Increased apoptosis of peripheral blood lymphocytes in children with nephrotic syndrome

Nephrotic syndrome is accompanied by and probably related to abnormal T-lymphocyte function. Decreased stimulation of survival factors and increased levels of ”dead signals” may lead to the malfunction of many cells, including lymphocytes. In our study, we investigated the process of apoptosis within T cells in children with a first attack of nephrotic syndrome (NS). We found that the number of apoptotic T cells is greater in these patients than in both children in remission from NS and in controls. The percentage of annexin-V-fluorescein isothiocyanate (FITC)-positive CD3+ cells was 27.30± 12.13% in children with a first attack of NS, 19.22± 15.16% (P=0.006) in children in remission and 16.20± 6.13% (P=0.004) in controls. The percentage of annexin-V-FITC-positive CD3+CD4+ cells was 7.35±7.72% in children with a first attack of NS, 3.80±3.75% (P=0.0001) in children in remission and 3.82±2.01% (P=0.0002) in controls. We conclude that abnormal number and function of T lymphocytes found in NS patients may be related to an increased apoptotic rate of circulating lymphocytes. Received: 30 May 2001 / Revised: 11 October 2001 / Accepted: 13 October 2001     

Naive CD4+ cells from cord blood can generate competent Th effector cells

BACKGROUND: Umbilical cord blood (UCB) cells have been increasingly used as a source of hematopoietic stem cells for allogeneic transplantation. Previous reports suggest that the low risk of graft-versus-host disease in patients that received cord blood cells seems related to the distinctive nature of cord blood T cells. METHODS: To analyze the maturation of CD4+CD45RA+ cord blood cells, we performed an in vitro differentiation assay to compare the generation of Th effector cells strictly from UCB and adult peripheral blood (APB) CD4+CD45RA+ cells. RESULTS: During the maturation into effector cells, UCB and APB cells acquired a comparable activation level determined by the expression pattern of CD69, CD40L, OX40 and CD62L as well as PD1 and CTLA-4 molecules. Moreover, the expression of CD45RO isoform was induced in most activated effector cells from both UCB and APB. OKT3-restimulated effector cells generated from naive UCB expressed higher levels of CD25 coinciding with the secretion of higher amounts of IL-2. Effector cells from both origins consisted of heterogeneous populations with similar frequencies of Th1 and Th2 cytokine producing cells, secreting equivalent levels of IL-4, IL-5 and IFNgamma. Although, higher levels of IL-10 were detected in the cytokine mRNA profile and in the supernatants of OKT3-restimulated UCB effector cells, blocking endogenous IL-10 with anti-IL-10 mAbs enhanced significantly the proliferative response of UCB as well as APB effector cells (P < 0.05). CONCLUSIONS: These results indicated that Th effector cells generated from naive UCB cells were intrinsically as competent as naive APB to respond to TCR-mediated stimulation. In addition, UCB effector cells produced higher IL-10 but its inhibitory effect on proliferation may be partially compensated by the higher production of IL-2 and enhanced expression of CD25.     

The number of circulating recent thymic emigrants is severely reduced 1 year after a single dose of alemtuzumab in renal transplant recipients

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 < 0.01), but the proportions of central memory, effector memory and terminally differentiated effector memory subsets among CD4+ cells were significantly increased. On the contrary, the proportion and the absolute number of naïve CD4+ T cells, although progressively increasing with time, were severely reduced. In particular, the absolute number of RTEs had only very slight increase with time (P = 0.049) and was dramatically low 1 year after the therapy (P < 0.01 vs. healthy controls; P < 0.05 vs. basiliximab‐treated transplant recipients). These data suggest that a prolonged defective thymic output after AL therapy in renal transplant recipients is one of the main causes of the persistent CD4+ T‐cell lymphopenia observed in these patients.     

Requirements for the induction of allospecific CD8+ suppressor T cells in the rat primary mixed lymphocyte response. CD4+, CD45R+ T cells, or supernatant factor

We examined the requirements for the induction of the MLR-generated allospecific CD8+ suppressor T cells in the rat. Depleting the responder population of CD4+ T cells before initiating the primary MLR abrogates the generation of day-5 CD8+ T suppressor effectors. Readdition of at least 10% CD4+ T cells to the CD4+ depleted primary MLR reconstitutes suppressor cell generation. Using the anti-CD45R monoclonal antibody OX22, we also show that the T suppressor inducer cells are CD4+ CD45R+. Using a dual chamber Transwell culture system, which allows cells to be co-incubated without direct cell-to-cell contact, we show that a soluble factor/s, produced during the course of the primary MLR, is capable of inducing naive CD8+ T cells to become suppressor effectors but only when these CD8 T cells are in direct contact with allogeneic stimulators. Allospecificity is conferred by the stimulator cells and not by the suppressor-inducer factor. The supernatant of day-5 primary MLR is also capable of inducing antigen-specific suppressor effectors from naive CD8+ T cells, and also only in the presence of allogeneic stimulator cells. Recombinant human IL-2, in doses that are up to five times the amount present in the supernatant cultures, is unable to induce suppressor-effector cells from naive CD8+ T cells. We conclude that, to become allospecific suppressor effectors, naive CD8+ T cells require contact with allogeneic stimulator cells and either CD4+ CD45R+ suppressor inducer cells or suppressor inducer factor/s produced during the course of the primary MLR.     

抗原特异性CD4+CD25+T细胞对同种异体胰岛移植影响及作用机制研究

目的:探讨抗原特异性CD4+CD25+Treg细胞免疫对同种异体胰岛急性移植排斥反应的影响和机制。方法:用MACS分选供体抗原特异性CD4+CD25+Treg细胞免疫糖尿病BALB/cByJ受体小鼠，以ICR小鼠胰岛为供体行同种异体胰岛移植。观察移植后小鼠的存活时间、移植前后外周血CD4+和CD8+T细胞亚群的变化和移植物中Th1/Th2细胞因子mRNA表达水平的变化。结果:抗原特异性Treg细胞联合胰岛移植组(C组)胰岛移植物平均生存期为(34.57±17.15)d，显著长于单纯胰岛移植组(B组)的(10.6±1.82) d (P<0.01)；移植后第3天，C组外周血CD4+/CD8+的值显著低于B组(P<0.01)；C组移植物中IL-10,TGF-β mRNA表达比B组显著增强。B组移植物中IL-1β，IL-2及IFN-γ mRNA表达明显强于C组。结论:抗原特异性CD4+CD25+ Treg细胞可通过调节Th2/Th1之间的反应平衡而延长同种异体胰岛移植物的存活时间。     

Decrease in phenotypically defined T helper inducer cells (T4+4B4+) and increase in T suppressor effector cells (T8+2H4+) in stable renal allograft recipients

Two monoclonal antibodies, anti-2H4 and anti-4B4, reciprocally divide the T4+ (CD4+) and T8+ (CD8+) lymphocytes into T4+2H4+, T4+4B4+, T8+2H4+ and T8+4B4+ subsets. The T4+2H4+, T4+4B4+ and T8+2H4+ subsets possess suppressor-inducer, helper-inducer, and suppressor-effector function, respectively, as previously defined in a system of B cell immunoglobulin production. Using monoclonal antibodies, including anti-2H4 and anti-4B4, and flow cytometry, we monitored lymphocyte subpopulations in 66 renal allograft recipients. We found that patients with stable allograft function have a decrease in the percentage of total T4+ lymphocytes from 41.9 +/- 9.5% pretransplant (pre-Tx) to 36.3 +/- 13.9% posttransplant (post-Tx) (P less than 0.05). This decrease was seen mainly in the T4+4B4+ or helper-inducer subset from 20.8 +/- 4.7% (pre-Tx) to 16.0 +/- 6.3% (post-Tx) (P less than 0.005). Patients with stable function were also noted to have an increase in the percentage of total T8+ lymphocytes from 21.3 +/- 10.7% (pre-Tx) to 30.9 +/- 15.4% (post-Tx) (P less than 0.02). Examination of T8 subsets revealed that a statistically significant increase was seen in the T8+2H4+ or suppressor effector subset from 15.5 +/- 9.2% (pre-Tx) to 21.5 +/- 10.2% (post-Tx) (P less than 0.01). Additionally, serial studies on 14 patients revealed an increase in the %T4+2H4+ suppressor-inducer subset from 9.31 +/- 3.64% (pre-Tx) to 15.71 +/- 6.41% (post-Tx) (P less than 0.0025). Since the role of these subsets has not been established in alloimmunity, in vitro allogeneic studies of 2H4-enriched (2H4+) and 2H4-depleted (2H4-) lymphocytes from normal peripheral blood were performed. In the mixed lymphocyte reaction, 2H4+ cells proliferated less than 2H4- cells (cpm ratio 2H4+/2H4-: 0.63-0.84), but 2H4+ cells generated twice as much suppressor activity as 2H4- cells (ratio % suppression 2H4+/2H4-: 1.9-2.3). These results suggest that 2H4+ cells play a role in the suppressor limb of the alloimmune response and that the increase in cells of this phenotype in our transplant population may be responsible for the maintenance of stable allograft function.     

Prediction of the clinical and histological severity of IgA nephropathy by flow cytometry of urinary mononuclear cells

Background. Urinary mononuclear cells (UMCs) were studied in IgA nephropathy (IgAN) in relation to clinical and histopathological parameters. Methods. A two-color flow-cytometry analysis was used to phenotype and quantitate UMCs in patients with IgAN (n = 37) and those with other kidney diseases (n = 27). Results. The IgAN patients had higher numbers of UMCs than the patients with other kidney diseases. Macrophages (CD14+; P < 0.0001) and T lymphocytes (CD3+; P = 0.0001; CD4+, P < 0.0001) were predominantly found. In the IgAN patients, the quantities of urinary CD3+, CD14+ cells correlated with impaired renal function; namely, serum creatinine, creatinine clearance, and urinary protein excretion. The UMCs were associated with histopathological changes such as glomerular segmental lesions (r = 0.511; P < 0.01 for CD3+; r = 0.623, P < 0.0001 for CD4+, and r = 0.552; P < 0.001 for CD14+) and the index of glomerular hypercellularity (r = 0.405; P = 0.01 for CD3+ T cells and r = 0.392, P = 0.01 for CD14+ macrophages). Moreover, the UMCs reflected the severity of pathology, as estimated by the glomerular score (r = 0.424; P < 0.01 for CD3+; r = 0.458; P < 0.01 for CD4+; r = 0.500, P = 0.001 for CD14+ cells). UMCs were weakly correlated with tubulointerstitial changes. Conclusion. UMCs were associated with the clinical stage of IgAN and mirrored the extent of histopathology in the kidney. Flow-cytometry analysis of UMCs may help to predict the course of the disease. Received: March 11, 1998 / Accepted: May 12, 1999     

Abnormal Bone Acquisition With Early‐Life HIV Infection: Role of Immune Activation and Senescent Osteogenic Precursors

Chronic immune activation associated with human immunodeficiency virus (HIV) infection may have negative consequences on bone acquisition in individuals infected with HIV early in life. Bone mineral density (BMD) and microarchitecture were characterized in 38 HIV‐infected men on antiretroviral therapy (18 perinatally‐infected, 20 adolescence‐infected) and 20 uninfected men age 20 to 25 years by dual‐energy X‐ray absorptiometry (DXA), high‐resolution peripheral quantitative computed tomography (HRpQCT). Flow cytometry was utilized to measure CD4+/CD8+ activation (HLADR+CD38+) and senescence (CD28–CD57+) and to quantify circulating osteogenic precursor (COP) cells in peripheral blood mononuclear cells using antibodies to RUNX2 and osteocalcin (OCN). Telomere lengths were measured in sorted COP cells using qPCR. DXA‐derived areal BMD Z‐scores and HRpQCT‐derived volumetric BMD (vBMD) measures were lower in HIV‐infected than uninfected men. Proportion of activated and senescent CD4+ and CD8+ T cells were higher in HIV‐infected than uninfected men. The percentage of COP cells (mean ± SE) was lower in HIV‐infected than uninfected (0.19% ± 0.02% versus 0.43% ± 0.06%; p < 0.0001) men, and also lower in perinatally‐infected than adolescence‐infected men (0.15% ± 0.02% versus 0.22% ± 0.03%; p < 0.04). A higher proportion of COP cells correlated with higher bone stiffness, a measure of bone strength, whereas a higher proportion of activated CD4+ T cells correlated with lower BMD and stiffness and lower proportion of COP cells. T cell activation with HIV‐infection was associated with decreased numbers of osteogenic precursors as well as lower peak bone mass and bone strength. © 2016 American Society for Bone and Mineral Research.     

Intracellular cytokine repertoire in different T cell subsets from patients with sarcoidosis

BACKGROUND: Pulmonary sarcoidosis is characterised by a mononuclear alveolitis with a predominance of CD4+ T cells and macrophages. We determined the intracellular expression of interferon (IFN)gamma, interleukin (IL)-2, tumour necrosis factor (TNF)alpha, IL-4, IL-5 and IL-10 in CD4+ and CD8+, naive and memory lymphocytes from blood and bronchoalveolar lavage (BAL) fluid using three colour flow cytometry. METHODS: Eighteen untreated patients with pulmonary sarcoidosis were evaluated and stratified according to whether they had acute or chronic disease. RESULTS: Significantly more T cells expressed Th1 than Th2 type cytokines in both BAL fluid and peripheral blood samples, regardless of clinical presentation. Significantly greater proportions of T cells secreted Th1 type cytokines in BAL fluid than in peripheral blood. Th1 type cytokines were more frequently expressed by peripheral and alveolar T cells in acute disease than in chronic disease. There were no significant differences between CD4+ and CD8+ T cells. Concerning naive and memory lymphocytes, significantly higher CD45RO:CD45RA ratios were found in BAL fluid than in blood, and increased expression of Th2 type cytokines was found in peripheral compared with alveolar memory T cells. CONCLUSIONS: Our data support the immunopathogenetic concept of Th1/Th2 imbalance and compartmentalisation in pulmonary sarcoidosis and suggest that the cytokine patterns change during the course of disease. Expression of Th2 type cytokines in memory lymphocytes is decreased in the alveolar compartment compared with peripheral blood.     

CD31+ Naïve Th Cells Are Stable during Six Months Following Kidney Transplantation: Implications for Post-transplant Thymic Function

Little is known about thymus function in transplant patients. Until recently, the phenotype of T cells that recently emigrated the thymus was unknown. Now it has been demonstrated that CD4(+) recent thymus emigrants coexpress CD31 and CD45RA. Here, we investigated whether uremia and immunosuppression influence CD31(+) CD45RA(+) Th cells before and after kidney transplantation, respectively. Forty-eight renal transplant patients were included receiving either standard triple/quadruple (n = 35) immunosuppression, OKT-3 induction (n = 7) or FTY-720 (n = 6), respectively. Peripheral CD31(+) CD45RA(+) Th cells were quantified flowcytometrically before and at week 1, 4, 12 and 24 post-transplantation. Thirty-nine healthy adults served as controls. CD31(+) CD45RA(+) Th cells correlated inversely with age in patients and controls and were comparable in patients before transplantation and age-matched controls. Importantly, CD31(+) CD45RA(+) Th cell frequencies remained stable during 6 months post-transplantation. In conclusion, CD31(+) CD45RA(+) Th cells are not significantly altered by uremia before and during 6 months of immunosuppressive therapy after kidney transplantation. Implications for thymus function are discussed.     

Comparison of Blood and Peritoneal Lymphocytes from Continuous Ambulatory Peritoneal Dialysis Patients,Asymptomatic and with Peritonitis

Objective. The purpose of this study was to compare the characteristics of the blood immunophenotype of CAPD patients with and without peritonitis and to compare the phenotypes of peripheral blood lymphocytes (PBL) and peritoneal lymphocytes (PL) in CAPD patients with peritonitis. Methods. Fifty-seven CAPD patients (20 with peritonitis and 37 without peritonitis) were recruited in the study (mean age 66,88 ± 13,48, male/female 16/21). Lymphocyte subsets (CD2+, CD3+, CD3+/4+, CD3+/8+, CD3-/16 + 56+, CD4/CD8 ratio) were quantitated by using monoclonal antibodies and dual-color flow cytometric analysis. With the above method we measured PBL in patients with and without peritonitis. In patients with peritonitis we also measured PL. Results. CD2 were slightly decreased in patients with peritonitis. Those patients also had more intense CD3 + /CD4+ lymphopenia (p < 0.05) and larger expansion of NK cells (p < 0.05). Patients with peritonitis appeared to have a lower ratio of CD4/CD8 (p < 0.05). All the above results are shown to . Following the onset of peritonitis, a consistent finding in all patients was a significant increase in CD2 population of PL compared with PBL (85.71 ± 9.20 versus 82.60 ± 7.34, p < 0.05) as well as in CD3 population (77.01 ± 13.09 versus 68.74 ± 13.43, p < 0.05). An increased number of CD3/8 in PL compared with PBL (33.70 ± 9.34 versus 27.98 ± 10.77, p < 0.05) was also noted. Conclusions. In the present study, we found important immune activation in asymptomatic CAPD patients. The activation increases during peritonitis. The causes and the clinical consequences of chronic activation remain unknown.     

Differentiation of human alloreactive CD4+ and CD8+ T cells in vitro

BACKGROUND: Recent studies have revealed that, during viral infection, ordered phenotypic and functional changes occur in human antigen-specific T cells. We analyzed whether a similar differentiation program is induced after alloantigen stimulation in vitro. METHODS: Peripheral blood mononuclear cells and purified CD4(+)CD45RA+, CD4(+)CD45RO+, and CD8+ T cells from healthy individuals were labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). Cells were co-cultured with allogeneic irradiated cells. Flow cytometric analysis was performed on days 3, 5, and 7 of culture using surface CD45RA, CD27, CD28, CCR7, and intracellular perforin and granzyme B markers in relation to CFSE dilution. RESULTS: Based on the decrease in CFSE fluorescence, both CD4+ and CD8+ T cells showed an early and vigorous response to allogeneic stimulation. Loss of CD45RA expression and up-regulation of CD27 and CD28 costimulatory molecules was an early event occurring in the first generations of dividing cells. Differentiation at later stages of proliferation was characterized by loss of CCR7 homing receptor expression, more pronounced in CD4+ than in CD8+ T cells, indicating the decreased ability of these cells to traffic to secondary lymphoid organs. Production of the cytotoxic effector molecules perforin and granzyme B increased with the number of cell divisions. CONCLUSIONS: Our data thus show that short-term phenotypic and functional changes of alloreactive T cells follow the differentiation pattern seen in the early stages of an antiviral immune response.     

Inhibition of osteoclast differentiation and matrix metalloproteinase production by CD4+CD25+ T cells in mice

Summary

To reveal the mechanism of inflammation-accompanied osteoporosis, mouse CD11b cells and CD4+CD25+ T cells were cocultured. Our results showed that CD11b+ monocytes cocultured with CD4+CD25+ T cells generated significantly less osteoclasts than those cocultured with CD4+CD25? T cells. The levels of MMP-7 and 9 in coculture supernatant were decreased significantly when CD11b+ monocytes and CD4+CD25+ T cells were cocultured comparing to the CD4+CD25? T cell group. These results highlight the role of CD4+CD25+ T cells on rheumatic osteoporosis.

Introduction

The purpose of this study was to reveal the mechanism of inflammation-accompanied osteoporosis by examining the effect of CD4+CD25+ T cells on osteoclast formation, as well as the production of matrix metalloproteinases (MMP)-2, 3, 7, and 9 from osteoclasts.

Methods

Mouse CD11b cells and CD4+CD25+ T cells were isolated using the microbead-based kits. Activated CD11b cells and CD4+CD25+ T were cocultured. CD4+CD25? T cells were used as controls. Osteoclasts were evaluated by counting the average of tartrate-resistant acid phosphatase-positive multinucleated large cells among five high-power fields (×200). After 2 days of coculture, supernatants were harvested for MMP measurement (by ELISA).

Results

At day 7, the CD11b+ monocytes cocultured with CD4+CD25+ T cells generated significantly (both p?<?0.01) less osteoclasts (3.4?±?0.8) than those cocultured with CD4+CD25? T cells (16.2?±?1.3) and those cultured alone (16.2?±?2.7). The supernatant levels of MMP-7 and 9 were decreased significantly (p?<?0.01) when CD11b+ monocytes and CD4+CD25+ T cells were cocultured compared to the other two conditions.

Conclusions

The decreased CD4+CD25+ T cells may cause an overproduction of MMP-7 and 9 from osteoclasts, which highlight the role of CD4+CD25+ T cells on rheumatic osteoporosis.     

Immune Reconstitution Following Rabbit Antithymocyte Globulin

Depletional induction therapies are routinely used to prevent acute rejection and improve transplant outcome. The effects of depleting agents on T‐cell subsets and subsequent T‐cell reconstitution are incompletely defined. We used flow cytometry to examine the effects of rabbit antithymocyte globulin (rATG) on the peripheral T‐cell repertoire of pediatric and adult renal transplant recipients. We found that while rATG effectively depleted CD45RA+CD27+ naïve and CD45RO+CD27+ central memory CD4+ T cells, it had little effect on CD45RO+CD27? CD4+ effector memory or CD45RA+CD31?, CD45RO+CD27+ and CD45RO+CD27? CD8+ T cell subsets. When we performed a kinetic analysis of CD31+ recent thymic emigrants and CD45RA+/RO+ T cells, we found evidence for both thymopoiesis and homeostatic proliferation contributing to immune reconstitution. We additionally examined the impact of rATG on peripheral CD4+Foxp3+ T cells. We found that in adults, administration of rATG‐induced peripheral expansion and new thymic emigration of T cells with a Treg phenotype, while CD4+Foxp3+ T cells of thymic origin predominated in children, providing the first evidence that rATG induces Treg in vivo. Collectively our data indicate that rATG alters the balance of regulatory to memory effector T cells posttransplant, providing an explanation for how it positively impacts transplant outcome.     

Th1/Th2 balance in childhood idiopathic nephrotic syndrome

AIMS: In view of the conflicting evidence of helper T cell type 1 (Th1) or type 2 (Th2) pattern of cytokine synthesis in childhood idiopathic nephrotic syndrome (INS) this study examined the balance of Th1 and Th2 which are characterized by intracellular cytokine production of interferon-gamma (IFNgamma) and interleukin-4 (IL-4), respectively. SUBJECTS AND METHODS: Sixteen children with steroid-sensitive INS (mean age 9.0 years) were included in this study, together with 15 healthy normal children (mean age 7.9 years) for the control group. Intracellular production of both IFNgamma and IL-4 in helper T cell (CD4+ cell) was investigated by a 3-color flow cytometry. RESULTS: The cross-sectional data showed no significant differences of percentages of Th0 (IFNgamma+ IL-4+ CD4+ cell), Th1 (IFNgamma+ lL-4- CD4+ cell) and Th2 (IFNgamma- IL-4+ CD4+ cell) in CD4+ cells (p > 0.05). The Th1/Th2 ratio during nephrotic relapse did not differ from those during nephrotic remission and in normal healthy children (p > 0.05). CONCLUSION: We conclude that there is no significant skew of Th1/Th2 balance in childhood INS and that the cardinal immunological abnormality does not lie in helper T cells but in other cells, such as suppressor/cytotoxic T cells, natural killer cells or monocytes/macrophage. To clarify the pathogenesis of INS, comprehensive studies for these cells would be worthwhile.     

Vitamin D deficiency, CD4+CD28null cells and accelerated atherosclerosis in chronic kidney disease

Aim: Cardiovascular disease (CVD) is the leading cause of death among chronic kidney disease (CKD) patients. The role of vitamin D remains controversial in this process. We evaluated the relationship between 25‐hydroxyvitamin D, abnormal T helper cells (CD4+CD28null cells), systemic inflammation and atherosclerosis in CKD patients. Methods: A total of 101 stage 4–5 non‐dialysis CKD patients and 40 healthy controls were studied. Common carotid artery intima media thickness (CCA‐IMT) was measured with an ultrasound system. 25(OH) vitamin D and highly sensitive C‐reactive protein (hsCRP) were measured in serum by enzyme linked immunosorbent assay. The frequency of circulating CD4+CD28null cells was evaluated by flowcytometry. Results: CKD subjects exhibited higher CCA‐IMT (0.71 ± 0.01 vs 0.56 ± 0.01 mm, P < 0.0001), hsCRP (90.7 ± 5.8 vs 50.1 ± 8.6 µg/mL, P < 0.0001), CD4+CD28null cell frequency (9.1 ± 0.9 vs 3.6 ± 0.5%, P < 0.0001) and lower 25(OH) vitamin D levels (17.9 ± 1.9 vs 26.9 ± 3.5 ng/mL, P < 0.0001). In CKD subjects, serum 25 (OH) vitamin D level showed a strong inverse correlation with CCA‐IMT (r = ?0.729, P < 0.0001) and correlated with CD4+CD28null cell frequency (r = ?0.249, P = 0.01) and hsCRP (r = ?0.2, P = 0.047). We also noted correlation of IMT with patient age (r = 0.291, P = 0.004) and CD4+CD28null cells (r = 0.34, P = 0.001). On multiple regression analysis, 25(OH) vitamin D level, diabetic status and CD4+CD28null cell frequency exhibited independent association with IMT in CKD subjects. Conclusions: Vitamin D deficiency, inflammatory activation and higher frequency of CD4+CD28null T lymphocyte population correlate with preclinical atherosclerotic changes in CKD population. These findings suggest possible linkage between vitamin D metabolism and T cell modulation – abnormalities that may contribute to development of atherosclerosis in CKD.     

Rat Renal Interstitial Fibroblasts Affect the Th1/Th2 Profile In Vitro

Background: T helper 1 (Th1)/T helper 2 (Th2) profile is pivotal in the development of fibrosis. Renal interstitial fibroblasts, which play a central role in the development of renal interstitial fibrosis, have an intimate relation with lymphocytes. However, there is little knowledge of the effect of fibroblasts on the profile of CD4 T-lymphocyte subsets. Methods: After coculture with rat renal interstitial fibroblasts, the proportions of Th1 and Th2 cells in CD4 T lymphocytes and the apoptosis rates of the two subsets were detected by flow cytometry. Galectin-9 expression in rat renal interstitial fibroblasts was detected by immunofluorescence, Western blotting, and enzyme-linked immunosorbent assay. Results: After 48 h of coculture, rat renal interstitial fibroblasts increased the proportion of Th2 cells, lowering the ratio of Th1/Th2. Meanwhile, interferon-gamma production in Th1 cells was inhibited and interleukin-4 production in Th2 was promoted. After coculture with activated rat renal interstitial fibroblasts for 24 h, apoptosis of Th1 was more highly promoted than that of Th2 cells. In addition, rat renal interstitial fibroblasts induced stronger Th2 cell differentiation than that of Th1 cells in vitro. Rat renal interstitial fibroblasts expressed but did not secrete galectin-9 (an apoptosis-inducing factor for Th1 cells) in vitro and the expression level decreased when cocultured with CD4 T lymphocytes. Conclusions: Rat renal interstitial fibroblasts shift the Th1/Th2 profile in vitro, and this may be another pathway by which renal interstitial fibroblasts promote fibrosis.     

Isolation, expansion and functional assessment of CD4+CD25+FoxP3+ regulatory T cells and Tr1 cells from uremic patients awaiting kidney transplantation

Background

The immunosuppressive properties of regulatory T cells have emerged as an attractive tool for the development of immunotherapies in various disease contexts, e.g. to treat transplantation induced immune reactions. This paper focuses on the process of obtaining and functionally characterizing CD4+CD25+FoxP3+ regulatory T cells and Tr1 cells from uremic patients awaiting kidney transplantation.

Methods

From October 2010 to March 2011 uremic patients awaiting living donor kidney transplantation, and their corresponding kidney donors, were enrolled in the study. A total of seven pairs were included. Isolation of CD4+CD25+FoxP3+ regulatory T cells was performed by magnetic activated cell sorting of peripheral blood mononuclear cells obtained from the uremic patients. Donor specific Tr1 cells were differentiated by repetitive stimulation of immature CD4+ T cells with immature dendritic cells, with the T cells coming from the future kidney recipients and the dendritic cells from the corresponding kidney donors. Cells were then expanded and functionally characterized by the one-way mixed leukocyte reaction and assessment of IL-10 production. Phenotypic analysis was performed by flow cytometry.

Results

The fraction of CD4+CD25+FoxP3+ regulatory T cells after expansion varied from 39.1 to 50.4% and the cells retained their ability to substantially suppress the mixed leukocyte reaction in all but one patient (3.8-19.2% of the baseline stimulated leukocyte activity, p < 0.05). Tr1 cells were successfully differentiated from all but one patient and produced high levels of IL-10 when stimulated with immature dendritic cells (1,275-11,038% of the baseline IL-10 secretion, p < 0.05).

Conclusion

It is practically feasible to obtain and subsequently expand CD4+CD25+FoxP3+ regulatory T cells and Tr1 cells from uremic patients without loss of function as assessed by in vitro analyses. This forms a base for adoptive regulatory T cell therapy in the setting of living donor kidney transplantation.     

Retinoic acid attenuates acute heart rejection by increasing regulatory T cell and repressing differentiation of Th17 cell in the presence of TGF‐β

Retinoic acid (RA), in a transforming growth factor beta (TGF‐β)‐dependent manner, promotes differentiation of regulatory T cells (Tregs) but inhibits the differentiation of Th17 cells in vitro from naive CD4⁺T cells. In addition, transfer of induced Tregs (iTregs) reduces rejection. We therefore examined whether RA could attenuate acute cardiac transplant rejection in vivo in a mouse model by regulating the reciprocal differentiation of Tregs and Th17 cells. The iTregs and naive T cells were respectively transferred into congenic mice. Two weeks later, the percentages of transferred cells and Forkhead box P3 (FoxP3)+ Tregs were measured in spleen. Mice with cardiac transplants were treated with TGF‐β alone, RA alone, both or none. The percentage of Tregs or Th17 cells in CD4⁺T cells, the level of FoxP3 protein or serous interleukin (IL)‐17A, or suppressive function of Tregs from recipient mice were assessed. The percentage of Th17 cells and level of serum IL‐17A both increased significantly during acute rejection. RA favored differentiation to Tregs over Th17 cells. Unlike naive T cells, only a few transferred iTregs remained after transfer. Treatment with RA plus TGF‐β prolonged graft survival, increased the percentage of Tregs, and decreased the percentage of Th17 cells in peripheral T cells. Tregs from all recipients had normal suppressive function. In conclusion, treatment with RA plus TGF‐β attenuates acute rejection by promoting the differentiation of Tregs and inhibiting the differentiation of Th17 cells.