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Despite long-lasting HIV replication control, a significant proportion of elite controller (EC) patients may experience CD4 T-cell loss. Discovering perturbations in immunological parameters could help our understanding of the mechanisms that may be operating in those patients experiencing loss of immunological control. Methods A case–control study was performed to evaluate if alterations in different T-cell homeostatic parameters can predict CD4 T-cell loss in ECs by comparing data from EC patients showing significant CD4 decline (cases) and EC patients showing stable CD4 counts (controls). The partial least-squares–class modeling (PLS-CM) statistical methodology was employed to discriminate between the two groups of patients, and as a predictive model. Results Herein, we show that among T-cell homeostatic alterations, lower levels of naïve and recent thymic emigrant subsets of CD8 cells and higher levels of effector and senescent subsets of CD8 cells as well as higher levels of exhaustion of CD4 cells, measured prior to CD4 T-cell loss, predict the loss of immunological control. Conclusions These data indicate that the parameters of T-cell homeostasis may identify those EC patients with a higher proclivity to CD4 T-cell loss. Our results may open new avenues for understanding the mechanisms underlying immunological progression despite HIV replication control, and eventually, for finding a functional cure through immune-based clinical trials., projects RD12/0017/0031, RD16/0025/ 0013, and SAF2015-66193-R as part of the Health Research and Development Strategy, State Plan for Scientific and Technical Research and Innovation (2008– 2011 and 2013–2016) and cofinanced by the Institute of Health Carlos III (ISCIII), Sub-Directorate General for Research Assessment and Promotion and European Regional Development Fund. NR is a Miguel Servet investigator from the ISCIII (CP14/00198), Madrid, Spain. C Restrepo was funded by project RD12/0017/ 0031 and is currently funded by project RD16/0025/0013. M García is a predoctoral student co-funded by grant CP14/00198 and an Intramural Research Scholarship from Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD).

An accumulating body of evidence suggests that development of autoimmune pathologies leads to thymic dysfunction and changes in peripheral T-cell compartment, which, in turn, perpetuate their pathogenesis. To test this hypothesis, thymocyte differentiation/maturation in rats susceptible (Dark Agouti, DA) and relatively resistant (Albino Oxford, AO) to experimental autoimmune encephalomyelitis (EAE) induction was examined. Irrespective of strain, immunization for EAE (i) increased the circulating levels of IL-6, a cytokine causally linked with thymic atrophy, and (ii) led to thymic atrophy reflecting partly enhanced thymocyte apoptosis associated with downregulated thymic IL-7 expression. Additionally, immunization diminished the expression of Thy-1, a negative regulator of TCR alpha beta-mediated signaling and activation thresholds, on CD4+CD8+ TCR alpha beta(lo/hi) thymocytes undergoing selection and thereby impaired thymocyte selection/survival. This diminished the generation of mature CD4+ and CD8+ single positive TCR alpha beta(hi) thymocytes and, consequently, CD4+ and CD8+ recent thymic emigrants. In immunized rats, thymic differentiation of natural regulatory CD4+Foxp3+CD25+ T cells (nTregs) was particularly affected reflecting a diminished expression of IL-7, IL-2 and IL-15. The decline in the overall thymic T-cell output and nTreg generation was more pronounced in DA than AO rats. Additionally, differently from immunized AO rats, in DA ones the frequency of CD28- cells secreting cytolytic enzymes within peripheral blood CD4+ T lymphocytes increased, as a consequence of thymic atrophy-related replicative stress (mirrored in CD4+ cell memory pool expansion and p16(INK4a) accumulation). The higher circulating level of TNF-alpha in DA compared with AO rats could also contribute to this difference. Consistently, higher frequency of cytolytic CD4+ granzyme B+ cells (associated with greater tissue damage) was found in spinal cord of immunized DA rats compared with their AO counterparts. In conclusion, the study indicated that strain differences in immunization-induced changes in thymopoiesis and peripheral CD4+CD28- T-cell generation could contribute to rat strain-specific clinical outcomes of immunization for EAE.

The peripheral naive T-cell pool is generally thought to consist of a subpopulation of recent thymic emigrants (RTEs) and a subpopulation of mature naive (MN) T cells with different dynamics. Thymus transplantation and adoptive transfer studies in mice have provided contradicting results, with some studies suggesting that RTEs are relatively short-lived cells, while another study suggested that RTEs have a survival advantage. We here estimate the death rates of RTE and MN T cells by performing both thymus transplantations and deuterium labeling experiments in mice of at least 12 weeks old, an age at which the size of the T-cell pool has stabilized. For CD4(+) T cells, we found the total loss rate from the RTE compartment (by death and maturation) to be fourfold faster than that of MN T cells. We estimate the death rate of CD4(+) RTE to be 0.046 per day, which is threefold faster than the total loss rate from the MN T-cell compartment. For CD8(+) T cells, we found no evidence for kinetic differences between RTE and MN T cells. Thus, our data support the notion that in young adult mice, CD4(+) RTE are relatively short-lived cells within the naive CD4(+) T-cell pool.

The expression of the coinhibitor PD-1 on T cells is important for the establishment of immune homeostasis. We previously found that PD-1 is particularly critical for the control of self-tolerance during lymphopenia-induced proliferation of recent thymic emigrants (RTEs). Previous studies suggested that PD-1 modulates the generation of Treg cells, particularly peripherally induced Treg (pTreg) cells, and controls Th17 cells. However, these conclusions were derived indirectly from studies on the ligand PD-L1, and not PD-1 itself. Herein we directly tested whether T-cell PD-1 expression was needed for Treg cell generation and examined if a paucity of Treg cells or enhanced Th17 cells could explain the severe lymphopenia-potentiated autoimmunity caused by PD-1 KO RTEs. Employing the murine FoxP3(EGFP) reporter system to simultaneously monitor conversion of WT and PD-1 KO T cells to pTreg cells in the same animal, we found that PD-1 deficiency did not inhibit pTreg cell generation or lead to Th17-cell-mediated autoimmunity. Surprisingly, pTreg cell numbers were increased in PD-1 KO versus WT cell populations. Furthermore, we noted an increased conversion to pTreg cells by RTEs. Our data suggest that the primary role for PD-1 is to restrain T-cell activation/proliferation to self-Ags rather than promote generation of Treg cells.

To explore the TCR sensitivity of recent thymic emigrants (RTEs), we triggered T cells with altered peptide ligands (APLs). Upon peptide stimulation in vitro, RTEs exhibited increased TCR signal transduction, and following infection in vivo with APL-expressing bacteria, CD8 RTEs expanded to a greater extent in response to low-affinity Ags than did their mature T cell counterparts. RTEs skewed to short-lived effector cells in response to all APLs but also were characterized by diminished cytokine production. RTEs responding to infection expressed increased levels of VLA-4, with consequent improved entry into inflamed tissue and pathogen clearance. These positive outcomes were offset by the capacity of RTEs to elicit autoimmunity. Overall, salient features of CD8 RTE biology should inform strategies to improve neonatal vaccination and therapies for cancer and HIV, because RTEs make up a large proportion of the T cells in lymphodepleted environments.

Alterations of the frequencies of recent thymic emigrants (RTEs) in blood have been demonstrated to be associated with human immunodeficiency virus-1 (HIV-1) disease progression and immune reconstitution following antiretroviral therapy. Flow cytometry is a technology that offers many advantages over T-cell receptor excision circles (TRECs) quantification to determine the RTE subset in blood. However, the gating strategies for identifying RTEs by flow cytometry have not been evaluated and compared in much detail. In the present study, we compared the frequencies and the senescence levels of RTEs in 54 HIV-1 seronegative controls (HC) and 70 HIV-1 seropositive (HIV-1+) subjects using two different gating strategies by flow cytometry. Our analysis indicated that the CD4+ RTE population determined by the expressing pattern CD45RO-CD31+ contained the terminal effector memory T-cell population after HIV-1 infection, which could significantly affect the further phenotypic and functional studies. Our data demonstrate the necessity of including an additional marker such as CCR7 to distinguish better CD4+RTE subset in HIV-1+ subjects.

αβ T-cell development and selection proceed while thymocytes successively migrate through distinct regions of the thymus. For γδ T cells, the interplay of intrathymic migration and cell differentiation is less well understood. Here, we crossed C-C chemokine receptor (CCR)7-deficient (Ccr7(-/-) ) and CCR9-deficient mice (Ccr9(-/-) ) to mice with a TcrdH2BeGFP reporter background to investigate the impact of thymic localization on γδ T-cell development. γδ T-cell frequencies and numbers were decreased in CCR7-deficient and increased in CCR9-deficient mice. Transfer of CCR7- or CCR9-deficient BM into irradiated C57BL/6 WT recipients reproduced these phenotypes, pointing toward cell-intrinsic migration defects. Monitoring recent thymic emigrants by intrathymic labeling allowed us to identify decreased thymic γδ T-cell output in CCR7-deficient mice. In vitro, CCR7-deficient precursors showed normal γδ T-cell development. Immunohistology revealed that CCR7 and CCR9 expression was important for γδ T-cell localization within thymic medulla or cortex, respectively. However, γδ T-cell motility was unaltered in CCR7- or CCR9-deficient thymi. Together, our results suggest that proper intrathymic localization is important for normal γδ T-cell development.

The goal of this study was to understand how immune reconstitution through ART in HIV-1 infected patients affects CD4(+) recent thymic emigrants (identified as CD31(+) naïve T cells). We performed FACS analysis of CD4(+) CD31(+) naïve T cells from PBMCs in a cross-sectional age-matched cohort, including 25 healthy controls (HC), 18 untreated HIV-1 infected viremic progressors (VP), 10 untreated HIV-1 infected viral controllers (VC), and 24 HIV-1 infected immune responders (IR) following ART. Our data reveal that 37.5% of IR failed to restore their CD4(+) CD31(+) naïve T cell counts. In addition, significantly higher expressions of Ki67, CD57, and CD95 were observed in CD4(+) CD31(+) naïve T cells of both VP and IR comparing to HC and VC. The significantly elevated CD57 and CD95 expressions are observed in IR with low CD4(+) CD31(+) naïve T cell counts. Therefore, our data indicate an incomplete immune reconstitution of CD4(+) CD31(+) naïve T cells in more than one third of IR, which is associated with HIV-1 driven immunological phenotypic alterations.

The changes that occur in the immune system with aging are commonly termed immunosenescence. Immunosenescence affects almost all components and functions of the immune response. The most commonly described change is a decrease in numbers and proportions of naïve T cells combined with the increase of terminally differentiated T lymphocytes, mainly affecting CD8+ T cells. The changes in the naïve T cell compartment are principally attributed to thymic involution and lifelong chronic antigen stimulation, among other triggers. Several strategies such as hormonal products, thymic peptides, or cytokines have been proposed for the restoration of the immune system. Here we show the effects of Biomodulina T (BT) on several populations of the immune system when administered to elderly patients diagnosed with recurrent respiratory infections. BT is a polypeptide fraction of bovine thymus, a Cuban product that obtained sanitary registration in 1994 for its immunomodulatory effects. We found that CD4+ naïve T, CD8+ stem cell-like memory (SCM) T, CD4+ recent thymic emigrants (RTE) T and CD4+ CD31+ naïve T cells increased with the administration of BT, whereas CD4+ and CD8+ T cells expressing PD1 decreased after the treatment with BT. Additionally, the proliferative capacity of CD4+ T cells measured by Ki67 expression, and the CD4+ T cell ability to produce IFN-γ were also improved by BT. Moreover, BT did not increase CD4+ Tregs. Altogether, these findings suggest that BT administration is a promising strategy for immune restoration in elderly patients and improvement of immunotherapeutic potential in cancer patients.

Delayed T cell recovery after allogeneic hematopoietic stem cell transplant (allo-HSCT) is an important contributor to transplant-related morbidity and mortality due to infection and malignant relapse. Optimal immune reconstitution requires a functional thymus, however, at the present time strategies to improve thymic reconstitution are limited. Zinc (Zn) is the second most abundant trace metal in the body, binding to more than 300 proteins. Zn deficiency is a clinical condition causing immunosuppression and thymic atrophy with a consequent reduction in the number of circulating recent thymic emigrants (RTEs). Furthermore, the role of Zn in tissue regeneration after damage has been clearly demonstrated in liver, skin, and intestinal diseases. In a pilot clinical trial, we previously demonstrated that patients receiving oral Zn supplementation after autologous HSCT showed increased thymic-dependent T cell reconstitution in the absence of adverse clinical events. Although a clear clinical benefit was observed, the mechanisms underlying this process are poorly understood. Thus, we used a murine model to evaluate the effect of Zn supplementation in thymic reconstitution after acute damage. Using a model of thymic damage caused by sub-lethal total body irradiation (SL-TBI, 550 cGy), we found that mice that received Zn supplementation demonstrated increased thymic cellularity when compared to untreated age-matched mice (Fig. 1a). Importantly, this finding was also confirmed in a clinically-applicable model of MHC-matched allogeneic HSCT (Fig. 1b). We have previously demonstrated endothelial cells (EC), which are extremely resistant to damaged, are able to trigger thymic endogenous reconstitution after damage by producing regenerative factors such as BMP4, which targets thymic epithelial cells (TECs), a key population crucial for T cell development. Interestingly, in our model of Zn administration, we found an increase in the number of regeneration-initiating ECs (Fig. 1c), and increased proliferation of TECs (Fig. 1d), which can occur in response to BMP4. Consistent with the hypothesis that Zn supplementation is activating the BMP4 pathway, thymic levels of BMP4 were increased at day +10 in zinc-treated mice (Fig. 2a, 2b), and, when stimulated in vitro for 24 hours with Zn sulfate, ex vivo propagated ECs (exECs) were directly induced to produce BMP4 (Fig. 2c), suggesting a likely mechanism by which Zn supplementation promotes thymic reconstitution. EC also express on their surface the Zn receptor GPR39 (Fig 3a), which is able to translate extracellular Zn signals in intracellular changes, and upregulate GPR39 expression after damage (Fig 3b). In conclusion, we demonstrate a mechanism by which zinc supplementation can improve thymic function and offers an innovative therapeutic strategy to improve T cell reconstitution in patients receiving allo-HSCT.

Objective: Disease progression and response to antiretroviral therapy (ART) in HIV-infected children is different to that of adults. Immune reconstitution in adults is mainly from memory T cells, whereas in children it occurs predominantly from the naive T-cell pool. It is unclear however what proportion of reconstituted CD4\+ T cells comes from thymic export and homeostatic proliferation in the periphery. Thymic output is often estimated by measuring T-cell receptor excision circles and markers such as CD31 expressed on recent thymic emigrants but these are confounded by peripheral T-cell division and cannot in themselves be used as quantitative estimates of thymic output.Design: To compare thymic output in HIV-infected children on ART, HIV-infected children not on ART and uninfected children of different ages.Method: Combined T-cell receptor excision circle (TREC) and proliferation data are used with a recently described mathematical model to give explicit measures of thymic output. Results: We found that age-adjusted thymic output is reduced in untreated children with HIV, which increases significantly with length of time on ART. Conclusion: Our results suggest that a highly active thymus in early childhood may contribute to better immune reconstitution if ART is initiated early in life. © 2014 Wolters Kluwer Health.

Objective: Considering a causal role of androgens in thymic involution, age-related remodeling of peripheral T-cell compartments in the absence of testicular hormones was evaluated. Methods: Rats were orchidectomized (ORX) at the age of 1 month, and T-peripheral blood lymphocytes (PBLs) and splenocytes from young (75-day-old) and aged (24-month-old) rats were examined for differentiation/activation and immunoregulatory marker expression. Results: In ORX rats, following the initial rise, the counts of CD4+ and CD8+ PBLs diminished with aging. This reflected the decline in thymic export as shown by recent thymic emigrant (RTE) enumeration. Orchidectomy increased the count of both of the major T-splenocyte subsets in young rats, and they (differently from controls) remained stable with aging. The CD4+:CD8+ T-splenocyte ratio in ORX rats shifted towards CD4+ cells compared to age-matched controls. Although in the major T-cell subsets in the blood and spleen from aged ORX rats the numbers of RTEs were comparable to the corresponding values in age-matched controls, the numbers of mature naïve and memory/activated cells substantially differed. Compared with age-matched controls, in aged ORX rats the numbers of CD4+ mature naïve PBLs and splenocytes were reduced, whereas those of CD4+ memory/activated cells (predictive of early mortality) were increased. Additionally, in spleens from aged ORX rats, despite unaltered thymic export, CD4+CD25+FoxP3+ and natural killer T cell counts were greater than in age-matched controls. Conclusion: (i) Age-related decline in thymopoietic efficacy is not dependent on androgen presence, and (ii) androgens are involved in the maintenance of peripheral T-cell (particularly CD4+ cell) homeostasis during aging.

The study of T cell biology has been accelerated by substantial progress at the technological level, particularly through the continuing advancement of flow cytometry. The conventional approach of observing T cells as either T helper or T cytotoxic is overly simplistic and does not allow investigators to clearly identify immune mechanisms or alterations in physiological processes that impact on clinical outcomes. The complexity of T cell sub-populations, as we understand them today, combined with the immunological and functional diversity of these subsets represent significant complications for the study of T cell biology. In this article, we review the use of classical markers in delineating T cell sub-populations, from "truly naive" T cells (recent thymic emigrants with no proliferative history) to "exhausted senescent" T cells (poorly proliferative cells that display severe functional abnormalities) wherein the different phenotypes of these populations reflect their disparate functionalities. In addition, since persistent infections and chronological aging have been shown to be associated with significant alterations in human T cell distribution and function, we also discuss age-associated and cytomegalovirus-driven alterations in the expression of key subset markers.

The generation of T cell receptor excision circle (TREC)–positive recent thymic emigrants (RTEs) in humans declines progressively with increasing age. Homeostatic proliferation is possibly an extrathymic mechanism for the generation of new T cells, and lymphopenia and common γ-chain cytokines appear to be the main driving force (1). However, thymic generation of TREC-positive RTEs can be restimulated throughout adult life if an increased supply of T cells is required under conditions of lymphopenia. Rheumatoid arthritis (RA) is associated with phenotypic alterations of T helper lymphocytes reminiscent of premature immunosenescence (2). In addition, RA is characterized by an age-inappropriate decrease in the number of CD4+ naive T cells and TREC-positive T cells (3), indicating decreased thymic output, diluting effects due to increased homeostatic maintenance proliferation, or both. Accelerated homeostatic proliferation of CD4+ T cells has also been observed in individuals who were thymectomized in early childhood, resulting in premature aging of T cells (4). In theory, thymic output in RA could be insufficient due to a shortage of thymus-seeding precursor cells. In the human system, those precursors were initially characterized in bone marrow as lineage-negative (Lin−) CD34+CD10+ common lymphoid progenitors (CLPs) (5), and their phenotype was subsequently refined to Lin−CD34highCD45RA+CD10+ (6). Six et al showed that CD34+CD10+CD24− progenitor cells are capable of migrating from the bone marrow and seeding the thymus (7). CLPs have recently been shown to have robust T cell potential regardless of CD7 expression, which appears to be a less important marker (8). Therefore, we decided to use CD10 expression as a marker defining the lymphoid commitment of human cells, in order to analyze the frequency of the best-characterized lymphoid-restricted progeny of hematopoietic stem cells (HSCs) (i.e., Lin−CD34+CD10+ CD24− CLPs) in the peripheral blood of patients with RA and healthy control subjects. In order to simultaneously determine thymic output, we measured the frequency of CD4+CD31+CD45RA+ T cells, which represents a well-established surrogate marker for TREC-positive RTEs (9). The results of the current study show a strong correlation between the frequencies of CLPs and RTEs in healthy control subjects. Compared with control subjects, patients with RA had a deficiency of RTEs despite a significantly increased number of thymic progenitors. Therapy with the tumor necrosis factor (TNF) inhibitor etanercept increased the frequency of thymic progenitors even further and almost normalized the deficient thymic output.

Objective: To assess pediatric patients with multiple sclerosis (MS) for early signs of homeostatic and functional abnormalities in conventional (T con ) and regulatory T cells (T reg ). Methods: We studied the composition of the peripheral T-cell compartment and T reg function in a cross-sectional study with 30 pediatric MS (pMS) patients by multicolor flow cytometry and proliferation assays. Data were compared to those obtained from adult patients (n = 26) and age-matched control donors (n = 67). Results: Proportions of naive T cells were 10%–20% higher in children than in adults, reflecting the age-related decline. pMS patients, however, had clearly lower numbers of naive T cells, among them recent thymic emigrants (RTE), whereas percentages of memory T cells were increased. In the T reg compartment, reduced RTE numbers coincided with markedly dampened suppressive capacities of total T reg . These homeostatic changes in circulating T cells precisely paralleled the pattern seen in adult MS. As in adults, treatment with immunomodulatory drugs attenuated these alterations. Conclusion: The homeostatic changes detected in the T-cell compartment in pMS are similar to those in adult-onset disease. With ratios between naive and memory T-cell subsets matching those of 20- to 30-years-older controls, signs of early thymic involution are already found in pMS, suggesting that an intrinsic compromise in thymic-dependent T-cell neogenesis might contribute to MS pathogenesis.

Recent thymic emigrants (RTEs) are the youngest T cells in the lymphoid periphery and exhibit phenotypic and functional characteristics distinct from those of their more mature counterparts in the naive peripheral T cell pool. We show in this study that the Il2 and Il4 promoter regions of naive CD4+ RTEs are characterized by site-specific hypermethylation compared with those of both mature naive (MN) T cells and the thymocyte precursors of RTEs. Thus, RTEs do not merely occupy a midpoint between the thymus and the mature T cell pool, but represent a distinct transitional T cell population. Furthermore, RTEs and MN T cells exhibit distinct CpG DNA methylation patterns both before and after activation. Compared with MN T cells, RTEs express higher levels of several enzymes that modify DNA methylation, and inhibiting methylation during culture allows RTEs to reach MN T cell levels of cytokine production. Collectively, these data suggest that the functional differences that distinguish RTEs from MN T cells are influenced by epigenetic mechanisms and provide clues to a mechanistic basis for postthymic maturation.

The immune reconstitution after allogeneic hematopoietic stem cell transplantation comprises thymus-dependent and thymus-independent pathways. We wanted to improve the understanding of this complex process using two different measurements at definite checkpoints of T-cell neogenesis. We therefore assessed the thymus-dependent pathway by combining measurements of single joint T-cell receptor excision circles (sjTREC) and β T-cell receptor excision circles (βTREC) in an improved quantitative light-cycler hybridization polymerase chain reaction assay. In a subgroup of patients, we additionally assessed the proliferation kinetics of the CD31(+) thymic naïve cell population, which corresponds to recent thymic emigrants by six-color immunostaining. After the establishment of normal values in 22 healthy volunteers, we applied our polymerase chain reaction to 66 patients undergoing allogeneic hematopoietic stem cell transplantation at a median age of 44 years. It took more than 2 years after transplant to restore the pre-transplant thymic proliferation capacity. Only one third of the patients in our longitudinal study reached age-adjusted normal values for both sjTREC and βTREC at a median follow-up of 558 days, with acute graft-versus-host disease being the most prominent negative factor by univariate analysis. We observed several patterns of sjTREC and βTREC recovery suggesting different mechanisms of thymic damage in individual patients. In a comparison of CD31(+) thymic naïve cells between volunteers and patients after transplant we found a significantly higher peak proliferation rate within the latter population in the first year after transplantation. The combination of measurements of sjTREC and βTREC by our simplified polymerase chain reaction assay provides insight about the stage of T-cell development affected by different types of damage and may help to choose the correct therapeutic intervention. Besides the sole thymic T-cell neogenesis, proliferation within the CD31(+) thymic naïve cell compartment contributed to the replenishment of the naïve T-cell pool after transplantation.

© 2013 National Academy of Sciences., Most Forkhead box P3(+) (Foxp3(+)) CD4 regulatory T cell (Treg) precursors are newly formed thymocytes that acquire Foxp3 expression on antigen encounter in the thymus. Differentiation of Treg, however, can also occur in the periphery. What limits this second layer of self- and nonself-reactive Treg production in physiological conditions remains to be understood. In this work, we tested the hypothesis that, similarly to thymic Treg, the precursors of peripheral Treg are immature T cells. We show that CD4(+)CD8(-)Foxp3(-) thymocytes and recent thymic emigrants (RTEs), contrarily to peripheral naïve mature cells, efficiently differentiate into Treg on transfer into lymphopenic mice. By varying donor and recipient mice and conducting ex vivo assays, we document that the preferential conversion of newly formed T cells does not require intrathymic preactivation, is cell-intrinsic, and correlates with low and high sensitivity to natural inhibitors and inducers of Foxp3 expression, such as IL-6, T-cell receptor triggering, and TGF-β. Finally, ex vivo analysis of human thymocytes and peripheral blood T cells revealed that human RTE and newly developed T cells share an increased potential to acquire a FOXP3(bright)CD25(high) Treg phenotype. Our findings indicating that RTEs are the precursors of Tregs differentiated in the periphery should guide the design of Treg-based therapies., This work was funded by the Portuguese Research Council (FCT)under Grant PTDC/SAU-IMU/113541/2009 (to I.C.), fellowships (to R.S.P., A.C.L., and M.-L.B.), and a contract (to I.C.) and the European Union’s 7th Framework Programme (FP7/2007-2013) under Grant 241447 [Natural Immunomodulators as Novel Immunotherapies for Type 1 Diabetes (NAIMIT); to J.D.].

The generation of the TCRαβ lineage of T cells occurs in the thymus through a series of orchestrated developmental events that result in a carefully selected population of CD4 or CD8 lineage–committed TCR+ thymocytes capable of recognizing foreign antigen in the context of self MHC. T cells first exit the thymus in a phenotypically and functionally immature state and require an approximately 3-week period of post-thymic maturation before transitioning into the mature T cell compartment. A greater understanding of recent thymic emigrant biology has come with the development of methods to exclusively identify and isolate this population for further characterization. I now review current knowledge about the phenotype and function of this key but understudied population of peripheral T cells.

Objective To define changes in phenotype and functional responses of reconstituting T cells in patients with aggressive multiple sclerosis (MS) treated with ablative chemotherapy and autologous hematopoietic stem cell transplantation (HSCT). Methods Clinical and brain magnetic resonance imaging measures of disease activity were monitored serially in patients participating in the Canadian MS HSCT Study. Reconstitution kinetics of immune-cell subsets were determined by flow cytometry, whereas thymic function was assessed using T-cell receptor excision circle analyses as well as flow cytometry measurements of CD31+ recent thymic emigrants (RTEs). Functional assays were performed to track central nervous system–autoreactive antigen-specific T-cell responses, and the relative capacity to generate Th1, Th17, or Th1/17 T-cell responses. Results Complete abrogation of new clinical relapses and new focal inflammatory brain lesions throughout the 2 years of immune monitoring following treatment was associated with sustained decrease in naive T cells, in spite of restoration of both thymic function and release of RTEs during reconstitution. Re-emergence as well as in vivo expansion of autoreactive T cells to multiple myelin targets was evident in all patients studied. The reconstituted myelin-specific T cells exhibited the same Th1 and Th2 responses as preablation myelin-reactive T cells. In contrast, the post-therapy T-cell repertoire exhibited a significantly diminished capacity for Th17 responses. Interpretation Our results indicate that diminished Th17 and Th1/17 responses, rather than Th1 responses, are particularly relevant to the abrogation of new relapsing disease activity observed in this cohort of patients with aggressive MS following chemoablation and HSCT. ANN NEUROL 2013;73:341–354

Background: The Bacillus Calmette-Guérin vaccine (BCG) has been associated with beneficial nonspecific effects on infant health. We aimed to examine the effect of BCG at birth on thymic size and the associations between thymic output, circulating lymphocytes, risk of infection, and thymic size. Methods: In infants randomized to BCG or no BCG, thymic index (TI), and thymic/weight index (TWI) were measured by ultrasound at birth and at the age of 3 mo. T cell subpopulations including CD4+ T cells, CD8+ T cells, and recent thymic emigrants (RTEs) were assessed by flow cytometry. Infections up to age 3 mo were parent-reported. Results: BCG vaccination did not affect thymic size at age 3 mo, measured as TI. At birth, the number of lymphocytes, CD4+ T cells, CD8+ T cells, and RTEs were positively associated with TI and TWI. Furthermore, a reduced risk of infections up to age 3 mo was associated with a large thymic size at birth. Conclusion: We found no effect of BCG vaccination on thymic size. The positive association between thymic output, lymphocytes, reduced risk of infections, and TI/TWI suggests that assessment of TI/TWI by ultrasound may be a predictor of the immunological capacity in the newborn.

T lymphopoiesis in the thymus was thought to be completed once it reaches the single positive (SP) stage, a stage when T cells are “fully mature” and waiting to be exported at random or follow a “first-in-first-out” manner. Recent evidence, however, has revealed that the newly generated SP thymocytes undergo a multistage maturation program in the thymic medulla. Such maturation is followed by a tightly regulated emigration process and a further postthymic maturation of recent thymic emigrants (RTEs). This review summarizes recent progress in the late stage T cell development. The regulation of this developmental process is discussed.

The factors involved in thymus regeneration after chemotherapy has not been sufficiently explored. This study was aimed to identify the clinical characteristics and single-nucleotide polymorphisms in the gene (IL7R) encoding IL-7Rα associated with thymus renewal after chemotherapy in Chinese Han individuals with lymphoma. The dynamics of thymic activity in 134 adults with Hodgkin lymphoma (HL) and B cell lymphoma from baseline to 12 months post-chemotherapy were analyzed by assessing thymic structural changes using serial computed tomography scans and correlating these with measurements of thymic output by concurrent analysis of single-joint T-cell receptor excision circles (sjTREC) and CD31+ recent thymic emigrants (RTE) in peripheral blood. The association of clinical variables and IL7R polymorphisms with the occurrence of rebound thymic hyperplasia (TH) and the recovery of thymic output following chemotherapy were evaluated. Thymic regeneration was observed, with the evidence that TH occurred in 38/134 (28.4%) cases, and thymic output, assessed by CD31+ RTE numbers and sjTREC content, recovered to baseline levels within 1 year after the end of therapy. The frequencies of the T allele and TT + GT genotype of rs7718919 located in the promoter of IL7R were significantly higher in patients with TH compared with those without TH (P = 0.031 and 0.027, respectively). In contrast, no significant difference was found between two groups with respect to the distribution of allele and genotype frequencies of rs6897932. By general linear models repeated-measure analysis, rs7718919 and rs6897932 were determined to exert no significant effects on the recovery of thymic output after therapy. Univariate analysis revealed host age under 30, the diagnosis of HL, baseline thymic index and CD31+ RTE counts, and rs7718919 genotype as potential predictors for TH after chemotherapy (P

Naïve T cells develop in the thymus and coordinate immune responses to new antigens; however, mechanisms for their long-term persistence over the human lifespan remain undefined. Here, we investigated human naïve T cell development and maintenance in primary and secondary lymphoid tissues obtained from individual organ donors aged 3 months-73 years. In the thymus, the frequency of double-positive thymocytes declined sharply in donors over age 40 coincident with reduced recent thymic emigrants (RTE) in lymphoid tissues, while naïve T cells were functionally maintained predominantly in lymph nodes (LN). Analysis of TCR clonal distribution by CDR3 sequencing of naïve CD4+ and CD8+ T cells in spleen and LNs reveal site-specific clonal expansions of naïve T cells from individuals >40 years of age with minimal clonal overlap between lymphoid tissues. We also identified biased naïve T cell clonal distribution within specific lymph nodes based on VJ usage. Together these results suggest prolonged maintenance of naïve T cells through in situ homeostasis and retention in lymphoid tissue.

Background X-linked agammaglobulinaemia (XLA) is a genetic disorder affecting B cell maturation, which is characterised by a low number of B cells, agammaglobulinaemia and increased susceptibility to a variety of bacterial infections. This study was performed to assess T cell subpopulations in a group of children with XLA in association with chronic respiratory disease (CRD). Methods Numbers of T cell subpopulations (CD3+, CD4+, CD8+, CD3+DR+, naive, memory, recent thymic emigrants (RTE), regulatory T cells, follicular T helpers) were measured by eight-colour flow cytometry in 22 XLA patients and 50 controls. BAFF level was measured by ELISA. Results XLA patients with CRD had a significantly lower percentage of RTE numbers and Tregs, while significantly higher absolute counts of lymphocytes, CD3+, CD8+, CD3+DR+ and CD4+CD45RO+ T cells were detected as compared with healthy controls. In patients with XLA without CRD, the number of follicular T helper cells was altered significantly (percentage and absolute), as compared with healthy controls. Additionally, they had significantly higher counts (percentage and absolute) of CD4+CD45RA+ cells and lower percentage of CD4+CD45RO+ cells in comparison with healthy controls. Conclusions Our study affords new information concerning CRD and T cell subsets that differentiate or are maintained in the absence of B cells in children with XLA. T cell's homeostasis depends on the presence of chronic respiratory disease that may be caused by the delay in diagnosis.

The study examined the influence of age, sex and peripubertal gonadectomy on a set of T-cell phenotypic parameters. Rats of both sexes were gonadectomised at the age of 1 month and peripheral blood and spleen T lymphocytes from non-gonadectomised and gonadectomised 3- and 11-month-old rats were examined for the expression of differentiation/activation (CD90/CD45RC) and immunoregulatory markers. Peripheral blood T lymphocytes from non-gonadectomised rats showed age-dependent sexual dimorphisms in (1) total count (lower in female than male 11-month-old rats); (2) CD4+:CD8 + cell ratio (higher in female than male rats of both ages); (3) the proportion of recent thymic emigrants in CD8 + T cells (lower in female than male 3-month-old rats) and (4) the proportions of mature na lt ve and memory/activated cells (irrespective of age, the proportion of na lt ve cells was higher, whereas that of memory/activated cells was lower in females). Gonadectomy influenced magnitudes or direction of these sex differences. Additionally, sex differences in peripheral blood T-lymphocyte parameters did not fully correspond to those observed in T-splenocyte parameters, suggesting the compartment-specific regulation of the major T-cell subpopulations' and their subsets' composition. Furthermore, there was no sexual dimorphism in the proportion of either CD25 + Foxp3 + cells among CD4 + or CD161+ (NKT) cells within CD8 + T lymphocytes. However, there was gonadal hormone-independent age-associated sexual dimorphism in the proportion of CD161 + cells (NKT cells) in CD8 + T splenocytes. Overall, the study revealed age-dependent variations in sexual dimorphisms in T-cell parameters relevant for immune response efficacy and showed that they are T-cell compartment-specific and partly gonadal hormone-related.

Background: Reduced thymic function causes poor immunological reconstitution in human immunodeficiency virus (HIV)-positive patients on combined antiretroviral therapy (cART).The association between immune activation and thymic function in asymptomatic HIV positive treatment-naive individuals has thus far not been investigated. Aims and objectives: To optimise a five-colour flow cytometric assay for measurement of thymic function by measuring recent thymic emigrants (RTEs) in treatment-naive HIV-infected patients and healthy controls and correlate results with levels of immune activation, CD4 counts and viral load. Methods: Blood obtained from 53 consenting HIV-positive individuals and 32 controls recruited from HIV prevention and testing clinic in Cape Town, South Africa. RTEs were measured (CD3 + /CD4 + /CD45RA + /CD31 + /CD62L + ) and levels were correlated with CD4 counts of HIV-infected individuals, log viral load and levels of immune activation (CD8 + /CD38 + T-cells). Results: HIV-infected individuals had reduced frequencies of RTEs when compared to controls ( p = 0.0035). Levels of immune activation were inversely correlated with thymic function( p = 0.0403), and the thymic function in HIV-infected individuals showed no significant correlation with CD4 counts ( p = 0.31559) and viral load ( p = 0.20628). Conclusions: There was impaired thymic function in HIV-infected individuals, which was associated with increased levels of immune activation. The thymic dysfunction was not associated with CD4 counts and viral load. Immune activation may result in inflammatory damage to the thymus and subsequent thymic dysfunction, and CD4 counts and viral load may not necessarily reflect thymic dysfunction in HIV.

The adaptive immune system utilizes multiple mechanisms linked to innate immune cell functions to respond appropriately to pathogens and commensals. Here we discover further aspects of this connectivity by demonstrating that naïve T cells as they emerge from the thymus (recent thymic emigrants, RTEs) express complement receptors (CR1 and CR2), the bacterial pathogen recognition receptor TLR1 and an enzyme that deactivates bacterial lipopolysaccharide (AOAH) and following activation secrete the anti-microbial cytokine IL-8. CR2+ naïve T cells also express a selection of genes associated with tissue migration, consistent with the hypothesis that following emigration from the thymus RTEs seed peripheral compartments where some pursue their anti-microbial potential by becoming IL-8-producing CR2+ memory cells while others undergo homeostatic expansion. CR2+ naïve and memory cells are abundant in children but decrease with age, coinciding with the involution of the thymus. The ability of CR2, which is also a receptor for Epstein-Barr Virus (EBV), to identify recent thymic emigrants will facilitate assessment of thymic function during aging and aid investigations of multiple clinical areas including the occurrence of T cell lymphomas caused by EBV.

Supranormal thymic output up to 2 decades after HIV-1 infection Christian R. Aguilera-Sandoval a , Otto O. Yang a,b , Nebojsa Jojic c , Pietro Lovato d , Diana Y. Chen a , Maria Ines Boechat a , Paige Cooper a , Jun Zuo a , Christina Ramirez e , Marvin Belzer f , Joseph A. Church f and Paul Krogstad a Objectives: AIDS is caused by CD4 þ T-cell depletion. Although combination anti- retroviral therapy can restore blood T-cell numbers, the clonal diversity of the recon- stituting cells, critical for immunocompetence, is not well defined. Methods: We performed an extensive analysis of parameters of thymic function in perinatally HIV-1-infected (n ¼ 39) and control (n ¼ 28) participants ranging from 13 to 23 years of age. CD4 þ T cells including naive (CD27 þ CD45RA þ ) and recent thymic emigrant (RTE) (CD31 þ /CD45RA þ ) cells, were quantified by flow cytometry. Deep sequencing was used to examine T-cell receptor (TCR) sequence diversity in sorted RTE CD4 þ T cells. Results: Infected participants had reduced CD4 þ T-cell levels with predominant depletion of the memory subset and preservation of naive cells. RTE CD4 þ T-cell levels were normal in most infected individuals, and enhanced thymopoiesis was indicated by higher proportions of CD4 þ T cells containing TCR recombination excision circles. Memory CD4 þ T-cell depletion was highly associated with CD8 þ T-cell activation in HIV-1-infected persons and plasma interlekin-7 levels were corre- lated with naive CD4 þ T cells, suggesting activation-driven loss and compensatory enhancement of thymopoiesis. Deep sequencing of CD4 þ T-cell receptor sequences in well compensated infected persons demonstrated supranormal diversity, providing additional evidence of enhanced thymic output. Conclusion: Despite up to two decades of infection, many individuals have remarkable thymic reserve to compensate for ongoing CD4 þ T-cell loss, although there is ongoing viral replication and immune activation despite combination antiretroviral therapy. The longer term sustainability of this physiology remains to be determined. Copyright s 2016 Wolters Kluwer Health, Inc. All rights reserved. AIDS 2016, 30:701–711 Keywords: adolescents, CD4 þ T cells, immune reconstitution, perinatal HIV infection, recent thymic emigrant, T-cell receptor, thymopoiesis, T-cell receptor recombination excision circles a David Geffen School of Medicine, University of California, b AIDS Healthcare Foundation, Los Angeles, California, USA, Microsoft Corporation, d Department of Computer Science, University of Verona, Verona, Italy, e Fielding School of Public Health, University of California, and f Children’s Hospital Los Angeles and the Keck School of Medicine at University of Southern California, Los Angeles, California, USA. Correspondence to Paul Krogstad, MD, UCLA AIDS Institute, David Geffen School of Medicine at UCLA, 615 Charles E. Young Drive South, BSRB 173, Los Angeles, CA, USA. Tel: +1 310 825 5235; fax: +1 310 206 4764; e-mail: pkrogstad@mednet.ucla.edu Received: 2 September 2015; revised: 24 November 2015; accepted: 16 December 2015. c DOI:10.1097/QAD.0000000000001010 ISSN 0269-9370 Copyright Q 2016 Wolters Kluwer Health, Inc. All rights reserved. Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.

Delayed immune recovery is a characteristic feature of allogeneic hematopoietic stem cell transplantation in adult recipients. Although recipient thymic T-cell neogenesis contributes to T-cell regeneration after transplantation, thymic recovery in the transplant recipient decreases with increasing age, and is diminished by intensive preconditioning regimens and graft-versus-host disease. In adult recipients, most events that determine transplant success or failure occur during the period when the majority of circulating T cells is derived from the donor's post thymic T-cell repertoire. As a result, the make-up of the donor lymphocyte compartment may strongly influence immune recovery and transplant outcomes. The aim of this study was to examine donor lymphocyte counts in a series of patients undergoing an allogeneic hematopoietic stem cell transplant to identify the potential contribution of donor regulatory and conventional T lymphocyte populations to immune recovery and transplant outcomes. We examined donor lymphocyte subset counts in relation to post-transplant lymphocyte recovery and transplant events in 220 consecutive myeloablative, T-cell-depleted, HLA-identical sibling hematopoietic stem cell transplant recipients with hematologic malignancies. In a multivariate analysis, absolute numbers of donor CD4(+) recent thymic emigrants were associated with overall survival (P=0.032). The donors' absolute lymphocyte count and thymic production of regulatory T cells were both associated with extensive chronic graft-versus-host disease (P=0.002 and P=0.022, respectively). In conclusion, these results identify donor immune characteristics that are associated with lymphocyte recovery, extensive chronic graft-versus-host disease, and survival in the recipient following allogeneic hematopoietic stem cell transplantation. The study reported here was performed using peripheral blood samples drawn from donors and patients enrolled in the ClinicalTrials.gov-registered trials NCT00001623, NCT00001873, NCT00353860, NCT00066300, NCT00079391, and NCT00398346.

The genetic modification of T cells with a suicide gene grants a mechanism of control of adverse reactions, allowing safe infusion after partially incompatible hematopoietic stem cell transplantation (HSCT). In the TK007 clinical trial, 22 adults with hematologic malignancies experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the HSV thymidine kinase suicide gene (TK+ cells). After a first wave of circulating TK+ cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naive lymphocytes, suggesting the thymus-dependent development of T cells, occurring only upon TK+-cell engraftment. Accordingly, after the infusions, we documented an increase in circulating TCR excision circles and CD31(+) recent thymic emigrants and a substantial expansion of the active thymic tissue as shown by chest tomography scans. Interestingly, a peak in the serum level of IL-7 was observed after each infusion of TK+ cells, anticipating the appearance of newly generated T cells. The results of the present study show that the infusion of genetically modified donor T cells after HSCT can drive the recovery of thymic activity in adults, leading to immune reconstitution. (Blood. 2012;120(9):1820-1830) The genetic modification of T cells with a suicide gene grants a mechanism of control of adverse reactions, allowing safe infusion after partially-incompatible Hematopoietic Stem Cell Transplantation (HSCT). In the TK007 clinical trial 22 adults with hematologic malignancies experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the Herpes Simplex Virus Thymidine Kinase suicide gene (TK(pos) cells). After a first wave of circulating TK(pos) cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naïve lymphocytes, suggesting the thymus-dependent development of T cells, occurring only upon TK(pos) cell engraftment. Accordingly, after the infusions we documented an increase in circulating T cell Receptor Excision Circles and CD31+ recent thymic emigrants, and a substantial expansion of the active thymic tissue at chest tomography scans. Interestingly, a peak in the serum level of interleukin-7 was observed after each infusion of TK(pos) cells, anticipating the appearance of newly generated T cells. Taken together, our data show that the infusion of genetically modified donor T cells after transplantation can drive the recovery of thymic activity in adults, leading to immune reconstitution.

Immunophenotyping of blood lymphocyte subpopulations is an important tool in the diagnosis of immunological and haematological diseases. Paediatric age-matched reference values have been determined for the major lymphocyte populations, but reliable reference values for the more recently described T lymphocyte subpopulations, like different types of memory T lymphocytes, recent thymic emigrants, regulatory T cells and CXCR5(+) helper T lymphocytes, are not sufficiently available yet. We determined reference values for the absolute and relative sizes of T lymphocyte subpopulations in healthy children using the lysed whole blood method, which is most often used in diagnostic procedures. When the absolute numbers of some or all T lymphocyte subpopulations fall outside these reference ranges, this may indicate disease. The reference values show the course of T lymphocyte development in healthy children. Absolute T lymphocyte numbers increase 1.4-fold during the first months of life, and after 9-15 months, they decrease threefold to adult values; this is mainly caused by the expansion of recent thymic emigrants and naive cells. Helper and cytotoxic T lymphocytes show the same pattern. Regulatory T cells increase in the first 5 months of life and then gradually decrease to adult values, although the absolute numbers remain small. The relative number of CXCR5(+) cells within the CD4(+) CD45RO(+) T lymphocytes increases during the first 6 months of life and then remains more or less stable around 20%.

T cell maturation was once thought to occur entirely within the thymus. Now, evidence is mounting that the youngest peripheral T cells in both mice and humans comprise a distinct population from their more mature, yet still naive, counterparts. These cells, termed recent thymic emigrants (RTEs), undergo a process of post-thymic maturation that can be monitored at the levels of cell phenotype and immune function. Understanding this final maturation step in the process of generating useful and safe T cells is of clinical relevance, given that RTEs are over-represented in neonates and in adults recovering from lymphopenia. Post-thymic maturation may function to ensure T cell fitness and self tolerance.

Progressive loss of renal function is associated with a dysregulation of circulating T cells that may underlie their impaired T-cell immunity. Here we tested whether end-stage renal disease (ESRD)-related T-cell alterations are compatible with the concept of premature immunological aging. Younger patients (25-45 years old) with ESRD were found to resemble older healthy controls (60-80 years old) as they had a significant loss of naive T cells and a relative increase of memory T cells showing progressive terminal differentiation. A significant decrease in the content of T-cell receptor excision circles and telomere length in patients with ESRD confirmed these phenotypic data. The loss of naive T cells in patients with ESRD was associated with an excessive age-related decrease of recent thymic emigrants, indicating a premature decline in thymic function. Additionally, increased homeostatic proliferation of naive T cells was found in patients with ESRD, similar to that of older healthy individuals, with an increased susceptibility for activation-induced apoptosis. Therefore, both decreased thymic output and increased susceptibility of naive T cells for apoptosis may play a role in the loss of naive T cells in ESRD patients. Thus, our results are compatible with premature aging of the T-cell system of patients with ESRD comparable with that of healthy individuals 20-30 years older. Kidney International (2011) 80, 208-217; doi:10.1038/ki.2011.110; published online 27 April 2011

The transcriptional repressor NKAP drives T cell maturation after positive selection in the thymus, with NKAP deficiency resulting in functionally immature peripheral T cells that maintain the phenotype of recent thymic emigrants., Newly generated T cells are unable to respond to antigen/MHC. Rather, post-selection single-positive thymocytes must undergo T cell maturation to gain functional competency and enter the long-lived naive peripheral T cell pool. This process is poorly understood, as no gene specifically required for T cell maturation has been identified. Here, we demonstrate that loss of the transcriptional repressor NKAP results in a complete block in T cell maturation. In CD4-cre NKAP conditional knockout mice, thymic development including positive selection occurs normally, but there is a cell-intrinsic defect in the peripheral T cell pool. All peripheral naive CD4-cre NKAP conditional knockout T cells were found to be functionally immature recent thymic emigrants. This defect is not simply in cell survival, as the T cell maturation defect was not rescued by a Bcl-2 transgene. Thus, NKAP is required for T cell maturation and the acquisition of functional competency.

Children with Down syndrome (DS) have low numbers of naive T cells and abnormal thymus development and function. Because next to thymic production, peripheral proliferation greatly contributes to naive T cell generation in healthy children, we examined the cause of reduced naive T cell numbers in children with DS. Compared with aged matched controls, the total number of signal joint TCR excision circles (sjTREC) per ml blood was reduced in DS. Reduced frequencies and absolute numbers of protein tyrosine kinase 7-positive recent thymic emigrants, but similar levels of naive T cell apoptosis and Ag-driven activation in DS, suggested that reduced thymic output and not increased peripheral loss of naive T cells caused the reduced sjTREC numbers. We found no support for defective peripheral generation of naive T cells in DS. In DS the naive T cells responded to IL-7 and, based on Ki-67 expression, had similar proliferation rates as in healthy controls. sjTREC content per naive CD8+ T cells was not increased, but even decreased, pointing to increased survival or peripheral generation of naive T cells in DS. In conclusion, we show in this study that reduced thymic output, but not reduced peripheral generation nor increased loss of naive T cells, results in the low naive T cell numbers found in DS.

Recent thymic emigrants (RTEs) are the youngest subset of peripheral T cells, and they differ functionally and phenotypically from the rest of the naïve T-cell pool. RTEs are present in the peripheral T-cell pool throughout life but are the most common subset of T cells in neonates and adults recovering from lymphoablation. Using a murine model to study the homeostasis of RTEs, we show that under lymphoreplete conditions, RTEs are at a competitive disadvantage to already established mature naïve (MN) T cells. This disadvantage may be caused by a defect in survival, because RTEs may transduce homeostatic signals inefficiently, and their ability to survive is enhanced with increased expression of IL-7 receptor or B-cell lymphoma 2 (Bcl-2). Conversely, under lymphopenic conditions, enhanced proliferation by RTEs allows them to out-compete their MN T-cell counterparts. These results suggest that in times of need, such as in neonates or lymphopenic adults, RTEs perform well to fill the gaps in the peripheral T-cell pool, but when the periphery already is full, many RTEs are not incorporated into the pool of recirculating lymphocytes.

Although autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, are frequently associated with premature aging of the thymus, a direct link is missing between autoimmunity and thymic atrophy. Here we monitored the progression of thymic involution in Aire-deficient mice, in which defective negative selection causes spontaneous and progressive development of autoimmunity. In young and middle-aged mice, Aire deficiency appeared to be protective as supported by the reduced β-gal+ epithelial cells and the enhanced thymic output. However, once the autoimmune phenotype was fully developed in aged Aire-deficient mice, their thymuses underwent accelerated involution. In comparison to the age-matched wildtype littermates, old Aire-deficient mice showed lower numbers of total thymocytes and recent thymic emigrants but more β-gal+ thymic epithelial cells. This phenomenon may partly be attributable to the increased number of activated Th1 cells homing to the thymus. This speculation was further supported by the enhanced thymic aging following repeated challenges with complete Freund’s adjuvant immunization. Taken together, the present study highlights a unique mechanism by which autoimmunity facilitates the senescence of thymic epithelial cells through returning Th1 cells.

After intrathymic development, T cells exit the thymus and join the peripheral T-cell pool. Such recent thymic emigrants (RTEs) undergo both phenotypic and functional maturation during the first 3 weeks they reside in the periphery. Using a well-controlled in vitro polarization scheme, we now show that CD4+ RTEs are defective in T-helper (Th) type 0 (Th0), Th1, Th17, and regulatory T-cell lineage commitment, with dampened cytokine production and transcription factor expression. In contrast, CD4+ RTES are biased toward the Th2 lineage both in vitro and in vivo, with more robust interleukin-4, interleukin-5, and interleukin-13 production than their mature naive counterparts. Coculture experiments demonstrate that mature naive T cells influence neighboring RTEs in their Th responses. In adoptive hosts, CD4+ RTEs drive production of the Th2-associated antibody isotype immunoglobulin G1 and mediate airway inflammatory disease. This bias in RTEs likely results from dampened negative regulation of the Th2 lineage by diminished levels of T-bet, a key Th1 transcription factor. CD4+ RTEs thus represent a transitional population with a distinct interpretation of, and response to, immunologic cues. These characteristics may be beneficial during the postthymic maturation period by leading to the avoidance of inappropriate immune responses, particularly in lymphopenic neonates and adults.

We have previously shown that regulatory T cells (Treg) accumulate dramatically in aged animals and negatively impact the ability to control persistent infection. However, the mechanisms underlying the age-dependent accrual of Treg remain unclear. In this study, we show that Treg accumulation with age is progressive and likely not the result of increased thymic output, increased peripheral proliferation, or from enhanced peripheral conversion. Instead, we found that Treg from aged mice are more resistant to apoptosis than Treg from young mice. Although Treg from aged mice had increased expression of functional IL-7Rα, we found that IL-7R signaling was not required for maintenance of Treg in vivo. Notably, aged Treg exhibit decreased expression of the proapoptotic molecule Bim compared with Treg from young mice. Furthermore, in the absence of Bim, Treg accumulate rapidly, accounting for >25% of the CD4+ T cell compartment by 6 mo of age. Additionally, accumulation of Treg in Bim-deficient mice occurred after the cells left the transitional recent thymic emigrant compartment. Mechanistically, we show that IL-2 drives preferential proliferation and accumulation of Bimlo Treg. Collectively, our data suggest that chronic stimulation by IL-2 leads to preferential expansion of Treg having low expression of Bim, which favors their survival and accumulation in aged hosts.

The pathogenetic mechanisms of organ-specific autoimmune diseases remain obscured by the complexity of the genetic and environmental factors participating in the breakdown of tolerance. A unique opportunity to study the pathogenesis of human autoimmunity is provided by autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a rare inherited autoimmune disease caused by mutations in Autoimmune Regulator (AIRE) gene. Loss of AIRE function disrupts the deletion of autoreactive T cells and impairs the suppressive function of regulatory T (Treg) cells. Here we show by multiparameter flow cytometry that in healthy controls the peripheral naive Treg cell subset forms a slowly dividing, persistent reservoir of recent thymic emigrants (RTEs). In APECED patients the RTE Treg cells show accelerated turnover and shift to the activated pool and the RTE reservoir is depleted. Moreover, the activated Treg cell population in the patients expresses significantly less Forkhead box protein P3 (FOXP3) than in the healthy controls, consistent with the impairment of peripheral activation. Our results indicate that in addition to their thymic effects, loss-of-function mutations in AIRE disrupt the peripheral homeostasis and activation of Treg cells. This may synergize with failed negative selection to cause APECED.

Mice lacking suppressor of cytokine signaling 1 (SOCS1) accumulate CD8(+) T lymphocytes in the thymus and in the periphery. Whereas IL-7 and IL-15 promote the generation of CD8 single positive (SP) thymocytes, IL-15 drives the expansion of CD8 T cells in the periphery. Here, we investigated whether increased production of CD8 SP thymocytes is accompanied by their increased export in SOCS1-deficient mice. In vivo labeling with bromodeoxyuridine showed increased cycling of CD8 SP thymocytes in SOCS1-deficient mice. However, SOCS1-deficient thymi contained increased proportion of CD24(lo)CD69(lo) SP thymocytes as well as increased expression of Qa-2 in both CD4 and CD8 SP compartments. Analysis of recent thymic emigrants (RTE) following intrathymic labeling with fluorescein isothiocyanate revealed less efficient export of CD8 RTEs from SOCS1-deficient thymi and comparable CD4:CD8 ratio among RTEs in SOCS1-null and control mice. These findings show that the rate of export of CD8 SP thymocytes is not proportional to their generation in SOCS1-deficient thymi and suggest the existence of homeostatic mechanisms controlling the egress of CD8 T cells.

Several studies have shown an increased accumulation of terminally differentiated T cells during HIV infection, suggestive of exhaustion/senescence, causing dysregulation of T cell homeostasis and function and rapid HIV disease progression. We have investigated whether long-term antiretroviral therapy (ART), which controls viremia and restores CD4 T cell counts, is correlated with reduction in terminally differentiated T cells, improved ratios of naïve to memory and function of T cells in 100 virologically controlled HIV-infected patients. We show that while the median frequencies of terminally differentiated CD4+ and CD8+ T cells (CD28-, CD27-, CD57+ and CD28-CD57+), were higher in the virologically controlled HIV-infected patients' cohort compared with uninfected individuals' cohort, the frequencies of these cells significantly decreased with increasing CD4 T cell counts in HIV-infected patients. Although, the naïve CD4+ and CD8+ T cells were lower in HIV patients' cohort than uninfected cohort, there was a significant increase in both naïve CD4+ and CD8+ T cells with increasing CD4 T cell counts in HIV-infected patients. The underlying mechanism behind this increased naïve CD4+ and CD8+ T cells in HIV-infected patients was due to an increase in recent thymic emigrants, CD4+CD31+, as compared to CD4+CD31-. The CD4+ T cells of HIV-infected patients produced cytokines, including IL-2, IL-10 and IFN-γ comparable to uninfected individuals. In conclusion, virologically controlled HIV-infected patients on long-term ART show a significant reduction in terminally differentiated T cells, suggestive of decreased exhaustion/senescence, and improvement in the ratios of naïve to memory and function of T cells.

Although human naturally occurring regulatory T cells (Tregs) may express either CD45RA or CD45RO, we find in agreement with previous reports that the (∼80%) majority of natural Tregs in adults are CD45RO+. The proportion of CD45RA+ Tregs decreases, whereas CD45RO+ Tregs increase significantly with age. Nevertheless, a small proportion of CD45RA+ Tregs are found even in old (>80 y) adults and a proportion of these express CD31, a marker for recent thymic emigrants. We found that CD45RO+ Tregs were highly proliferative compared with their CD45RA+ counterparts. This was due in part to the conversion of CD45RA Tregs to CD45RO expression after activation. Another difference between these two Treg populations was their preferential migration to different tissues in vivo. Whereas CD45RA+ Tregs were preferentially located in the bone marrow, associated with increased CXCR4 expression, CD45RO+ Tregs were preferentially located in the skin, and this was associated with their increased expression of CLA and CCR4. Our studies therefore show that proliferation features strongly in maintenance of the adult Treg pool in humans and that the thymus may make a minor contribution to the maintenance of the peripheral pool of these cells, even in older adults. Furthermore, the different tissue compartmentalization of these cells suggests that different Treg niches exist in vivo, which may have important roles for their maturation and function.

Summary It is widely accepted that aging is accompanied by remodelling of the immune system including thymic atrophy and increased frequency of senescent T cells, leading to immune compromise. However, physical activity, which influences immunity but declines dramatically with age, is not considered in this literature. We assessed immune profiles in 125 adults (55–79 years) who had maintained a high level of physical activity (cycling) for much of their adult lives, 75 age‐matched older adults and 55 young adults not involved in regular exercise. The frequency of naïve T cells and recent thymic emigrants (RTE) were both higher in cyclists compared with inactive elders, and RTE frequency in cyclists was no different to young adults. Compared with their less active counterparts, the cyclists had significantly higher serum levels of the thymoprotective cytokine IL‐7 and lower IL‐6, which promotes thymic atrophy. Cyclists also showed additional evidence of reduced immunesenescence, namely lower Th17 polarization and higher B regulatory cell frequency than inactive elders. Physical activity did not protect against all aspects of immunesenescence: CD28−ve CD57+ve senescent CD8 T‐cell frequency did not differ between cyclists and inactive elders. We conclude that many features of immunesenescence may be driven by reduced physical activity with age.

CD31+ is a marker for recent thymic emigrants. Nevertheless it is present in a proportion of memory cells. We looked at the distribution of CD31 on CD4 T-cell subpopulations. In cord blood, CD31 was present in the majority of the CD45RA high and 60% of the CD45RA low cells, and in adults over 70% of "true" naive and in 5-10% of all memory sub- populations (central memory, effector memory, follicular helper and T regulatory cells). No major differences were seen in the distribution of chemokine receptors between CD31+ and CD31- populations within the naive cells nor the memory populations except for CCR3 and CCR9, which were preferentially expressed in the CD31+ memory cells. The CD31 distribution and cytokine receptors was similar between HIV negative and positive individuals, and between adult blood and tonsils. There was a correlation between the levels of TRECs and the percentages of CD31 in all samples studied.

Summary To better understand the kinetics of the delayed reconstitution of peripheral CD4+ T-cells after depletion with a single administration of alemtuzumab (AL) for renal transplantation, we evaluated in these patients the percentage and absolute number of recent thymic emigrants (RTEs) CD4+ T cells, together with naive and memory subsets, defined by the analysis of CD31, CD45RA and CCR7 expression, and compared with patients treated with a nondepleting protocol based on basiliximab, and with healthy controls. In AL-treated patients, the number of circulating CD4+ T cells was greatly reduced 1 year after the infusion (P

The immune system undergoes a process of profound remodelling during aging, referred to as immunosenescence, and characterized by complex modifications of several components. In this review, we discuss recent developments and observations regarding the generation of T cells in the thymus during aging and longevity, and the regulation and maintenance of peripheral blood lymphocytes. The generation of new T cells is indeed crucial to maintain a functional immune system, and is a fundamental step to avoid unsuccessful aging, thus reaching longevity in good health. Mechanisms will be described that are related to the production and maintenance of those lymphocytes defined "recent thymic emigrants", and to the detection of the so called "T cell receptor rearrangement excision circles (TREC)", along with the presence in the periphery of naïve and memory T cells, that can be influenced and regulated by several different mechanisms. Several strategies aimed at improving thymic functionality are currently receiving a growing interest, and some of them are based on molecules that are produced by, and/or act on immune cells. Data on the possible use of these molecules, including cytokines like interleukin (IL)-7, IL-15 and keratinocyte growth factor, to restore thymic function are reviewed and discussed.