Frequency Of Recent Thymic Emigrants Research Articles

Type 1 Diabetes Brazilian patients exhibit reduced frequency of recent thymic emigrants in regulatory CD4+CD25+Foxp3+T cells

To control immune responses, regulatory CD4+CD25+Foxp3+ T cells (Treg) maintain their wide and diverse repertoire through continuous arrival of recent thymic emigrants (RTE). However, during puberty, the activity of RTE starts to decline as a natural process of thymic involution, introducing consequences, not completely described, to the repertoire. Type 1 diabetes (T1D) patients show quantitative and qualitative impairments on the Treg cells. Our aim was to evaluate peripheral Treg and RTE cell frequencies, in T1D patients from two distinct age groups (young and adults) and verify if HLA phenotypes are concomitant associated. To this, blood samples from Brazilian twenty established T1D patients (12 young and 8 adults) and twenty-one healthy controls (11 young and 10 adults) were analyzed, by flow cytometry, to verify the percentages of CD4, Treg (CD4+CD25+Foxp3+) and the subsets of CD45RA+ (naive) and CD31+(RTE) within then. Furthermore, the HLA typing was also set. We observed that the young established T1D patients feature decreased frequencies in total Treg cells and naive RTE within Treg cells. Significant prevalence of HLA alleles, associated with risk, in T1D patients, was also identified. Performing a multivariate analysis, we confirmed that the cellular changes described offers significant variables that distinct T1D patients from the controls. Our data collectively highlight relevant aspects about homeostasis imbalances in the Treg cells of T1D patients, especially in young, and disease prognosis; that might contribute for future therapeutic strategies involving Treg cells manipulation.

Thymic Atrophy and Immune Dysregulation in Infants with Complex Congenital Heart Disease

Congenital heart disease (CHD) is the most common birth defect, and up to 50% of infants with CHD require cardiovascular surgery early in life. Current clinical practice often involves thymus resection during cardiac surgery, detrimentally affecting T-cell immunity. However, epidemiological data indicate that CHD patients face an elevated risk for infections and immune-mediated diseases, independent of thymectomy. Hence, we examined whether the cardiac defect impacts thymus function in individuals with CHD. We investigated thymocyte development in 58 infants categorized by CHD complexity. To assess the relationship between CHD complexity and thymic function, we analyzed T-cell development, thymic output, and biomarkers linked to cardiac defects, stress, or inflammation. Patients with highly complex CHD exhibit thymic atrophy, resulting in low frequencies of recent thymic emigrants in peripheral blood, even prior to thymectomy. Elevated plasma cortisol levels were detected in all CHD patients, while high NT-proBNP and IL-6 levels were associated with thymic atrophy. Our findings reveal an association between complex CHD and thymic atrophy, resulting in reduced thymic output. Consequently, thymus preservation during cardiovascular surgery could significantly enhance immune function and the long-term health of CHD patients.

Thymic changes as a contributing factor in the increased susceptibility of old Albino Oxford rats to EAE development

The study was aimed to examine putative contribution of thymic involution to ageing-associated increase in susceptibility of Albino Oxford (AO) rats to the development of clinical EAE, and vice versa influence of the disease on the progression of thymic involution. To this end we examined (i) the parameters of thymocyte negative selection efficacy, the thymic generation of CD4+CD25+Foxp3+ T regulatory cells (Tregs) and thymic capacity to instruct/predetermine IL-17-producing T-cell differentiation, and thymopietic efficacy-associated accumulation of “inflammescent” cytotoxic CD28- T cells in the periphery, and (ii) the key underlying mechanisms in young and old non-immunised AO rats and their counterparts immunised for EAE (on the 16th day post-immunisation when the disease in old rats reached the plateau) using flow cytometry analysis and/or RT-qPCR. It was found that thymic involution impairs: (i) the efficacy of negative selection (by affecting thymocyte expression of CD90, negative regulator of selection threshold and the expression of thymic stromal cell integrity factors) and (ii) Treg generation (by diminishing expression of cytokines supporting their differentiation/maturation). Additionally, the results suggest that thymic involution facilitates CD8+ T-cell differentiation into IL-17-producing cells (previously linked to the development of clinical EAE in old AO rats). Furthermore, they confirmed that ageing-related decrease in thymic T-cell output (as indicated by diminished frequency of recent thymic emigrants in peripheral blood) resulted in the accumulation of CD28- T cells in peripheral blood and, upon immunisation, in the target organ. On the other hand, the development of EAE (most likely by increasing circulatory levels of proinflammatory cytokines) contributed to the decline in thymic output of T cells, including Tregs, and thereby to the progression/maintenance of clinical EAE. Thus, in AO rats thymic involution via multi-layered mechanisms may favour the development of clinically manifested autoimmunity, which, in turn, precipitates the thymus atrophy.

Distinct CD4+ T cell signature in ANA-positive young adult patients.

Failure of immune tolerance can lead to autoantibody production resulting in autoimmune diseases, a broad spectrum of organ-specific or systemic disorders. Immune tolerance mechanisms regulate autoreactive T and B cells, yet some lymphocytes escape and promote autoantibody production. CD4+ T cell dysregulation, characterized by decreased or impaired regulatory cells (Tregs) and/or accumulation of memory and effector T cells such as TH17, plays a crucial role in the pathogenesis of these diseases. Antinuclear antibody (ANAs) testing is used as a first step for the diagnosis of autoimmune disorders, although most ANA-positive individuals do not have nor will develop an autoimmune disease. Studying the differences of T cell compartment among healthy blood donors, ANA-negative patients and ANA-positive patients, in which loss of tolerance have not led to autoimmunity, may improve our understanding on how tolerance mechanisms fail. Herein, we report that ANA-positive patients exhibit a distinct distribution of T cell subsets: significantly reduced frequencies of recent thymic emigrants (RTE) and naïve T cells, and significantly increased frequencies of central memory T cells, TH2 and TH17 cells; modulations within the T cell compartment are most profound within the 18-40 year age range. Moreover, CD4+ T cells in ANA-positive patients are metabolically active, as determined by a significant increase in mTORC1 and mTORC2 signals, compared to ANA-negative patients and healthy blood donors. No significant impairment of Treg numbers or pro-inflammatory cytokine production was observed. These results identify a unique T cell signature associated with autoantibody production in the absence of autoimmune disease.

Age-related effects on thymic output and homeostatic T cell expansion following depletional induction in renal transplant recipients

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 naïve T (TNaïve ) cells emerged posttransplantation. Analysis by generalized estimating equation linear models revealed a strong negative linear association between reconstituting TNaïve 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 30years of age showed significantly higher levels of CD4+ CD31+ cells than patients over 55years 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.

Accelerated T Cell Senescence after Allogeneic Hematopoeitic Stem Cell Transplantation

Long-term survivors of allogeneic hematopoietic stem cell transplantation (HSCT) remain susceptible to opportunistic infections, relapse of primary malignancy and development of secondary malignancy, features that are consistent with continued immune deficiency. Reconstitution of T cell populations following HSCT and the preceding conditioning regimens occurs via two main routes: a thymic-dependent generation of naive T cells from progenitors via thymopoeisis, and a thymic-independent peripheral expansion of adoptively transferred, mature T cells in the graft. We hypothesized that dependence on peripheral expansion for reconstitution in individuals over forty years old leads to T cell populations that are prematurely senescent. In this retrospective study of patients who underwent matched, related allogeneic transplantation, we compared PBMCS from a cohort of long-term (4-5 years) survivors of transplant and their original donor infusion products (n=13), evaluating the expession of markers of immunosenescence and aging (CD28, KLRG1, CD57 and PD-1) at each time point. Using multi-parameter flow cytometry, we determined that CD8 cells expressed significantly higher levels of markers of T cell terminal differentiation and senescence post-transplant. Both the frequency and absolute number of Effector Memory and TEMRA, as well as the incidence of cells expressing senescence markers increased. Although there was no significant change in frequency of CD8+ T-cells actively proliferating (% Ki-67+), TREC analysis revealed that the frequency of recent thymic emigrants in the peripheral T-cell pool was reduced long-term. Additionally, we assessed the telomere lengths of CD8+ T-cells using Flow-FISH and, in line with our hypothesis that reliance on peripheral expansion results in replicative senescence, found that they were significantly shorter post-transplant. In conclusion, our study suggests that a contributory factor underlying the disposition for opportunistic infections in long-term survivors of transplant may be premature T cell dysfunction resulting from immune reconstitution-related senescence.Figure Captions:Figure I. Immune reconstitution post-transplant(A) Absolute T, B, and NK cell counts were measured at the donor infusion and post-transplant time points in 13 donor-recipient pairs. Lines indicate median values for each time point. (B) Individual CD4+ and CD8+ T cell changes post-transplant relative to the donor infusion time point. (C) Inversion of the normal CD4:CD8 ratio at the post-transplant time point. (D) Median values for absolute CD4 and CD8 cell counts per uL for the patient cohort throughout the post-transplant time period.Figure II. Alterations in T cell subsets post-transplant(A) Frequencies of Naive (CCR7+ CD45RA+), Central Memory (CCR7+ CD45RA-), Effector Memory (CCR7- CD45RA-), and Effector Memory cells that re-express CD45RA (TEMRA; CCR7- CD45RA+) subsets for both CD4 and CD8 cells at the donor infusion and post-transplant time points. Boxes indicate the 25th quartile, median, and 75th quartile values; whiskers indicate the minimum and maximum values. (B) Absolute cell numbers per uL for each CD4 and CD8 T cell subset. (C) Ratio of absolute cell number for Naïve/Central Memory + Effector Memory populations at the donor infusion and post-transplant time points. (D) TREC analysis of the CD4 and CD8 populations assessing thymic generation of naive T cells at the donor infusion and post-transplant time points (n=6). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

T-Cell Composition of the Lymph Node Is Associated with the Risk for Early Rejection after Renal Transplantation.

The T-cell composition within the lymph node (LN) of end-stage renal disease (ESRD) patients differs from the composition within the circulation. Activation of the alloreactive T-cell response within secondary lymphoid organs is important after organ transplantation. However, to date no data are present on LN T-cell subsets and the risk for acute rejection after kidney transplantation. T cells from LNs of ESRD patients were analyzed for frequency of recent thymic emigrants, relative telomere length, expression of differentiation markers, and were related to the development of early acute rejection (EAR), occurring within 3 months after renal transplantation (RT). Furthermore, the alloreactive potential of mononuclear cells isolated from the LN and peripheral blood of 10 patients was analyzed. Measures of alloreactive potential included proliferation, cytokine production, frequencies of interferon-gamma-producing cells, and the presence of cytotoxic molecules. Patients with EAR were younger (p = 0.019), cytomegalovirus-seropositive (p = 0.037) and usually received dialysis prior to RT (p = 0.030). Next to this, patients with EAR showed a lower CD4:CD8 ratio (p = 0.027) within the LN. T cells from the LN were similar with regard to alloreactive capacity compared with those within the circulation. Univariate regression analysis showed that the CD4:CD8 ratio (OR: 0.67, p = 0.039), patient age (OR: 0.93, p = 0.024), and preemptive RT (OR: 0.11, p = 0.046) were associated with EAR. After a multivariate analysis, only the CD4:CD8 ratio (OR: 0.58, p = 0.019) and preemptive RT (OR:0.05, p = 0.012) were associated with EAR. A lower CD4:CD8 ratio in the LN is associated with a higher risk for the development of rejection within 3 months after RT.

Lymph node and circulating T cell characteristics are strongly correlated in end-stage renal disease patients, but highly differentiated T cells reside within the circulation.

Ageing is associated with changes in the peripheral T cell immune system, which can be influenced significantly by latent cytomegalovirus (CMV) infection. To what extent changes in circulating T cell populations correlate with T cell composition of the lymph node (LN) is unclear, but is crucial for a comprehensive understanding of the T cell system. T cells from peripheral blood (PB) and LN of end-stage renal disease patients were analysed for frequency of recent thymic emigrants using CD31 expression and T cell receptor excision circle content, relative telomere length and expression of differentiation markers. Compared with PB, LN contained relatively more CD4+ than CD8+ T cells (P < 0·001). The percentage of naive and central memory CD4+ and CD8+ T cells and thymic output parameters showed a strong linear correlation between PB and LN. Highly differentiated CD28null T cells, being CD27- , CD57+ or programmed death 1 (PD-1+ ), were found almost exclusively in the circulation but not in LN. An age-related decline in naive CD4+ and CD8+ T cell frequency was observed (P = 0·035 and P = 0·002, respectively) within LN, concomitant with an increase in central memory CD8+ T cells (P = 0·033). Latent CMV infection increased dramatically the frequency of circulating terminally differentiated T cells, but did not alter T cell composition and ageing parameters of LN significantly. Overall T cell composition and measures of thymic function in PB and LN are correlated strongly. However, highly differentiated CD28null T cells, which may comprise a large part of circulating T cells in CMV-seropositive individuals, are found almost exclusively within the circulation.

Antiretroviral therapy suppressed participants with low CD4+ T-cell counts segregate according to opposite immunological phenotypes.

The failure to increase CD4 T-cell counts in some antiretroviral therapy suppressed participants (immunodiscordance) has been related to perturbed CD4 T-cell homeostasis and impacts clinical evolution. We evaluated different definitions of immunodiscordance based on CD4 T-cell counts (cutoff) or CD4 T-cell increases from nadir value (ΔCD4) using supervised random forest classification of 74 immunological and clinical variables from 196 antiretroviral therapy suppressed individuals. Unsupervised clustering was performed using relevant variables identified in the supervised approach from 191 individuals. Cutoff definition of CD4 cell count 400 cells/μl performed better than any other definition in segregating immunoconcordant and immunodiscordant individuals (85% accuracy), using markers of activation, nadir and death of CD4 T cells. Unsupervised clustering of relevant variables using this definition revealed large heterogeneity between immunodiscordant individuals and segregated participants into three distinct subgroups with distinct production, programmed cell-death protein-1 (PD-1) expression, activation and death of T cells. Surprisingly, a nonnegligible number of immunodiscordant participants (22%) showed high frequency of recent thymic emigrants and low CD4 T-cell activation and death, very similar to immunoconcordant participants. Notably, human leukocyte antigen - antigen D related (HLA-DR) PD-1 and CD45RA expression in CD4 T cells allowed reproducing subgroup segregation (81.4% accuracy). Despite sharp immunological differences, similar and persistently low CD4 values were maintained in these participants over time. A cutoff value of CD4 T-cell count 400 cells/μl classified better immunodiscordant and immunoconcordant individuals than any ΔCD4 classification. Immunodiscordance may present several, even opposite, immunological patterns that are identified by a simple immunological follow-up. Subgroup classification may help clinicians to delineate diverse approaches that may be needed to boost CD4 T-cell recovery.

Uremia-Associated Premature Aging of T Cells Does Not Predict Infectious Complications After Renal Transplantation.

Patients with end-stage renal disease have prematurely aged T cell systems. We tested whether T cell aging parameters were associated with the risk of infections after renal transplantation (RTx). We studied 188 patients over 1 year. Peripheral T cells were analyzed before and at 3 and 6 mo after RTx for frequency of recent thymic emigrants, relative telomere length and differentiation status. These parameters were related to the occurrence of opportunistic and serious infections. Overall, 84 patients developed an infection. In this group, 50 developed an opportunistic infection and 53 developed a serious infection. T cell aging parameters assessed before RTx were not associated with infection risk. The memory T cells showed a decrease within the first 3 mo in both groups (p < 0.001). The CD4(+) memory T cells increased between 3 and 6 mo within the infection group (p = 0.015). The number of CD8(+) memory T cells increased in both groups (p < 0.001) but reached baseline levels only in the infection group. In the infection group, the CD8(+) CD28(null) T cell percentage increased between 3 and 6 mo (p = 0.024), tending to be higher than at baseline (p = 0.061). These differences in post-RTx dynamics resulted from infections. Parameters of uremia-associated premature aging of peripheral T cells do not predict posttransplant infections.

Regulatory T-cell deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked–like disorder caused by loss-of-function mutations in LRBA

A number of heritable immune dysregulatory diseases result from defects affecting regulatory T (Treg) cell development, function, or both. They include immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, which is caused by mutations in forkhead box P3 (FOXP3), and IPEX-like disorders caused by mutations in IL-2 receptor α (IL2RA), signal transducer and activator of transcription 5b (STAT5b), and signal transducer and activator of transcription 1 (STAT1). However, the genetic defects underlying many cases of IPEX-like disorders remain unknown. We sought to identify the genetic abnormalities in patients with idiopathic IPEX-like disorders. We performed whole-exome and targeted gene sequencing and phenotypic and functional analyses of Treg cells. A child who presented with an IPEX-like syndrome and severe Treg cell deficiency was found to harbor a nonsense mutation in the gene encoding LPS-responsive beige-like anchor (LRBA), which was previously implicated as a cause of common variable immunodeficiency with autoimmunity. Analysis of subjects with LRBA deficiency revealed marked Treg cell depletion; profoundly decreased expression of canonical Treg cell markers, including FOXP3, CD25, Helios, and cytotoxic Tlymphocyte-associated antigen 4; and impaired Treg cell-mediated suppression. There was skewing in favor of memory Tcells and intense autoantibody production, with marked expansion of T follicular helper and contraction of T follicular regulatory cells. Whereas the frequency of recent thymic emigrants and the differentiation of induced Treg cells were normal, LRBA-deficient T cells exhibited increased apoptosis and reduced activities of the metabolic sensors mammalian target of rapamycin complexes 1 and 2. LRBA deficiency is a novel cause of IPEX-like syndrome and Treg cell deficiency associated with metabolic dysfunction and increased apoptosis of Treg cells.

Frequent participation in high volume exercise throughout life is associated with a more differentiated adaptive immune response

Exercise induces changes in the immune system depending on its intensity and duration. For example, transient states of immunodepression can be induced after acute intense physical activity whereas beneficial anti-inflammatory effects of moderate chronic exercise on many diseases and longevity have been described. To study the impact of high volume exercise over a lifetime on aspects of immunity we compared immunological features of 27 young and 12 elderly athletes with 30 young and 26 elderly non-athletes stratified by their CMV serostatus. We characterized blood leukocyte and lymphocyte subpopulations by flow cytometry, quantified TREC content, and measured activation and proliferation ability of T-lymphocytes in the presence of anti-CD3. NK-cells functionality was determined in response to K-562, 721.221 and 721.221-AEH cell-lines. High volume physical activity reduced the total number of circulating leukocytes, neutrophils, and lymphocytes. In the lymphocyte compartment, athletes had higher frequencies of NK-cells and CD8+ T-lymphocytes, whereas CD4+ T-lymphocytes were present at significantly lower levels in CMV-seropositive athletes. We found, in the high volume physical activity individuals, a higher degree of differentiation in CD4+ T-lymphocytes. CD8+ T-lymphocytes from young athletes had reduced TREC content and lower frequencies of recent thymic emigrants. Furthermore, the functional ability of CD4+ and CD8+ T-lymphocytes was significantly impaired in young but not in elderly athletes, and may be compensated for significantly higher activation and degranulation of NK-cells. In conclusion, high volume exercise throughout life appears to be associated with increased levels of biomarkers that are associated with an aging immune system, which are partially reduced with physiological aging.

Brief Report: Deficient Thymic Output in Rheumatoid Arthritis Despite Abundance of Prethymic Progenitors

ObjectiveTo determine the frequencies of common lymphoid progenitors (CLPs) and recent thymic emigrants (RTEs) in patients with rheumatoid arthritis (RA) and healthy control subjects.MethodsFlow cytometry was performed to determine the frequencies of CLPs and RTEs in the peripheral blood of 101 control subjects and 51 patients with RA. Thirteen of these patients were also analyzed longitudinally for 6 months after initiation of treatment with a tumor necrosis factor (TNF) inhibitor.ResultsA significant correlation between the frequencies of CLPs and RTEs was observed in healthy control subjects. The frequencies of both CLPs and RTEs decreased with age and correlated inversely with absolute lymphocyte numbers in peripheral blood. In patients with RA, the frequencies of RTEs were significantly decreased compared with the frequencies in control subjects. Importantly, the frequencies of CLPs were significantly higher in patients with RA compared with control subjects. Therapeutic TNF blockade further increased the frequency of CLPs, thereby normalizing thymic output, as indicated by an increase in the number of RTEs.ConclusionThymic insufficiency in RA is not attributable to an inadequate supply of progenitor cells to the thymus. Thus, insufficient numbers of RTEs could result from inadequate thymic T cell neogenesis, or alternatively, could be a consequence of high CD4+ T cell turnover, homeostatic proliferation, and subsequent dilution of the RTE population.

Impact of Thymic Function On Chronic Graft-Versus-Host Disease (cGVHD).

Abstract 3052The initial analysis of the impact of cGVHD on thymic function used TREC analysis and demonstrated a sustained decrease in thympoiesis in hematopoietic stem cell transplant (HSCT) recipients with a history of cGVHD (Blood 97:1458, 2011). We have reported that HSCT recipients, who had the clinical resolution of their cGVHD, had increases in their resting regulatory T lymphocytes (rTreg) as compared to HSCT recipients with active cGVHD [ASH 2011]. The increase in rTreg lymphocytes suggested that clinical resolution of cGVHD might be due to improved thymic function, which resulted in the increased production of rTreg lymphocytes.To measure the thymic function of pediatric HSCT recipients with cGVHD, we determined the frequency of recent thymic emigrants (RTE; CD4+, CD45RA+, CD31+) in the whole CD4 T lymphocyte population and the CD4 regulatory T (Treg; CD127-, CD25+) and CD4 conventional T lymphocytes (Tcon; CD127+, CD25+) subpopulations of normal individuals, HSCT recipients without a history of cGVHD (n =16) and HSCT recipients with a history of cGVHD (n=20). The frequency of RTE in total, Treg, and Tcon lymphocytes of the HSCT recipients with cGVHD was decreased (P=0.001) compared to HSCT recipients without a history of cGVHD but was the same as normal individuals. Thus, pediatric HSCT recipients with cGVHD have thymic function that is similar to normal individuals, but have decreased thymic reserves compared to HSCT recipients without cGVHD.However, since rTreg lymphocytes (CD4+, CD127-, CD25+, CD45RA+, FoxP3+) represent only 20% of total Treg lymphocytes, we specifically determined the thymic contribution to rTreg lymphocytes. The RTE content of rTreg lymphocytes was determined in HSCT recipients with active or resolved cGVHD. The RTE content of the rTreg lymphocytes (as a percentage of total CD4 T lymphocytes) was similar in HSCT patients with both active and resolved cGVHD. Thus, the increase in rTreg lymphocytes present in HSCT recipients with the clinical resolution of their cGVHD is not due to an increase in the frequency of RTE in their rTreg lymphocyte population. Therefore, the increase in rTreg lymphocytes, that is associated with the clinical resolution of cGVHD, may be due to either their decreased conversion to Th17 lymphocytes or their decreased apoptosis. Disclosures:No relevant conflicts of interest to declare.

Characterization of recent thymic emigrants (RTEs), transitional B and Th17 cells in multiple sclerosis (MS)

The finding that APECED syndrome is due to a failure in negative thymic selection has suggested that central tolerance could play a role also in polygenic autoimmunity and that autoimmune patients should have an increase in RTEs. The aim of this work was to assess the frequency of RTEs, transitional B cells and Th17 lymphocytes in MS. PBLs from 55 MS patients [20 relapsing-remitting (RR) with disease modifying drugs (DMD), 20 RRMS without DMD, 10 progressive MS and 5 RRMS patients on relapse] and 32 healthy controls were analysed by FACS for RTEs, naive and memory T cells as well as Th17 cells and transitional B cells. No differences in the percentage of RTEs (CD45RA+CD31+PTK7+) (3.30±2.94 vs 3.05±1.54, 0.933) or B transitional cells (CD19+CD24hiCD38hiCD27-) (8.67±3.62 vs 8.59±3.8, p=0.45) were found between controls and MS. A significant decrease in the percentage of CD4 effector memory (CD45RA-CCR7-CD27+) (23.13±8.76 vs 20.8±6.1, p=0.043), as well as an increase in Th17 cells (6.9±3.2 vs5.49±2.33, p=0.007) was observed in MS vs controls. There were no differences in lymphocyte populations among the clinical forms of MS. RRMS under treatment had similar results to RRMS without DMD. Patients on relapse did show an increased proportion of CD4 effector memory cells, Th17 cells and RTEs and patients with higher EDSS had a higher percentage of Th17 cells (OR 2.3,p=0.045). The results of this preliminary study do not favour a failure in central tolerance in MS but confirm involvement of Th17 cells which are associated with a higher degree of disability.

Plasma Levels of Soluble Interleukin-7 Receptor Alpha (sIL-7Ra) and IL-7Ra Polymorphism After Allogeneic Stemcell Transplantation,

Abstract 4012 Background:Interleukin-7 (IL-7) is a cytokine essential for T cell development in the thymus and maintenance of peripheral T cells. IL-7 binds to cellular IL-7 receptors (IL-7Ra-common g chain heterodimer), in competition with a soluble form of the receptor, shed by the cells (sIL-7Ra). We have identified single nucleotide polymorphisms in the exons of the gene encoding IL-7Ra (+510T/C rs1494558, +1237 G/C rs1494555, 2087 C/T rs6897932), and previous results by us and by others indicate that IL-7R SNPs are associated with aGvHD and mortality after SCT. Moreover, the biological significance of +2087 C/T SNP has been suggested by the finding of elevated serum levels of sIL-7Ra in healthy individuals with 2087CC (Haplotype 4), also associated with increased alternative splicing of exon 6 and a higher frequency of recent thymic emigrants. We hypothesized that sIL-7Ra levels during SCT are influenced by genetic polymorphism and may play a role in immune reconstitution after SCT. Aims:1) To investigate sIL-7Ra levels during SCT along with IL-7Ra genotypes and 2) to evaluate associations between sIL-7Ra levels and immune reconstitution and outcome in SCT. Patients and Methods:122 patients undergoing SCT for haematological malignancies after either myeloablative (n= 52) or reduced intensity conditioning (n=70) were included. Donors were either matched siblings (n=68) or matched unrelated donors (n=54), and the patient age at the time of transplant was 50 years (6–67) (median (range)). sIL-7Ra levels were measured in plasma by a quantitative bead capture assay. IL-7Ra SNPs were determined by a SSP-PCR system and IL-7 was tested by ELISA (R&D) (n=77). Results:sIL-7Ra levels decreased during the course of transplantation from 113 ng/ml (32–558) at day −15 to 48 ng/ml (32–195) at day +14 (p=0.0001), and reached baseline levels again at day +60. sIL-7Ra levels were not associated with the intensity of the conditioning regimen. This pattern appeared to be inversely mirrored by the IL-7 levels, which increased from baseline values at day –14 of 2.4 pg/ml (0.3–17.6) to 11.3 pg/ml (2.0–30.2) (p<0.0001) at day +14, followed by a gradual decline down to baseline values at day +60.sIL-7Ra levels at day +90 were reduced in patients transplanted with donors carrying IL-7Ra 2087T allele, in line with our previous findings in healthy individuals (105 ng/ml (42–274) vs. 152 ng/ml (20–971), p=0.0015). In addition, post-transplant sIL-7Ra levels correlated with pre-transplant sIL-7Ra levels (r=0.39 p=0.0032), indicating that patient related factors in addition to the genotype of the donor lymphocytes may play a role in the regulation of sIL-7Ra levels post-transplant.sIL-7Ra appeared to be predictive of the rate of immune reconstitution by the finding that sIL-7Ra at day +14 correlated significantly with total lymphocyte counts post-transplant (day +90: r=0.28 (p=0.031) and day +180: r=0.55 (p<0.0001)). In contrast, IL-7Ra genotypes were not associated with immune lymphocyte counts post-transplant and early post-transplant IL-7 levels did not correlate significantly with lymphocyte counts at any stage. Outcomes:There was a trend towards an association between high sIL-7Ra levels and increased overall survival (p=0.057), but sIL-7Ra levels were unrelated to the occurrence of aGvHD. However, the IL-7/sIL-7Ra ratio at day +14 was significantly higher in patients with grade 2–4 aGvHD compared to grade 0–1 aGvHD (0.29 (0.04–0.73) vs 0.19 (0.01–0.8), p=0.033). Conclusion:The data of the present study indicates that sIL-7Ra levels after SCT are determined by the IL-7R 2087 genotype of the donor in addition to patient related factors. sIL-7Ra levels in the early phase post transplant is associated with the rate of lymphocyte recovery post-SCT. Thereby this study adds to the growing evidence suggesting the importance of the IL-7 axis in SCT. The study further suggests that monitoring sIL-7Ra levels post-transplant may help to guide the clinical use of IL-7. Disclosures:No relevant conflicts of interest to declare.

Haplotype 4 of the multiple sclerosis-associated interleukin-7 receptor alpha gene influences the frequency of recent thymic emigrants

The receptor for the homeostatic T cell cytokine interleukin-7 (IL-7Ralpha) has recently shown genetic association to multiple sclerosis (MS). To investigate the functional contribution of IL-7Ralpha polymorphisms to the pathogenesis of MS, we correlated the IL-7Ralpha haplotypes with different T cell parameters in a group of MS patients and healthy controls. We show that carriers of one of the four IL-7Ralpha haplotypes (Hap4) show a higher expression of IL-7Ralpha (CD127) on their CD4(+) T cells, compared with noncarriers (P=0.04). Moreover, Hap4 carriers possess higher frequencies of recent thymic emigrants (RTEs, CD31(+)) in both the regulatory T cell (Treg; P=0.007) and conventional T cell (Tconv) population (P=0.0001). This effect is most pronounced within the MS population (Treg, P=0.0077; Tconv, P=0.0007), whereas in healthy controls significance was only reached for Tconv (P=0.043; Treg, P=0.11). Because previous studies showed a decreased RTE-Treg frequency in MS patients compared to healthy subjects, we here conclude that this decrease is localized within the MS population of non-Hap4 carriers. In conclusion, our findings suggest that IL-7Ralpha polymorphisms can influence T cell development and homeostasis, and thereby contribute to the altered immune regulation associated with disease development in patients with MS.

Murine neonatal recent thymic emigrants are phenotypically and functionally distinct from adult recent thymic emigrants

In contrast to adults, the murine neonatal CD4+ compartment contains a high frequency of recent thymic emigrants (RTEs). However, the functional capabilities of these cells in neonates are relatively unknown. Moreover, it has not been determined whether RTEs from neonates and adults are comparable. Here we have directly compared neonatal and adult CD4+ RTEs for the first time, using a transgenic mouse strain that allows for the identification and purification of RTEs. Our data demonstrate that RTEs from murine neonates and adults are phenotypically and functionally distinct. In particular, although the magnitude of RTEs cytokine responses from both age groups is dependent on the conditions of activation, neonatal RTEs always exhibited higher levels of effector Th1/Th2 cytokine production than adult RTEs. In addition, neonatal, but not adult, RTEs showed early proliferation in response to stimulation with interleukin-7 alone. This was associated with faster kinetics of interleukin-7Ralpha down-regulation and higher levels of pSTAT5 in neonatal RTEs. These quantitative and qualitative differences in the neonatal and adult RTEs populations may at least partially explain the diverse responses that are elicited in vivo in neonates in response to different conditions of antigen exposure.

Impact of Post-Thymic Cellular Longevity on the Development of Age-Associated CD4+ T Cell Defects

Elderly people are at higher risk for infections due to declining cellular and humoral immune responses. Central to this dysfunction is the reduced responsiveness of the naive CD4(+) T cell compartment. Previous data from our laboratory suggest that although defects in the aged naive CD4(+) T cell response are apparent in recent thymic emigrant populations, additional defects develop during extended post-thymic longevity in the periphery. To further investigate the factors that lead to aging defects, we took advantage of the OT-II TCR-transgenic (Tg) mouse model. We show that because of an apparent superantigen-mediated loss of naive Vbeta5(+) Tg CD4(+) T cells from the periphery of aging OT-II mice, this compartment becomes enriched for cells of reduced post-thymic longevity, resulting in a frequency of recent thymic emigrants in aged mice that is similar to that of young mice. Purification and functional analysis of aged OT-II cells with reduced post-thymic longevity reveal that they have an age-associated decrease in expansion and IL-2 production in response to Ag in vitro. However, the in vivo expansion, IL-2 production, and cognate B cell helper ability of these cells are similar to those of cells from young mice. In contrast, T cells from aged HNT Tg mice demonstrate extended post-thymic longevity and exhibit severe defects in the same in vitro and in vivo models. These data support a correlation between the requirement for increased post-thymic longevity and the development of the most severe naive CD4(+) T cell-aging defects.

Neonatal recent thymic emigrants (RTE): phenotypic and functional comparison to adult RTE (85.16)

Abstract Neonatal CD4+ T cells typically produce high levels of IL-4, resulting in Th2-biased responses. The neonatal CD4+ compartment contains a high frequency of recent thymic emigrants (RTE), in contrast to the small proportion found in adults. Using intrathymic labeling to identify RTE, it has been described that adult RTE produce high levels of IL-4. Thus, neonatal Th2 biased responses may result from their over-represented RTE population. Here we have compared neonatal and adult CD4+ RTE for the first time, using a mouse strain that allows for the identification and purification of RTE from the periphery. In contrast to the intrathymic labeling system, adult RTE from these mice produced less IL-4 and IFNγ than the resident cells. Similarly, neonatal RTE produced less of both IL-4 and IFNγ than resident neonatal cells. The phenotypic changes accompanying post-thymic maturation of RTE were also similar in neonates and adults. Thus, both neonatal and adult RTE appear to require a period of post-thymic maturation to acquire mature Th2 and Th1 function and phenotype. Importantly, despite these similarities, neonatal RTE produced more IL-4 than adult RTE. Together, these results indicate that neonatal and adult RTE differ functionally but the subsequent processes of post-thymic maturation are similar in early development and adult life. This work was supported by NIAID grant number R01 AI44923-02 (B.A.)