hematopoietic stem cell transplantation. Thymus involutes as part of the aging process and several factors, including previous chemotherapy treatments, conditioning regimen used in preparation to the allograft, occurrence of graft-versus-host disease, and steroid therapy that impair the integrity of the thymus, thus affecting its role in supporting T-cell neogenesis. Although the pathways governing its regeneration are still poorly understood, the thymus has a remarkable capacity to recover its function after damage. Measurement of both recent thymic emigrants and T-cell receptor excision circles is valuable tools to assess thymic output and gain insights on its function. In this review, we will extensively discuss available data on factors regulating thymic function after allogeneic hematopoietic stem cell transplantation, as well as the strategies and therapeutic approaches under investigation to promote thymic reconstitution and accelerate immune recovery in transplanted patients, including the use of cytokines, sex-steroid ablation, precursor T-cells, and thymus bioengineering. Although none of them is routinely used in the clinic, these approaches have the potential to enhance thymic function and immune recovery, not only in patients given an allograft but also in other conditions characterized by immune deficiencies related to a defective function of the thymus.

Background. Regulatory T cells (Tregs) and recent thymic emigrants (RTEs) have an essential role in the regulation of allogeneic immune responses. However, their mechanisms of action in chronic antibody-mediated rejection (cAMR) are still unclear. In this study, we aimed to compare Treg and RTE levels between stable graft function (SGF) patients and cAMR subjects after kidney transplantation. Method. Mononuclear cells (MNs) were separated from peripheral blood, and flow cytometry analysis was performed for detection of CD4+ and CD25high as Treg markers and CD4+, CD31+, and CD45RA+ as RTE immunophenotyping markers. Result. The level of peripheral Treg cells was significantly lower in cAMR subjects in comparison to stable graft function patients. Moreover, SGF patients who had received cyclosporine A had a higher level of Treg in comparison to the tacrolimus recipients. Nevertheless, the RTE level between SGF and cAMR patients did not show any significant differences. Conclusion. It seems that Treg cells are significantly associated with transplant outcomes in cAMR patients, and prescribed immunosuppressive drugs can influence the frequency of this crucial subset of T cells. Although these drugs are beneficial and inevitable for allograft maintenance, more investigations are needed to elucidate their complete effects on different immune cell subsets which some of them like Tregs are in favor of transplant tolerance. Besides, the thymic output is seemingly not a beneficial biomarker for predicting cAMR; however, more in vivo and in vitro studies are needed for revealing the precise role of Tregs and RTEs in the transplantation context.

Paediatric heart transplantation recipients suffer an increased incidence of infectious, autoimmune and allergic problems. The relative roles of thymus excision and immunosuppressive treatments in contributing to these sequelae are not clear. We compared the immunological phenotypes of 25 heart transplant recipients (Tx), 10 children who underwent thymus excision during non-transplantation cardiac surgery (TE) and 25 age range-matched controls, in two age bands: 1-9 and 10-16 years. Significant differences from controls were seen mainly in the younger age band with Tx showing lower CD3 and CD4 cell counts whilst TE showed lower CD8 cell counts. Naïve T cell and recent thymic emigrant proportions and counts were significantly lower than controls in both groups in the lower age band. T cell recombination excision circle (TREC) levels were lower than controls in both groups in both age bands. There were no differences in regulatory T cells, but in those undergoing thymus excision in infancy, their proportions were higher in TE than Tx, a possible direct effect of immunosuppression. T cell receptor V beta spectratyping showed fewer peaks in both groups than in controls (predominantly in the older age band). Thymus excision in infancy was associated with lower CD8 cell counts and higher proportions of Tregs in TE compared to Tx. These data are consistent with thymus excision, particularly in infancy, being the most important influence on immunological phenotype after heart transplantation.

Pyroptosis cell death in recent thymus emigrants (RTE) CD4+ T lymphocytes plays an important role on HIV-1 infection as a cause of CD4+ T cell depletion, being influenced by several factors, among them, the sex. Thus, the aim of this study was evaluated pyroptosis levels in RTE CD4+ T lymphocytes of individuals under antiretroviral therapy (ART) stratified by sex. Thirty-seven ART-treated HIV-positive patients (22 females and 15 males) and 12 (seven females and five males) clinically health subjects were recruited. Analysis by flow-cytometry of RTE CD4+ cells (CD4+ CD31+ /fluorescent-labeled inhibitors of caspases-Caspase-1+) were performed. Clinical and sociodemographic aspects were also evaluated from medical records. We observed statistically higher levels of pyroptosis RTE CD4+ T cells in male individuals (69.3%) compared with female group (39.1%) (P = 0.0356). Pre- and post-treatment CD4+ T cell counts were also higher in women than men (P = 0.004 and P = 0.012, respectively). Our data provides important evidence of the sex as a potential predictor of immunological reconstitution in ART-treated individuals.

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 naive 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.

Background: Prolonged immunosuppression and hypoinflammation, termed compensatory anti-inflammatory response syndrome (CARS), contribute to high morbidity and mortality in the late phase of sepsis. Although apoptosis is a well-known cause of lymphopenia in sepsis, the contribution of thymic output to immune alterations in sepsis and potential compensatory mechanisms are largely unknown. Methods: We investigate the release of CD4+ T cells from the thymus and their peripheral proliferation by evaluating T-cell receptor excision circles (TREC) and the expression of CD31 as markers for recent thymic emigrants (RTE) and their proliferative offspring in septic patients with relevant lymphopenia in the CARS phase. Moreover, we determine the aging of T cells by measuring telomere characteristics. Results: In septic patients, we found decreased CD4+ T-helper cell numbers, while CD8+ T cell numbers were unchanged. As a possible cause, we detected increased apoptosis of CD4+ T-helper cells and decreased levels of IL-7, which promotes the maturation of T cells in the thymus. Accordingly, the relative number of mature CD4+ T cells, TREC-containing CD4+ T cells, and CD31+ RTEs (characteristic of thymic output) was decreased, while the relative number of CD31-T cells (peripherally expanded naïve T cells) was increased. Furthermore, the telomere length decreased, although telomerase activity and markers for the shelterin complex were increased specifically in CD4+ but not in CD8+ T cells. Conclusion: We thus conclude that, in addition to T-cell apoptosis, decreased thymic output and increased aging of CD4+ T cells may contribute to lymphopenia and immunosuppression in sepsis. Increased proliferation of peripheral T cells cannot compensate for these effects.

Recent thymic emigrants are the youngest subset of peripheral T cells and their involvement in combating persistent bacterial infections has not been explored. Here, we hypothesized that CD4+ recent thymic emigrants are essential immune mediators during persistent Salmonella infection. To test this, we thymectomized adult mice either prior to, or during, persistent Salmonella infection. We found that thymic output is crucial in the formation of protective immune responses during the early formation of a Salmonella infection but is dispensable once persistent Salmonella infection is established. Further, we show that thymectomized mice demonstrate increased infection-associated mortality and bacterial burdens. Unexpectedly, numbers of Salmonella-specific CD4+ T cells were significantly increased in thymectomized mice compared to sham control mice. Lastly, we found that T cells from thymectomized mice may be impaired in producing the effector cytokine IL-17 at early time points of infection, compared to thymically intact mice. Together, these results imply a unique role for thymic output in the formation of immune responses against a persistent, enteric pathogen.

Thymic involution is an important factor leading to the aging of the immune system. Most of what we know regarding thymic aging comes from mouse models, and the nature of the thymic aging process in humans remains largely unexplored due to the lack of a model system that permits longitudinal studies of human thymic involution. In this study, we sought to explore the potential to examine human thymic involution in humanized mice, constructed by transplantation of fetal human thymus and CD34+ hematopoietic stem/progenitor cells into immunodeficient mice. In these humanized mice, the human thymic graft first underwent acute recoverable involution caused presumably by transplantation stress, followed by an age-related chronic form of involution. Although both the early recoverable and later age-related thymic involution were associated with a decrease in thymic epithelial cells and recent thymic emigrants, only the latter was associated with an increase in adipose tissue mass in the thymus. Furthermore, human thymic grafts showed a dramatic reduction in FOXN1 and AIRE expression by 10 weeks post-transplantation. This study indicates that human thymus retains its intrinsic mechanisms of aging and susceptibility to stress-induced involution when transplanted into immunodeficient mice, offering a potentially useful in vivo model to study human thymic involution and to test therapeutic interventions.

Accelerated thymic involution is a main feature of end-stage renal disease (ESRD)-associated immune senescence. Recent evidences suggest that ESRD-associated immune senescence is associated with adverse outcomes in dialysis patients. However, no study focused on the association between pre-transplant thymic function and patient survival after transplantation. We conducted a prospective, multicenter study to assess whether pre-transplant thymic function measured by recent thymic emigrants (RTE) may predict death after first kidney transplantation. Results were tested in a validation cohort. Nine hundred and sixty-seven incident kidney transplant recipients were included in the prospective study. Mean follow up was 5.1 + 2.9 years. Eighty two patients (8.5%) died during follow up. Lower RTE levels were associated with a higher risk of death (2.53; 95%CI, 1.54–4.39 for each decrease of 1 log in RTE; p < 0.001). Cancer-related death was particularly increased in patients with low RTE levels (4.23; 95%CI, 1.43–12.13; p = 0.007). One hundred and thirty-six patients having received a first kidney transplantation were included in the validation cohort. Lower TREC levels were associated with higher risk of death (1.90; 95%CI, 1.11–3.51 for each decrease of 1 log in RTE; p = 0.025). RTE were not associated with death-censored graft loss. Pre-transplant thymic function is strongly associated with death after transplantation. Attempt to reverse ESRD-related thymic loss may prevent premature death.

Eighty-six infants born without a thymus have been treated with allogeneic cultured thymus tissue implantation (CTTI). These infants, who lack T cells and are profoundly immunodeficient at birth, after CTTI from an unmatched donor develop T cells similar to those of recipient that are tolerant to both their own major histocompatibility antigens and those of the donor. We tested use of CTTI with the goal of inducing tolerance to unmatched heart transplants in immunocompetent rats. We thymectomized and T cell-depleted Lewis rats. The rats were then given cultured thymus tissue from F1 (Lewis × Dark Agouti ) under the kidney capsule and vascularized Dark Agouti (DA) heart transplants in the abdomen. Cyclosporine was administered for 4 months. The control group did not receive CTTI. Recipients with CTTI showed repopulation of naive and recent thymic emigrant CD4 T cells; controls had none. Recipients of CTTI did not reject DA cardiac allografts. Control animals did not reject DA grafts, due to lack of functional T cells. To confirm donor-specific unresponsiveness, MHC-mismatched Brown Norway (BN) hearts were transplanted 6 months after the initial DA heart transplant. LW rats with LWxDA CTTI rejected the third-party BN hearts (mean survival time 10 days); controls did not. CTTI recipients produced antibody against third-party BN donor but not against the DA thymus donor, demonstrating humoral donor-specific tolerance. Taken together, F1(LWxDA) CTTI given to Lewis rats resulted in specific tolerance to the allogeneic DA MHC expressed in the donor thymus, with resulting long-term survival of DA heart transplants after withdrawal of all immunosuppression.

Autoimmune hemolytic anemia (AIHA) leads to accelerated destruction of autologous red blood cells (RBCs) by autoantibodies. AIHA is a severe and sometimes fatal disease. While there are several therapeutic strategies available, there are currently no licensed treatments for AIHA and few therapeutics result in treatment-free durable remission. The etiology of primary AIHA is unknown; however, secondary AIHA occurs concurrently with lymphoproliferative disorders and infections. Additionally, AIHA is the second most common manifestation of primary immunodeficiency disorders and has been described as a side effect of checkpoint inhibitor therapy. Given the severity of AIHA and the lack of treatment options, understanding the initiation of autoimmunity is imperative. Herein, we utilized a well-described model of RBC biology to dissect how RBC-specific autoreactive T cells become educated against RBC autoantigens. We show that, unlike most autoantigens, T cells do not encounter RBC autoantigens in the thymus. Instead, when they leave the thymus as recent thymic emigrants (RTEs), they retain the ability to positively respond to RBC autoantigens; only after several weeks in circulation do RTEs become nonresponsive. Together, these data suggest that any disruption in this process would lead to breakdown of tolerance and initiation of autoimmunity. Thus, RTEs and this developmental process are potential targets to prevent and treat AIHA.

The thymus is a primary lymphoid organ where T lymphocyte proliferation and selection takes place. The different subsets of lymphocytes leave the thymus as recent thymic emigrants. Peripheral dendritic cells migrate to the thymus. In addition to the homing of hematopoietic progenitor cells to the thymus, there is evidence for lymphocyte entry from peripheral lymphoid tissues mainly into the medulla. The entry sites are the venules in the medullary part near to the cortex with a higher endothelium. Furthermore, there are also B lymphocytes in the thymus. The thymus is not only a primary lymphoid organ but is well integrated in lymphocyte traffic as shown in several different species.

Background: Visceral leishmaniasis/HIV-co-infected patients (VL/HIV) accounts for around 8% of VL reported cases in Brazil. Relapses of Leishmania infection after anti-leishmanial treatment constitute a great challenge in the clinical practice because of the disease severity and drug resistance. We have shown that non-relapsing-VL/HIV (NR-) evolved with increase of CD4+ T-cell counts and reduction of activated CD4+ and CD8+ T cells after anti-leishmanial treatment. This immune profile was not observed in relapsing-VL/HIV patients (R-), indicating a more severe immunological compromising degree. Elevated activation status may be related to a deficient immune reconstitution and could help to explain the frequent relapses in VL/HIV co-infection. Our aim was to evaluate if this gain of T cells was related to changes in the peripheral TCRVβ repertoire and inflammatory status, as well as the possible thymus involvement in the replenishment of these newly formed T lymphocytes.Methods: VL/HIV patients, grouped into non-relapsing (NR- = 6) and relapsing (R- = 12) were evaluated from the active phase up to 12 months post-treatment (mpt). HIV-infected patients (non-VL) and healthy subjects (HS) were included. The TCRVβ repertoire was evaluated ex vivo by flow cytometry, whereas the plasmatic cytokine levels were assessed by Luminex assay. To evaluate the thymic output, DNA was extracted from PBMCs for TCR rearrangement excision circles (TREC) quantification by qPCR.Results: VL/HIV cases presented an altered mobilization profile (expansions or retractions) of the TCRVβ families when compared to HS independent of the follow-up phase (p < 0.05). TCRVβ repertoire on CD4+ T-cells was more homogeneous in the NR-VL/HIV cases, but heterogeneous on CD8+ T-cells, since different Vβ-families were mobilized. NR-VL/HIV had the inflammatory pattern reduced after 6 mpt. Importantly, VL/HIV patients showed number of TREC copies lower than controls during all follow-up. An increase of recent thymic emigrants was observed in NR-VL/HIV individuals at 10 mpt compared to R- patients (p < 0.01), who maintained lower TREC contents than the HIV controls.Conclusions: VL/HIV patients that maintain the thymic function, thus generating new T-cells, seem able to replenish the T lymphocyte compartment with effector cells, then enabling parasite control.

22q11.2 deletion syndrome (DiGeorge), CHARGE syndrome, Nude/SCID and otofaciocervical syndrome type 2 (OTFCS2) are distinct clinical conditions in humans that can result in hypoplasia and occasionally, aplasia of the thymus. Thymic hypoplasia/aplasia is first suggested by absence or significantly reduced numbers of recent thymic emigrants, revealed in standard-of-care newborn screens for T cell receptor excision circles (TRECs). Subsequent clinical assessments will often indicate whether genetic mutations are causal to the low T cell output from the thymus. However, the molecular mechanisms leading to the thymic hypoplasia/aplasia in diverse human syndromes are not fully understood, partly because the problems of the thymus originate during embryogenesis. Rodent and Zebrafish models of these clinical syndromes have been used to better define the underlying basis of the clinical presentations. Results from these animal models are uncovering contributions of different cell types in the specification, differentiation, and expansion of the thymus. Cell populations such as epithelial cells, mesenchymal cells, endothelial cells, and thymocytes are variably affected depending on the human syndrome responsible for the thymic hypoplasia. In the current review, findings from the diverse animal models will be described in relation to the clinical phenotypes. Importantly, these results are suggesting new strategies for regenerating thymic tissue in patients with distinct congenital disorders.

Recent thymic emigrants (RTEs) are peripheral T cells that have most recently completed selection and thymic egress and constitute a population that is phenotypically and functionally distinct from its more mature counterpart. Ag-activated RTEs are less potent effectors than are activated mature T cells, due in part to reduced aerobic glycolysis (correctable by exogenous IL-2), which in turn impacts IFN-γ production. Mitochondria serve as nodal regulators of cell function, but their contribution to the unique biology of RTEs is unknown. In this study, we show that activated mouse RTEs have impaired oxidative phosphorylation, even in the presence of exogenous IL-2. This altered respiratory phenotype is the result of decreased CD28 signaling, reduced glutaminase induction, and diminished mitochondrial mass in RTEs relative to mature T cells. These results suggest an uncoupling whereby IL-2 tunes the rate of RTE glycolytic metabolism, whereas the unique profile of RTE mitochondrial metabolism is “hard wired.”

Premature aging of both CD4+ regulatory T (Treg) and CD4+ responder‐T (Tresp) cells in patients with end‐stage renal disease (ESRD) is expected to affect the success of later kidney transplantation. Both T‐cell populations are released from the thymus as inducible T‐cell co‐stimulator‐positive (ICOS +) and ICOS − recent thymic emigrant (RTE) Treg/Tresp cells, which differ primarily in their proliferative capacities. In this study, we analysed the effect of ESRD and subsequent renal replacement therapies on the differentiation of ICOS + and ICOS − RTE Treg/Tresp cells into ICOS + CD31− or ICOS − CD31− memory Treg/Tresp cells and examined whether diverging pathways affected the suppressive activity of ICOS + and ICOS − Treg cells in co‐culture with autologous Tresp cells. Compared with healthy controls, we found an increased differentiation of ICOS + RTE Treg/Tresp cells and ICOS − RTE Treg cells through CD31+ memory Treg/Tresp cells into CD31− memory Treg/Tresp cells in ESRD and dialysis patients. In contrast, ICOS − RTE Tresp cells showed an increased differentiation via ICOS − mature naive (MN) Tresp cells into CD31− memory Tresp cells. Thereby, the ratio of ICOS + Treg/ICOS + Tresp cells was not changed, whereas that of ICOS − Treg/ICOS − Tresp cells was significantly increased. This differentiation preserved the suppressive activity of both Treg populations in ESRD and partly in dialysis patients. After transplantation, the increased differentiation of ICOS + and ICOS − RTE Tresp cells proceeded, whereas that of ICOS + RTE Treg cells ceased and that of ICOS − RTE Treg cells switched to an increased differentiation via ICOS − MN Treg cells. Consequently, the ratios of ICOS + Treg/ICOS + Tresp cells and of ICOS − Treg/ICOS − Tresp cells decreased significantly, reducing the suppressive activity of Treg cells markedly. Our data reveal that an increased tolerance‐inducing differentiation of ICOS + and ICOS − Treg cells preserves the functional activity of Treg cells in ESRD patients, but this cannot be maintained during long‐term renal replacement therapy.

Recent thymic emigrants (RTEs) are those peripheral T cells that have most recently completed thymic development and egress. Over the past decade, significant advances have been made in understanding the cell-extrinsic and cell-intrinsic requirements for RTE maturation to mature naïve (MN) T cells and in detailing the functional differences that characterize these two T cell populations. Much of this work has suggested that RTEs are hypo-functional versions of more mature T cells. However, recent evidence has indicated that rather than being defective T cells, RTEs are exquisitely adapted to their cellular niche. In this review, we argue that RTEs are not flawed mature T cells but are adapted to fill an underpopulated T cell compartment, while maintaining self tolerance and possessing the capacity to mount robust immune responses.

Mature naive T cells circulate through the secondary lymphoid organs in an actively enforced quiescent state. Impaired cell survival and cell functions could be found when T cells have defects in quiescence. One of the key features of T cell quiescence is low basal metabolic activity. It remains unclear at which developmental stage T cells acquire this metabolic quiescence. We compared mitochondria among CD4 single-positive (SP) T cells in the thymus, CD4+ recent thymic emigrants (RTEs), and mature naive T cells in the periphery. The results demonstrate that RTEs and naive T cells had reduced mitochondrial content and mitochondrial reactive oxygen species when compared with SP thymocytes. This downregulation of mitochondria requires T cell egress from the thymus and occurs early after young T cells enter the circulation. Autophagic clearance of mitochondria, but not mitochondria biogenesis or fission/fusion, contributes to mitochondrial downregulation in RTEs. The enhanced apoptosis signal-regulating kinase 1/MAPKs and reduced mechanistic target of rapamycin activities in RTEs relative to SP thymocytes may be involved in this mitochondrial reduction. These results indicate that the gain of metabolic quiescence is one of the important maturation processes during SP–RTE transition. Together with functional maturation, it promotes the survival and full responsiveness to activating stimuli in young T cells.

To evaluate the immunological mechanisms associated with clinical outcomes after autologous hematopoietic stem cell transplantation (AHSCT), focusing on regulatory T- (Treg) and B- (Breg) cell immune reconstitution, 31 systemic sclerosis (SSc) patients underwent simultaneous clinical and immunological evaluations over 36-month posttransplantation follow-up. Patients were retrospectively grouped into responders (n = 25) and nonresponders (n = 6), according to clinical response after AHSCT. Thymic function and B-cell neogenesis were respectively assessed by quantification of DNA excision circles generated during T- and B-cell receptor rearrangements. At the 1-year post-AHSCT evaluation of the total set of transplanted SSc patients, thymic rebound led to renewal of the immune system, with higher T-cell receptor (TCR) diversity, positive correlation between recent thymic emigrant and Treg counts, and higher expression of CTLA-4 and GITR on Tregs, when compared with pretransplant levels. In parallel, increased bone marrow output of newly generated naive B-cells, starting at 6 months after AHSCT, renovated the B-cell populations in peripheral blood. At 6 and 12 months after AHSCT, Bregs increased and produced higher interleukin-10 levels than before transplant. When the nonresponder patients were evaluated separately, Treg and Breg counts did not increase after AHSCT, and high TCR repertoire overlap between pre- and posttransplant periods indicated maintenance of underlying disease mechanisms. These data suggest that clinical improvement of SSc patients is related to increased counts of newly generated Tregs and Bregs after AHSCT as a result of coordinated thymic and bone marrow rebound.