Cell Receptor Rearrangement Excision Circles Research Articles

Successful Immune Reconstitution in a Patient with a TYK2 Deficiency after Allogeneic Stem Cell Transplantation from Unrelated Donors.

A boy with primary immunodeficiency, caused by a tyrosine kinase 2 (TYK2) mutation, presented with immune defects and a lifelong history of severe infections. Our aim was to determine whether allogeneic hematopoietic stem cell transplantation (HSCT) could restore the patient's immune defenses and reduce susceptibility to infection. In the absence of a suitable HLA-matched blood relative to act as a donor, the patient received an allogeneic HSCT from unrelated donors. The patient's clinical data were analyzed in the Children's Hospital of Chongqing Medical University (Chongqing, China) before transplantation and during the 4-year follow-up period using a combination of western blotting (e.g., TYK2 and STAT levels), qRT-PCR (e.g., T cell receptor rearrangement excision circles, kappa deletion element recombination circles, and TYK2 transcript levels), and flow cytometry (e.g., lymphocyte subpopulations and CD107α secretion). We found that HSCT significantly reduced the incidence of severe infections, restored normal TKY2 levels, and reversed defects such as impaired JAK/STAT signaling in response to interferon-α or interleukin-10 treatment. Although the patient did not develop acute graft-versus-host disease (GVHD) after transplantation, he did experience chronic GVHD symptoms in a number of organs, which were effectively managed. Our findings suggest that HSCT is a feasible strategy for reconstituting the immune system in TYK2-deficient patients; however, the factors associated with GVHD and autoimmune thyroiditis development in TYK2-deficient patients undergoing HSCT warrant further investigation.

Bone Marrow-Derived Mesenchymal Stromal Cells Promote Thymopoiesis and Function Recovery in Acute Graft-Versus-Host Disease Murine Models

Background: The thymus is the primary lymphoid organ of T-cells development and plays a crucial role for immune tolerance following allogeneic hematopoietic stem cell transplantation (allo-HSCT). The toxicity of chemoradiotherapy conditioning regimen and acute graft-versus-host disease (aGVHD) damage the thymic structure and function, which result in the production of autoreactive T-cells and the reduction of natural regulatory T cells (nTregs) to aggravate the development of aGVHD. Nowadays, mesenchymal stromal cells (MSCs) as multipotent stem cells, have been widely recognized as a promising therapeutic strategy for aGVHD. However, the well-known mechanisms of MSCs ameliorating aGVHD mainly focus on their immunoregulatory effect on peripheral immune compartments. The contribution of MSCs to thymus, the central immunological organs, is rarely reported.Methods: In this study, we carried out studies in a murine aGVHD models of fully MHC-mismatched myeloablative bone marrow transplantation (C57BL/6 to BALB/c). We randomly divided aGVHD mice into two groups, including MSCs group and control group without MSCs treatment. In MSCs group, aGVHD mice were intravenously administrated GFP-labelled bone marrow (BM) derived MSCs at a dose of 5×105 cells/mice at 7 day after allo-HSCT. The mice were killed at 1d, 2d, 4d and 20d after MSCs injection in MSCs group and the corresponding time in control group, respectively. Thymic tissues were collected and manufactured into frozen sections or thymic single-cell suspension. And then we analyzed the distribution of GFP-MSCs in thymus and thymic size, the expression of thymic T cells subsets (CD4+CD8-, CD4-CD8+, CD4+CD8+ T, CD4+CD25+Foxp3+Tregs), thymic epithelial cells (TECs) substes (CD45-CD326+Ly51+ cortical TECs and CD45-CD326+UEA-1+ medullary TECs) and the level of T cell receptor rearrangement excision circles (TRECs) in thymus between the two groups to evaluate the repair effect of MSCs for thymus.Results: We found that MSCs treated mice have longer survival and alleviate the clinical signs of aGVHD compared to the control group (P<0.05). Next, we observed that GFP-MSCs can home to thymic tissue and mainly locate in the corticomedullary boundary at 2d, 4da after MSCs infusion. MSCs treated thymus exhibited a more organized corticomedullary architecture, compared with the conrol group. Significant increases in thymus size and weight, as well as the number of total thymocytes were seen in MSCs group. To further investigate whether MSCs promote thymopoiesis and ameliorate thymic functions after allo-HSCT, we found that in MSCs group the proportion of thymic T cells subets composition (CD4+CD8-, CD4-CD8+, CD4+CD8+), especially Foxp3+ Tregs, and the expression of TECs, especially cTECs, were significantly higher than control group at 20d after the MSCs infusion (P<0.001). Furthermore, MSCs administration resulted in a remarkable increase in the levels of TRECs in the thymocyte at 20d after the MSCs infusion compared with the control group (P=0.007).Conclusions: Collectively, our data proposed a new mechanism for MSCs ameliorating aGVHD by central immune organ, including promoting thymopoiesis and enhancing thymic output. DisclosuresNo relevant conflicts of interest to declare.

Thymectomy in Juvenile Myasthenia Gravis Is Safe Regarding Long Term Immunological Effects

Thymectomy is an established treatment in adult MG and also recommended for the treatment of post-pubertal onset juvenile MG. Whether the youngest children should be thymectomized is still debated. Signs of premature aging of the immune system have been shown in studies on early perioperative thymectomy in children with congenital heart defect. In this retrospective cohort study the objective was to investigate the long-term effects of treatment related thymectomy on T cell subsets and T cell receptor rearrangement excision circles (TRECs) in peripheral blood of juvenile myasthenia gravis (MG) patients, as well as clinical occurrence of autoimmune disorders, malignancies and infectious diseases. Forty-seven patients with onset of myasthenia gravis before the age of 19 years were included; 32 (68.1%) had been thymectomized and 15 (31.8%) had not. They were studied at varying times after thymectomy (7–26 years). We found a significant lower number of naïve helper T cells (CD4+CD45RA+) with an increased proportion of memory helper T cells (CD4+CD45RO+), and a significant lower number of naïve cytotoxic T cells (CD8+CD27+CD28+) in the thymectomized patients. In addition they showed a significant reduction in the number of TRECs and proportion of recent thymic emigrants (RTE) compared to non-thymectomized patients. In none of them an increased frequency of malignancies or infections was found. Our findings indicate a premature aging of the immune system after thymectomy in juvenile MG, but associated clinical consequences could not be verified.

Decrease in naïve T cell production due to HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) development

Human T-lymphocytic virus 1 (HTLV-1) is mainly associated with adult T-cell leukemia/lymphoma (ATLL) and HTLV-1–associated myelopathy/tropical spastic paraparesis (HAM/TSP). Patients with HAM/TSP exhibit significant changes in their immune response, and HTLV-1 infection can interfere in cytokine production and perhaps in T cell production. The aims of this study were to evaluate thymic function in HAM/TSP patients and HTLV-1 healthy carriers (HCs) and correlate it to age and interleukin 7 (IL-7) gene expression. Thymic function in 21 HAM/TSP patients and 12 HCs was evaluated by quantifying T cell receptor rearrangement excision circle (TREC) particles and IL-7 gene expression, both measured by quantitative polymerase chain reaction. HAM/TSP patients presented lower TREC particle counts (p = 0.0112) and lower IL-7 expression (p = 0.0102) than HCs. Both TREC particles and IL-7 gene expression were separately analyzed in two age groups: ≤ 59 years and ≥60 years, The ≤59-year-old HAM/TSP patients had a lower TREC count compared with the ≤59-year-old HCs (p = 0.0476). In conclusion, HAM/TSP development could interfere with thymic function because the results showed TREC particle reduction in HAM/TSP patients in relation to HCs, and it could be associated with a concomitant reduction in IL-7 expression.

Mesenchymal Stem Cells Improve the Structure and Function of the Graft-Versus-Host Disease Receptor Thymus: CCR9 Plays an Important Role in Its Homing Thymus

Background: Insufficient thymic function of allogeneic hematopoietic stem cell transplantation (allo-HSCT) receptors results in continuous production of alloreactive T cells, which leads to the development of graft-versus-host disease (GVHD), especially chronic GVHD (cGVHD). We have previously found that patients with acute GVHD (aGVHD) treated with mesenchymal stem cells (MSCs) have increased thymic output and decreased incidence of cGVHD, thus hypothesized that MSCs may reduce the incidence of cGVHD by remodeling the thymus. Chemokine receptor 9 (CCR9), the receptor that specifically guides migration of T-lineage precursors into thymus, is also expressed on MSCs, and thus may be a key factor mediating MSCs homing to the thymus. This in turn allows MSCs to reduce GVHD by repairing thymus tissue structure and saving thymus function. Methods: We carried out studies in a murine GVHD model of fully MHC-mismatched myeloablative bone marrow transplantation (C57BL/6 to BALB/c), a model that can observe the prolongation of aGVHD to cGVHD. We randomly divided GVHD mice into four groups, including three MSCs treated groups and one untreated group. CCR9 over-expressed (MSC/CCR9+), knocked-down (MSC/CCR9-) and empty-load MSCs (MSC/Control) were generated and administrated intravenously at dose of 5 × 105 cells/infusion at 7th and 21th day post HCT to the treated groups respectively to compare their thymic homing ability, and therapeutic effects of GVHD with the untreated group. Clinical scores were recorded once every five days to evaluate GVHD symptoms. Mice of MSCs treated groups and the untreated group were sacrificed at 30d, 45d and 60d after HCT. Thymuses of each group were collected and assessed for size and weight before being manufactured into frozen sections or thymic single-cell suspension. We then analyzed the number and distribution of MSCs in the thymus of the treated groups to assess the role of CCR9 in thymic homing, and analyzed the expression of thymic T cells subsets (CD4+CD8-, CD4-CD8+, CD4+CD8+ T, CD4+CD25+Foxp3+Tregs), thymic epithelial cells (TECs) substes (CD45-CD326+Ly51+ cortical TECs and CD45-CD326+UEA-1+ medullary TECs) and the level of T cell receptor rearrangement excision circles (TRECs) in thymus among the four groups to evaluate the repair effect of MSCs for thymus. Radiation-pretreated murine TECs were cultured alone or co-cultured with murine MSCs in vitro to assess the effect of MSCs on damaged TECs. Results: The infusion of MSC/CCR9+ potently alleviated the clinical signs of GVHD and prolonged the survival of GVHD mice (P&lt;0.05 versus MSC/CCR9- and untreated group). Significant increases in thymus size and weight were observed in the MSC/CCR9+ group, as well as the number of total thymocytes and the more organized cortical medullary structure compared to the other groups. MSCs enter the thymus from the microvascular region at the cortex-medium junction. MSC/CCR9+ were found to appear in the cortex-medium junction of thymus in a greater amount 24 hours after the first infusion, then distribute throughout the whole thymus and relocate in proximity with TECs 48 hours thereafter. MSC/Control could be observed in the cortical and cortex-medium junction, whereas MSC/CCR9- was observed only in the cortex-medium junction with a small amount of distribution. Immunofluorescence of thymus frozen sections showed that, compared with other groups, TECs had decreased apoptosis and significantly increased proliferation and maturation levels in MSC/CCR9+ group, indicating MSCs potently repaired injured TECs and promoted their proliferation and maturation. The number of TECs and its proportion of thymus stroma were significantly improved, including cortical TEC and medullary TECs. As for thymocyte, MSC/CCR9+ infusion significantly increased the number and proportion of CD4+CD8+T cells and Tregs, which were reported deficiency in GVHD thymus. Furthermore, MSC/CCR9+ administration resulted in a remarkable increase in the levels of TRECs in the thymocyte at 45d and 60d after HCT (P&lt;0.05 versus MSC/CCR9- and untreated group). In vitro study showed co-cultured TECs had a decreased apoptosis and increased proliferation compared to TECs cultured alone. Conclusion: This study demonstrates that CCR9 plays an important role in guiding migration of MSCs to thymus and thus highly intensify their issue repair and immunomodulatory effect to rescue thymus function in GVHD model. Disclosures No relevant conflicts of interest to declare.

T cell receptor rearrangement excision circles (TRECs) and CD31+ regulatory T cells for assessing recent thymic output in patients with chronic hepatitis B

Objective To evaluate the clinical value of combined detection of T cell receptor rearrangement excision circles (TRECs) and CD31+ regulatory T (Treg) cells for accessing the recent thymic output in patients with chronic hepatitis B. Methods Four groups involving 135 subjects were set up in this study as follows: mild chronic hepatitis B (Mild CHB, n=35), moderate chronic hepatitis B (Moderate CHB, n=35), severe chronic hepatitis B (Severe CHB, n=35) and healthy control (HCs, n=30) groups. CD4+ CD25+ Treg cells in these subjects were sorted out using magnetic cell separation. The ratio of peripheral CD31+ Treg cells to Treg cells in each group was analyzed by flow cytometry. Real-time PCR was performed to detect TRECs in CD4+ CD25+ Treg cells. The percentages of CD3+ , CD4+ and CD8+ T cell subsets were also measured. Results The ratios of CD31+ Treg/Treg cells and the numbers of TRECs in peripheral blood of the Moderate CHB and Severe CHB groups were significantly lower than those of the Mild CHB and HCs groups (P 0.05). No significant difference in the percentages of CD3+ , CD4+ or CD8+ T cell subsets was observed between the four groups (P>0.05). CD31+ Treg/Treg cell ratio had a positive correlation with the number of TRECs (r=0.551, P=0.014). Conclusions Both CD31+ Treg/Treg cell ratio and the number of TRECs were reduced in the peripheral blood of patients with moderate or severe CHB. CD31+ Treg/Treg cell ratio and the number of TRECs were positively correlated and could be used as new indices to evaluate recent thymus output. Key words: Chronic hepatitis B; CD4+ CD25+ regulatory T cell; CD31+ regulatory T cell; T cell receptor rearrangement excision circles

Neonatal and adult recent thymic emigrants produce IL-8 and express complement receptors CR1 and CR2.

The maintenance of peripheral naive T lymphocytes in humans is dependent on their homeostatic division, not continuing emigration from the thymus, which undergoes involution with age. However, postthymic maintenance of naive T cells is still poorly understood. Previously we reported that recent thymic emigrants (RTEs) are contained in CD31+CD25− naive T cells as defined by their levels of signal joint T cell receptor rearrangement excision circles (sjTRECs). Here, by differential gene expression analysis followed by protein expression and functional studies, we define that the naive T cells having divided the least since thymic emigration express complement receptors (CR1 and CR2) known to bind complement C3b- and C3d-decorated microbial products and, following activation, produce IL-8 (CXCL8), a major chemoattractant for neutrophils in bacterial defense. We also observed an IL-8–producing memory T cell subpopulation coexpressing CR1 and CR2 and with a gene expression signature resembling that of RTEs. The functions of CR1 and CR2 on T cells remain to be determined, but we note that CR2 is the receptor for Epstein-Barr virus, which is a cause of T cell lymphomas and a candidate environmental factor in autoimmune disease.

In Virologically Suppressed HIV+ Patients, High CD4/CD8 Ratio and Length of Treatment Independently Correlate to the Intracellular Content of Proviral HIV-DNA in Different Types of CD4+ T Cells

Introduction:Potent antiretroviral drugs block the progression of HIV infection and inhibit virus integration into the host DNA. Response to therapy is typically monitored by counting CD4+ T cells and measuring plasma viral load, which becomes undetectable in most patients. However, at present HIV cannot be eradicated and persists in the host. Robust data are needed to understand the importance and clinical meaning of the residual activity of the virus, and it is crucial to investigate the role of intracellular HIV reservoirs in patients with undetectable viremia. Since HIV establishes latent infection at different degrees within central memory (CM), effector memory (EM) or naïve (TN) CD4+ T cells, measuring the content of HIV-DNA in different lymphocyte populations is a novel approach to follow the infection. Similarly, the residual capacity of the host to reconstitute the immune system can be accurately monitored by measuring: i) the amount of cells expressing signal-joint (sj) T cell receptor (TCR) rearrangement excision circles (sjTREC), that are a marker of homeostatic proliferation, and ii) telomere length, for evaluating cell senescence. Thus, using flow cytometry and cell sorting along with a molecular biology approach based on droplet digital PCR (ddPCR), we have quantified proviral HIV DNA, the amount of sjTREC+ cells and telomere length in different subsets of CD4+ T cells, such as TN, CM and EM.Methods:According to the Declaration of Helsinki, after informed consent and approval by the local Ethical Committee, we enrolled 32 HIV+ patients (mean age 49.0±7.2 years, 11 females) successfully treated for >2 years, with a CD4+ T cell count >500 cells/uL and plasma viremia undetectable from at least one year. After staining with fluorochrome-labeled mAbs, TN, CM and EM CD4+ T cells were sorted with a S3e sorter (Bio-Rad, CA, USA) equipped with a specifically designed biosafety containment hood (Biobubble, UK). Cell purity was always >95%. Proviral HIV-DNA and sjTREC were quantified in each lymphocyte subset with QX200 droplet digital PCR (Bio-Rad); telomere length, expressed as relative T/S (the ratio between telomere length and single gene copy, related to CD178, i.e., Fas Ligand) was quantified with CFX9600 Real time PCR (Bio-Rad). Viro-immunological parameters such as CD4+ and CD8+ T cell counts and percentages, and viral load were collected for each withdrawal together with the clinical characteristics of patients.Results:Considering all patients, HIV proviral DNA, measured as LTR copies/1,000 cells, was significantly lower in TN cells (mean±SEM: 0.77±0.23) compared to CM (2.42±0.38) or to EM (2.34±0.33), with p<0.0001 in both cases. Conversely, TN cells contained a higher number of sjTREC copies/1,000 cells (11.62±1.54) compared to CM (0.99±0.23) or to EM (1.26±0.44); p<0.0001 in both comparisons. No appreciable changes were observed in T/S ratio among CD4+ subsets. The stratification of patients revealed that HIV content was significantly higher in TN and CM T cells of patients with shorter time of treatment. Similarly, stratifying for CD4/CD8 ratio revealed that those with lower ratio had more intracellular HIV. A similar trend was observed also in EM cells, even if it was not statistically significant. The CD4+ T cell count pre-therapy or CD4+ T cell nadir did not influence the HIV reservoir.Conclusions: The importance of recovering a good CD4/CD8 ratio after successful antiretroviral therapy is further underlined by the fact that different amounts of virus were present and well measurable in different CD4+ T cell populations from patients with high or low ratio. The length of treatment was also significantly correlated to a better virological outcome. It is to note that in our patients homeostatic proliferation or cell senescence likely did not affect HIV reservoir. Linking highly purified cell sorting to a molecular biology approach based on ddPCR is crucial to understand what happens to the HIV reservoir when plasma viremia is suppressed. The measure of HIV DNA in selected lymphocyte populations not only will lead to a better comprehension of the features of HIV reservoir and its monitoring, but could also be useful for the development of eradication strategies. DisclosuresNo relevant conflicts of interest to declare.

The impact of immunosenescence on humoral immune response variation after influenza A/H1N1 vaccination in older subjects.

BackgroundAlthough influenza causes significant morbidity and mortality in the elderly, the factors underlying the reduced vaccine immunogenicity and efficacy in this age group are not completely understood. Age and immunosenescence factors, and their impact on humoral immunity after influenza vaccination, are of growing interest for the development of better vaccines for the elderly.MethodsWe assessed associations between age and immunosenescence markers (T cell receptor rearrangement excision circles – TREC content, peripheral white blood cell telomerase – TERT expression and CD28 expression on T cells) and influenza A/H1N1 vaccine-induced measures of humoral immunity in 106 older subjects at baseline and three timepoints post-vaccination.ResultsTERT activity (TERT mRNA expression) was significantly positively correlated with the observed increase in the influenza-specific memory B cell ELISPOT response at Day 28 compared to baseline (p-value=0.025). TREC levels were positively correlated with the baseline and early (Day 3) influenza A/H1N1-specific memory B cell ELISPOT response (p-value=0.042 and p-value=0.035, respectively). The expression and/or expression change of CD28 on CD4+ and/or CD8+ T cells at baseline and Day 3 was positively correlated with the influenza A/H1N1-specific memory B cell ELISPOT response at baseline, Day 28 and Day 75 post-vaccination. In a multivariable analysis, the peak antibody response (HAI and/or VNA at Day 28) was negatively associated with age, the percentage of CD8+CD28low T cells, IgD+CD27- naïve B cells, and percentage overall CD20- B cells and plasmablasts, measured at Day 3 post-vaccination. The early change in influenza-specific memory B cell ELISPOT response was positively correlated with the observed increase in influenza A/H1N1-specific HAI antibodies at Day 28 and Day 75 relative to baseline (p-value=0.007 and p-value=0.005, respectively).ConclusionOur data suggest that influenza-specific humoral immunity is significantly influenced by age, and that specific markers of immunosenescence (e.g., the baseline/early expression of CD28 on CD4+ and/or CD8+ T cells and T cell immune abnormalities) are correlated with different humoral immune response outcomes observed after vaccination in older individuals, and thus can be potentially used to predict vaccine immunogenicity.

Low-dose growth hormone for 40 weeks induces HIV-1-specific T cell responses in patients on effective combination anti-retroviral therapy

Recombinant human growth hormone (rhGH) administered to combination anti-retroviral therapy (cART)-treated human immunodeficiency virus-1 (HIV-1)-infected individuals has been found to reverse thymic involution, increase total and naive CD4 T cell counts and reduce the expression of activation and apoptosis markers. To date, such studies have used high, pharmacological doses of rhGH. In this substudy, samples from treated HIV-1(+) subjects, randomized to receive either a physiological dose (0·7 mg) of rhGH (n = 21) or placebo (n = 15) daily for 40 weeks, were assessed. Peptide-based enzyme-linked immunospot (ELISPOT) assays were used to enumerate HIV-1-specific interferon (IFN)-γ-producing T cells at baseline and week 40. Individuals who received rhGH demonstrated increased responses to HIV-1 Gag overlapping 20mer and Gag 9mer peptide pools at week 40 compared to baseline, whereas subjects who received placebo showed no functional changes. Subjects with the most robust responses in the ELISPOT assays had improved thymic function following rhGH administration, as determined using CD4(+) T cell receptor rearrangement excision circle (TREC ) and thymic density data from the original study. T cells from these robust responders were characterized further phenotypically, and showed decreased expression of activation and apoptosis markers at week 40 compared to baseline. Furthermore, CD4 and CD8 T cell populations were found to be shifted towards an effector and central memory phenotype, respectively. Here we report that administration of low-dose rhGH over 40 weeks with effective cART resulted in greater improvement of T lymphocyte function than observed with cART alone, and provide further evidence that such an approach could also reduce levels of immune activation.

Effects of recombinant human granulocyte colony-stimulating factor on central and peripheral T lymphocyte reconstitution after sublethal irradiation in mice

Granulocyte colony-stimulating factor (G-CSF) is one of the most critical cytokines used for the treatment of acute radiation syndrome (ARS). In addition to the hematopoietic effects of G-CSF on the differentiation and proliferation of myeloid progenitor cells, G-CSF is also known to have immunomodulatory effects. The aim of the present study was to investigate whether G-CSF could accelerate central and peripheral T lymphocyte recovery after a sublethal dose of irradiation. Female BALB/c mice were subjected to 6 Gy of total body irradiation and then were treated with either 100 μg/kg G-CSF or an equal volume of PBS once daily for 14 days. Percentages of thymocyte subpopulations including CD4 − CD8 − , CD4 + CD8 + , CD4 + CD8− and CD4 − CD8+ T cells, peripheral CD3 + , CD4+ and CD8+ cells were analyzed by flow cytometry. Recent thymic emigrants (RTEs) were assessed by real-time polymerase chain reaction (PCR) using primers specific to the 257-bp T cell receptor rearrangement excision circles (sjTRECs). The proliferative capacity of splenic mononuclear cells upon exposure to ConA was measured by using the Cell Count Kit-8 (CCK-8). G-CSF treatment promoted thymocyte regeneration, accelerated the recovery of CD4 + CD8+ cells and increased the frequency of thymocyte sjTRECs. These effects were more prominent at early time points (Day 28) after irradiation. G-CSF also increased the rate of recovery of peripheral CD3 + , CD4+ and CD8+ cells and shortened the period of severe lymphopenia following irradiation. G-CSF also increased the splenic mononuclear cell mitotic responsiveness to ConA more than control-treated cells. Our results show that G-CSF accelerates T cell recovery through both thymic-dependent and thymic-independent pathways, which could be used to increase the rate of immune reconstitution after sublethal irradiation.

T cell receptor rearrangement excision circles (TREC) study as an approach to "in vivo" thymus gland function investigation

Age-dependent changes in immune system such as thymus gland involution, T-cells amount decrease, are typical both for autoimmune diseases (Rheumatoid arthritis - RA), and progressive atherosclerosis characterized as accelerated ageing. But till now processes of T-cell maturation were studied only by indirect methods. The introduction of T-cell receptor excision circle (TREC) PCR-assay seemed to enable direct detection of recent thymic emigrants in blood and therefore the quantification of thymic output [1]. High TREC levels were detected during childhood, and were decreasing with age, but TREC-expressing cells are not completely lost in the elderly. At the first stage of our investigation we studied TREC level in 3 groups of healthy donors: 16 people. 16 - 30 years old (group 1, TREC Median 0,156299 Units), 8 persons 30 - 45 years old (group 2, TREC Median 0,08782 Units) and 9 people over 45 years (group 3 TREC Median 0,051858 Units). Thereby we confirmed age-related decline of thymic output in healthy donors. In RA patients we found age-dependent statistical definite difference of TREC expression. In the 1-st group (n-12, age range 40,4+2,8 y) TREC amount was following: Median 0,00766 I/U lower level 0,00045, upper level 0,01961. In the 2-nd group (n = 16, age range 57,5+1,32) TREC were diminished (Median 0,00065, lower level 0,000002, upper level 0,00095). Detected high TREC amount in some young RA patients is not entirely consistent with the data of literature.TREC level in patients with chronic forms of coronary heart disease (age 55 - 70 years old) was lower but comparable with donors group 3 (TREC Median 0,0200 Units). Unexpectedly high level of TREC comparable with donors group 2 we detected in patients with Acute Myocardial Infarction (AMI) (10 patients, age range 48 - 71 y) (TREC Median 0,089845 Units). According to our viewpoint, the content of TREC in blood lymphocytes depends both on thymic output and peripheral factors, such as survival time of naive T cells in periphery. Recent data give evidence that the up-regulation of Th1 cell-functions and interferon-γ hyperproduction existed in patients with AMI after the onset of symptoms. This may participate in the immune-mediated ventricular remodeling after AMI. The slowing of naive-T-cells turnover and Th1/Th2 imbalance could be the reason of TREC increase in AMI patients. The work is done in framework of project 11-04-01670 sponsored by Russian Foundation of Basic Research. Project director Dr. Goloviznin M.V.

T-regs/Th17 function defect in systemic autoimmunity as a result of "recent thymic emigrants" maturation defect

T-regs and Th17 cells are the new generation of CD4+T-cells which play crucial role in autoimmunity. Both of subsets can influence each other and probably have common precursor. A key question for understanding the mechanism of autoimmunity is to recognize how T-regs and Th17 cells turn from self-protection to autoreactivity. Based on literature data and own observations, we have constructed a conception of age-dependent thymic T-cells maturation peripherialisation as cause of errors in Th17-T-reg cells interrelations. The connection of T-regs with thymus is determined currently. Connection of Th17 cells with thymus remains to be determined properly. Main, there may be naturally occurring Tregs of thymic origin that are resistant to cell death and serve as reserve pool for autoimmunity protective suppressors. This mechanism could be affected by external factors producing profound lymphopenia [1]. Previously we found that RA patients with numerous rheumatoid nodules (RN) and lymphopenia had statistically reliable decrease of CD3+T-cells level. We found definite negative correlation between CD3+PBL amount and RN number (p = 0,029). In all RA patients with and without RN we didn't found the decrease of CD4 receptor. Hereby we expected to find unusual CD3-4+ and CD3-8+ cells in RA. Otherwise the percentage of CD3+4+ and CD3+8+ cells was normal in general. But in 4 RA patients after magnetic separation of CD3+T-cells we detected reliable amount of CD3-4+ lymphocytes (25-28%) These cells were not detected before separation. One of possible explanation of this phenomenon is CD3 molecule modulation after the contact with anti-CD3 antibodies conjugated with magnetic particles. So the presence of T-cells with unusual phenotype in peripheral blood of RA patients doesn't give absolute evidence of T-cells maturation disorders. According to our viewpoint recent thymic emigrants (RTE) fraction presence among T-regs and hypothetically among Th17-cells is the sign of normal Th17/T-regs function. Otherwise the absence of RTE among them leads to immunopathology. CD31 receptor and T cell receptor rearrangement excision circles (TREC) are now markers of RTE. We investigated the number of CD4+CD31+T-cells in RA patients. The preliminary results permit us to suggest the diminution of RTE in RA (less then 1%/ml) We also found the diminution of TREC amount in PBL of 22 rheumatoid arthritis patients, (Median 0,035539 units). FOXP3, RORγ, RORα and CD31 expression in RA will permit to establish role of RTE in autoimmunity.

Low thymic output in the 22q11.2 deletion syndrome measured by CCR9+CD45RA+ T cell counts and T cell receptor rearrangement excision circles

Thymic hypoplasia is a frequent feature of the 22q11.2 deletion syndrome, but we know little about patients' age-related thymic output and long-term consequences for their immune system. We measured the expression of T cell receptor rearrangement excision circles (TREC) and used flow cytometry for direct subtyping of recent thymic emigrant (RTE)-related T cells in 43 patients (aged 1-54 years; median 9 years) from all over Norway and in age-matched healthy controls. Thymic volumes were estimated by ultrasound in patients. TREC levels correlated well with RTE-related T cells defined by co-expression of CD3, CD45RA and CCR9 (r=0.84) as well as with the CD4+ and CD8+ T cell subtypes. RTE-related T cell counts also paralleled age-related TREC reductions. CD45RA+ T cells correlated well with absolute counts of CD4+ (r=0.87) and CD8+ (r=0.75) RTE-related T cells. Apart from CD45RA- T cells, all T cell subsets were lower in patients than in controls. Thymic volumes correlated better with RTE-related cells (r=0.46) than with TREC levels (r=0.38). RTE-related T cells and TREC levels also correlated well (r=0.88) in patients without an identifiable thymus. Production of RTEs is impaired in patients with a 22q11.2 deletion, and CCR9 appears to be a good marker for RTE-related T cells.

T Cell Homeostasis in Centenarians: From the Thymus to the Periphery

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.

Study of thymic size and function in children and adolescents with treatment refractory systemic sclerosis eligible for immunoablative therapy

Following hematopoietic stem cell transplantation (HSCT), thymic reconstitution of peripheral T lymphocytes is essential to avoid a chronically immunodeficient state and disease recurrence. The purpose of this study was to determine if children and adolescents with treatment refractory SSc, awaiting HSCT, have sufficient thymic function to reconstitute T lymphocyte function after transplantation. Thirteen children with systemic scleroderma were enrolled and assessed by physical exam, chest MRI, measurement of autoantibodies, B and T cell immuno-phenotyping, and quantization of T cell receptor rearrangement excision circles (TREC) as a marker of thymopoiesis. MRI detected thymic tissue in 9/13 children. TREC levels were detectable in all but one child but were significantly reduced (p<0.001) when compared to a control population. SSc patients also had a reduced percentage of naïve (CD45RA+CD31+) CD4+ T lymphocytes, further indicating diminished thymopoiesis. Our data suggest that thymic function in children with SSc might be insufficient for an adequate immunoreconstitution following transplantation in some patients. A thorough evaluation of immune and thymic functions to identify those patients prior to HSCT is recommended.

Immunological Reconstitution after Autologous Hematopoietic Stem Cell Transplantation in Patients with Systemic Sclerosis: Relationship Between Clinical Benefits and Intensity of Immunosuppression

To analyze the relationship between clinical benefits and immunological changes in patients with systemic sclerosis (SSc) treated with autologous hematopoietic stem cell transplantation (HSCT). Ten patients with SSc were treated with high-dose cyclophosphamide followed by highly purified CD34+ cells (n=5) or unpurified grafts (n=5). Two groups of patients were retrospectively constituted based on their clinical response (good responders, n=7; and poor responders, n=3). As well as clinical findings, immunological reconstitution through autologous HSCT was assessed by fluorescence-activated cell sorter analysis, quantification of signal joint T cell receptor rearrangement excision circles (sjTREC), reflecting the thymic function, and foxp3, a key gene of regulatory T cells, mRNA levels. Patients' clinical and immunological findings were similar between good and poor responders, or CD34-purified and unpurified groups at inclusion. The sjTREC values were significantly suppressed at 3 months after autologous HSCT in good responders compared with poor responders (p=0.0152). Reconstitution of CD4+CD45RO- naive T cells was delayed in good responders compared with poor responders. The phenotype of other lymphocytes, cytokine production in T cells, and foxp3 gene expression levels after autologous HSCT did not correlate with clinical response in good or poor responders. Clinical and immunological findings after autologous HSCT were similar between CD34-purified and unpurified groups. Our results suggest that immunosuppression intensity, sufficient to induce transient suppression of thymic function, is attributable to the feasible clinical response in patients with SSc treated with autologous HSCT. Appropriate monitoring of sjTREC values may predict clinical benefits in transplanted SSc patients after autologous HSCT.

Homeostatic Cytokines and Expansion of Regulatory T Cells Accompany Thymic Impairment in Children with Down Syndrome

Down syndrome (DS), the most common chromosomal abnormality in humans, is characterized by precocious immunologic aging that results, among other things, in alterations of B and T lymphocyte subsets and natural killer cells, defective phagocytosis, and chemotaxis of polymorphonuclear leukocytes. We studied 30 children affected by DS, compared them to 29 healthy controls, and evaluated the functionality of the thymus (by measuring the amount of lymphocytes that express the signal-joint T cell receptor rearrangement excision circles [sj-TREC+]), the plasma levels of interleukin (IL)-7 and IL-15, the proliferative T cell response to these cytokines, the expression of the alpha chain of the IL-7 receptor (CD127), the extrathymic differentiation of T lymphocytes, and the presence of natural regulatory T cells (Tregs) in peripheral blood. We found that DS children had a significantly lower number of sj-TREC+ lymphocytes, the levels of which were strongly correlated with age. We found higher plasma levels of IL-7 and IL-15 than in healthy controls, and a higher proliferative T cell response to IL-15. DS children also showed a lower percentage of CD4(+) cells and profound alterations of T cell differentiation, along with increased amount of Tregs and of cells expressing markers of apoptosis. We can thus hypothesize that the precocious thymic involution occurring in DS is mirrored by a high production of IL-7 and IL-15, which is crucial for cell survival and proliferation. The complex alterations present in the periphery are likely the result of a compensatory mechanism: the overproduction of homeostatic cytokines could be a reaction to the impaired intrathymic production of T lymphocytes and/or to the expansion of Treg in the periphery, and could be required to allow the survival of T cells.

CD7−T Cells are Late Memory Cells Generated from CD7+T Cells

CD7(-) T cells constitute a distinct subset within the CD4(+) and CD8(+) T cell populations; their developmental and functional relationship to the majority of CD7(+) T cells, however, remained so far unresolved. We here elucidate that CD7(-) cells represent aging T cells in late memory cell development characterized by a high activation threshold, low effector capacities, and high sensitivity to activation-induced cell death (AICD). In this regard, CD7(-) T cells highly express killer cell lectin-like receptor G1 (KLRG-1), harbor telomeres of shorter lengths, a decreased telomerase expression per cell, and less amounts of T cell receptor rearrangement excision circles (TRECs) compared to CD7(+) cells. CD7(-) T cells are generated in vitro from naive CD7(+) T cells upon repetitive TCR/CD28 engagement, a process that is unidirectional and requires multiple cell divisions. Consequently, clonal expansions of CD7(-) T cells in vivo are less frequent than of CD7(+) T cells, the former can be traced back to those of CD7(+) T cells.

Growth hormone enhances thymic function in HIV-1–infected adults

Growth hormone (GH) is an underappreciated but important regulator of T cell development that can reverse age-related declines in thymopoiesis in rodents. Here, we report findings of a prospective randomized study examining the effects of GH on the immune system of HIV-1-infected adults. GH treatment was associated with increased thymic mass. In addition, GH treatment enhanced thymic output, as measured by both the frequency of T cell receptor rearrangement excision circles in circulating T cells and the numbers of circulating naive and total CD4(+) T cells. These findings provide compelling evidence that GH induces de novo T cell production and may, accordingly, facilitate CD4(+) T cell recovery in HIV-1-infected adults. Further, these randomized, prospective data have shown that thymic involution can be pharmacologically reversed in humans, suggesting that immune-based therapies could be used to enhance thymopoiesis in immunodeficient individuals.