Altered balance between effector T cells and FOXP3+HELIOS+ regulatory T cells after thymoglobulin induction in kidney transplant recipients

This study examined the effect of thymoglobulin induction therapy on leukocyte population dynamics in kidney transplant patients. Patients receiving standard immunosuppression were compared with those who received additional thymoglobulin at the time of kidney transplantation. Thymoglobulin induction led to an immediate and significant decrease of all T cells and NK cells, but not B cells or monocytes. CD8⁺ T cells recovered to near pretransplant level by 4 weeks post‐transplant. CD4⁺ T cells remained at less than 30% of pretransplant level for the entire study period of 78 weeks. Both CD4⁺ and CD8⁺ T cells showed reduced cytokine production after recovery. Deletion of CD4⁺FOXP3⁺HELIOS⁺ regulatory T cells (Tregs) was less profound than that of CD4⁺FOXP3^? cells, thus the relative percentage of Tregs elevated significantly when compared with pretransplant levels in thymoglobulin‐treated patients. In contrast, the percentages of Tregs and their expression of FOXP3 in the standard immunosuppression group decreased steadily and by 12 weeks after transplant the average percentage of Tregs was 56% of the pretransplant level. Thus, thymoglobulin‐induced deletion of T cells led to significant and long‐lasting alterations of the T‐cell compartment characterized by a preservation of Tregs and long‐lasting reduction in CD4⁺, and potentially pathogenic, T cells.     

Melanoma induces immunosuppression by up-regulating FOXP3(+) regulatory T cells

BACKGROUND: The immune response to melanoma is rarely curative, suggesting the emergence of immunosuppression. FOXP3-expressing regulatory T cells (T(reg) cells) function to suppress immune responses. The objective of this study was to determine if melanoma evades immune surveillance, in part, by inducing T(reg) cells. MATERIAL AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated and exposed to melanoma-conditioned media (MCM) or control media for 1 week. The induction of T(reg) cells in these PBMCs was determined by measuring the proportion of CD25(+)FOXP3(+) T cells in all CD4(+) T cells by flow cytometry. FOXP3 expression was determined by mean fluorescence intensity (MFI) and Western blot. Supernatant cytokines were determined by ELISA. RESULTS: Normal PBMCs exposed to MCM revealed higher proportions of T(reg) cells than those exposed to control media after 6 days (3.4% versus 1.3%, respectively, P < 0.02). The expression of FOXP3 in T(reg) cells from PBMCs exposed to MCM increased over time by MFI and Western blot but was not significantly different than those exposed to control media. The level of IL-10 and TGF-beta in supernatants after 6 days growth was higher in MCM than control media, but this did not reach statistical significance. CONCLUSION: Exposure of PBMCs to melanoma results in induction of FOXP3(+) T(reg) cells.     

Impact of Basiliximab on regulatory T‐cells early after kidney transplantation: down‐regulation of CD25 by receptor modulation

Monoclonal anti‐CD25‐antibodies are successfully applied in organ transplantation to reduce the incidence of acute graft rejection. However, targeting the CD25 molecule might not only affect activated T‐cells but also regulatory T‐cells (T_regs) constitutively expressing the CD4⁺CD25⁺CD127^lowFoxP3⁺ phenotype. In this study, we investigated the influence of the anti‐CD25‐antibody Basiliximab on the frequency of T_regs early after kidney transplantation comparing individuals receiving/not receiving induction therapy (n = 14 and n = 7). Following Basiliximab administration, a distinct loss of CD4⁺CD25^high T‐cells was observed lasting for at least 6 weeks. This was not accompanied by a disappearance of the entire CD4⁺CD25⁺FoxP3⁺ T_regs but rather a decreased expression density of CD25 on the latter. In addition, a transient rise in CD4⁺CD25^?FoxP3⁺ T‐cells was found which expressed the CD127^low phenotype. Thus, a phenotypic shift of T_regs from the CD25⁺ to the CD25^? compartment was suggested. This was supported by in vitro findings showing that the disappearance of CD4⁺CD25^high cells in the presence of Basiliximab was due to down‐regulation of CD25 expression meanwhile the suppressive function of these cells was maintained. In conclusion, Basiliximab therapy directly affects CD4⁺CD25⁺CD127^lowFoxP3⁺ T_regs but does not seem to be associated with functional consequences. Thus, it is unlikely that Basiliximab treatment negatively influences strategies involving T_regs to promote tolerance after organ transplantation.     

Costimulatory blockade with mTor inhibition abrogates effector T‐cell responses allowing regulatory T‐cell survival in renal transplantation

The advent of novel immunosuppressive strategies in renal transplantation, with immunomodulatory properties, might facilitate long‐term allograft survival. T‐cell depletion, costimulation‐blockade and mTor inhibition have been shown to favour anti‐donor hyporesponsiveness. Recently, the combination of rATG, belatacept (Bela) and sirolimus (SRL) has been used in kidney transplantation, showing very low incidence of acute rejection and excellent 12‐month graft and patient survival. Herein, we have analysed the 1‐year evolution of memory/effector and regulatory T cells and assessed the donor‐specific T‐cell alloimmune response in a group of these patients and compared with others treated with a calcineurin‐inhibitor(CNI)‐based (rATG/tacrolimus/MMF), and two other Bela‐based regimens (rATG/Bela/MMF and basiliximab/Bela/MMF/steroids). During the first year after transplantation, patients receiving rATG/Bela/SRL had significantly higher percentage of Tregs upon the memory T‐cell compartment and showed a potent anti‐donor suppressive activity. In an in vitro naive and memory/effector T‐cell co‐culture, the combination of costimulation‐blockade and SRL could abrogate both antigen‐specific T‐cell responses as efficiently as using a CNI drug. The combination of T‐cell depletion, costimulation‐blockade and mTor inhibition seems to be able to allow Treg survival and inhibit donor‐specific alloreactive effector immune responses after kidney transplantation in humans.     

Hepatic encephalopathy and post‐transplant hyponatremia predict early calcineurin inhibitor‐induced neurotoxicity after liver transplantation

Early calcineurin inhibitor‐induced neurotoxicity (ECIIN) is considered when neurological symptoms occur within 4 weeks after liver transplantation (LT). Risk factors and clinical outcome of ECIIN remain largely unknown. We sought to estimate the incidence, risk factors, and outcome of ECIIN after LT. We retrospectively evaluated 158 patients that underwent LT in a 2‐year period and received immunosuppression with calcineurin inhibitors (CNI) and prednisone. ECIIN was considered when moderate/severe neurological events (after excluding other etiologies) occurred within 4 weeks after LT and improved after modification of CNI. Demographic and clinical variables were analyzed as risk factors. Twenty‐eight (18%) patients developed ECIIN and the remaining 130 patients were analyzed as controls. History of pre‐LT hepatic encephalopathy (OR 3.16, 95% CI 1.29–7.75, P = 0.012), post‐LT hyponatremia (OR 3.34, 95% CI 1.38–9.85, P = 0.028), and surgical time >7 h (OR 2.62, 95% CI 1.07–6.41, P = 0.035) were independent factors for ECIIN. Acute graft rejection and infections were more frequent in the ECIIN group. In addition, length of stay was longer in ECIIN patients. In conclusion, pre‐LT hepatic encephalopathy, surgical time >7 h, and post‐LT hyponatremia are risk factors for ECIIN. Clinical complications and a longer hospital stay are associated with ECIIN development.     

CD4+CD25+FOXP3+T细胞在肝癌肝移植肿瘤复发中的作用

目的 探讨肝癌肝移植病人移植前后外周血和肿瘤组织中CD4+CD25+FOXP3+T细胞比例变化及其临床意义.方法 用流式细胞仪检测肝癌肝移植病人和其他肝移植病人术后外周血中CD4+CD25+FOXP3+T细胞的比例,并采用正常人作对照.用免疫组化法检测肝癌病人和非肝癌病人肿瘤组织中FoxP3的表达及CD8+T细胞浸润的比例.观察肝癌肝移植病人术后及肿瘤复发后调节性T细胞的变化及其对肿瘤复发的影响.结果 流式细胞检测显示肝癌肝移植、非肝癌肝移植的病人术后外周血中CD4+CD25+FOXP3+T细胞占CD4+T细胞的比例较正常人明显升高,分别为(10.15±1.00)%、(5.30±1.64)%和(3.20±1.18)%,P<0.05.肝癌肝移植肿瘤复发病人较未复发病人外周血CD4+CD25+FOXP3+T比例明显升高,分别为(15.15±1.50)%和(6.80±1.50)%,P<0.01.免疫组化检测显示肿瘤组织中FOXP3+T细胞增多,CD8+T细胞浸润明显减少.结论 肝癌肝移植肿瘤复发的病人外周血中CD4+CD25+FOXP3+T细胞比例升高.调节性T细胞可能通过减少CD8+T细胞浸润,加速肿瘤复发.     

早期肝癌术后患者调节性T细胞数量及功能变化

目的通过分析早期肝癌患者肝癌切除手术前、后外周血中CD4+CD25+FOXP3+调节性T细胞(Treg)数量及功能的变化,从免疫抑制角度探讨手术对肝癌患者免疫功能的影响。方法采集肝癌患者(病例组,15例)手术治疗前、后及正常人群(对照组,5例)外周血,提取淋巴细胞,行细胞外(CD4、CD25)及细胞内(FOXP3)染色,应用流式细胞仪分析Treg的数量及功能。结果 病例组CD4+CD25+T细胞和CD25+FOXP3+T细胞在术前外周血中所占比例均明显高于正常对照组〔(12.43±2.57)%比(5.56±1.02)%,(5.14±1.4)%比(2.18±0.83)%,P<0.05〕,病例组以上两种细胞在术后1周﹝(10.56±2.13)%,(4.28±1.08)%﹞较术前均有所下降,但差异无统计学意义(P>0.05),而术后2周〔(7.30±0.89)%,(3.43±0.83)%〕较术前下降明显(P<0.05)。病例组CD8+T细胞和CD4+CD25-T细胞在术前外周血中所占比例明显低于正常对照组〔(23.42±1.80)%比(29.22±2.26)%,(36.14±1.12)%比(43.69±2.78)%,P<0.05〕,病例组以上两种细胞在术后2周〔(27.15±1.71)%,(40.30±2.00)%〕较术前均明显升高(P<0.05)。对Treg与AFP进行相关性分析发现二者具有低度相关关系(r=0.48,P<0.05)。结论肝癌切除术能够在一定程度上改善肝癌患者的免疫功能,且Treg在肝癌的诊治及预后的判断中,可能具有一定辅助意义。     

Foxp3 regulates human natural CD4+CD25+ regulatory T‐cell‐mediated suppression of xenogeneic response

Sun L, Yi S, O’Connell PJ. Foxp3 regulates human natural CD4+CD25+ regulatory T‐cell‐mediated suppression of xenogeneic response. Xenotransplantation 2010; 17: 121–130. © 2010 John Wiley & Sons A/S. Abstract: Backgrounds: Cellular rejection of xenografts is predominantly mediated by CD4+ T cells. Foxp3 expressing human naturally occurring CD4+CD25+ regulatory T cells (nTregs) have been shown to suppress pathological and physiological immune responses, including the CD4+ T‐cell‐mediated anti‐pig xenogeneic response in vitro. Although Foxp3 is required for nTreg development and their function, the precise role of Foxp3 in regulating Treg suppressive function in xenoimmune response remains to be identified. Methods: In vitro expanded human nTregs were transfected with fluorescein isothiocyanate ‐conjugated Foxp3 small interfering RNA (siRNA) by Lipofectamine 2000. Transfected nTregs were sorted by fluorescence‐activated cell sorting, and then analyzed for Foxp3 gene and protein expression as well as their phenotypic characteristics. Human CD4+CD25? T cells were stimulated with xenogeneic pig peripheral blood mononuclear cell in the presence or absence of nTregs in a coculture or transwell system for evaluation of nTreg suppressive activity. The production of effector cytokines by xenoreactive CD4+CD25? T cells as well as suppressive cytokine by nTregs in their cocultures was examined by ELISA. Results: The siRNA‐mediated Foxp3 knockdown resulted in impaired nTreg anergic state, downregulated expression of nTreg function associated molecules, and reduced production of suppressive cytokines by nTregs, which together leading to impaired nTreg‐mediated suppression of CD4+CD25? T‐cell proliferation and their effector cytokine production in response to xenogeneic stimulation. Conclusions: This study demonstrates that Foxp3 expression is required for human nTregs to maintain their suppressive function in the xenoimmune response.     

Uremia‐associated immunological aging is stably imprinted in the T‐cell system and not reversed by kidney transplantation

The uremia‐induced inflammatory environment in end‐stage renal disease (ESRD) patients is associated with premature T‐cell aging resulting in a defective T‐cell immunity. As kidney transplantation (KTx) reduces the pro‐inflammatory environment, we hypothesized that KTx would rejuvenate the aged T‐cell system. As aging parameters, we determined in 70 KTx recipients the differentiation status by immunophenotyping, thymic output by the T‐cell receptor excision circle (TREC) content together with CD31⁺ naïve T‐cell numbers and the relative telomere length (RTL) as a measure for proliferative history at pre‐KTx, 3, 6 and 12 months post‐KTx. In addition, T‐cell function was determined by measuring the proliferative capacity and percentages of cytokine‐producing cells. Directly post‐KTx, memory T‐cell numbers were diminished but restored to pre‐KTx values at 12 months, except for CD4⁺EM T cells. The RTL of (memory) CD4⁺ and CD8⁺ T cells did not change. In contrast, TREC content and CD31⁺ naïve T‐cell numbers were stable post‐KTx although the RTL of naïve CD4⁺ and CD8⁺ T cells decreased implying homeostatic proliferation of naïve cells, in response to a temporary decrease in memory cells. The T‐cell function was not improved post‐KTx. Our findings demonstrate that the uremia‐associated aged phenotype is stably imprinted in the T‐cell system and not reversed by KTx.     

Corticosteroid avoidance in adult kidney transplant recipients under rabbit anti‐T‐lymphocyte globulin,mycophenolate mofetil and delayed cyclosporine microemulsion introduction

We conducted the first prospective, randomized, open‐label multicenter study in low‐immunologic risk adult recipients of primary cadaver kidney transplants receiving rabbit anti‐T‐lymphocyte globulin, mycophenolate mofetil, cyclosporine microemulsion introduced on day 5, with and without corticosteroids. Patients were randomly assigned according to age and cold ischemia time to receive corticosteroids for at least 6 months or no corticosteroids at all. The main efficacy evaluation criterion was acute rejection (including all treated episodes and those biopsy‐confirmed) during the first year following transplantation. For this purpose, this report includes the actual results of the whole 12‐month follow‐up of all randomized patients. For efficacy analysis, 98 patients were evaluated in the Steroid avoidance group and 99 in the Steroid maintenance group. Taken as a whole, 81% of the patients (n = 159) never received anti‐rejection treatment. From the 38 patients who received anti‐rejection treatment, 25 (25.5%) were in the Steroid avoidance group and 13 (13.1%) in the Steroid maintenance group (P < 0.031), experiencing respectively 17 (17.3%) and 7 (7.1%) biopsy‐proven first episodes of acute rejection (P < 0.031). Borderline changes (6 vs. 3) were not considered as biopsy‐proven acute rejections. Onset of first rejection was significantly shorter in the Steroid avoidance group (P < 0.027). First‐line anti‐rejection treatment response, need for any rescue therapy, as well as histologic severity of rejection episodes did not statistically differ between the groups. One‐year post‐transplantation analysis showed no differences in delayed graft function, serum creatinine, creatinine clearance, 24‐h proteinuria, as well as serious adverse events between the groups. De novo diabetes (P < 0.07) or dyslipidemia (P < 0.01) as well as newly diagnosed malignancies (P < 0.059) were however more frequently observed in the Steroid maintenance group. At the end of the first post‐transplant year, 99% of patients in the Steroid avoidance group and 97% of patients in the Steroid maintenance group were respectively alive (P = 0.34), with respectively 95% and 93.2% of functioning kidney grafts (P = 0.62). Our results showed that total avoidance of corticosteroids from the day of transplantation was associated with a significantly increased number of clinically diagnosed and treated, and biopsy‐proven acute rejections during the first year of transplantation. Nevertheless, overall outcome, 1‐year patient and graft survival as well as renal function were similar, and the patients in the Steroid avoidance group exhibited a lower incidence of de novo dyslipidemia, diabetes mellitus and malignancies often associated with steroid treatment (Clinical Trials.gov NCT00200551).     

Predictive factors of allosensitization in renal transplant patients switched from calcineurin to mTOR inhibitors

Conversion of kidney‐transplant recipients from calcineurin inhibitors to mTOR inhibitors has been suggested to be a risk factor for increased alloimmune response. We have analyzed the development of new HLA‐antibodies (HLA‐Abs) early after conversion in 184 patients converted in stable phase at our hospital and compared with a control group of nonconverted comparable 63 transplants. Using single‐antigen solid‐phase immunoassay analysis, a preconversion and a 3–6 months postconversion sera were prospectively analyzed in every patient for the appearance of new HLA‐Abs. Renal function at 2 years postconversion and cumulative graft survival were compared between groups. In 16 patients, new HLA‐Abs (3‐DSA and 13‐NonDSA), not present at the moment of conversion, were detected (8.7% vs. 3.1% in the control group). The type of mTORi used, type of CNI preconversion, the presence of steroids, time of conversion, or indication for conversion did not have influence on this effect but the presence of HLA‐Abs before conversion highly correlated with the appearance of new specificities. Patients with de novo HLA‐Abs showed a trend to worst graft function and survival. In conclusion, conversion to mTORi can be followed by early appearance of de novo HLA‐Abs, especially in patients with HLA‐Abs preconversion, and this complication should be screened early after conversion.     

FOXP3+ regulatory T cells in normal prostate tissue,postatrophic hyperplasia,prostatic intraepithelial neoplasia,and tumor histological lesions in men with and without prostate cancer

Background

The tumor promoting or counteracting effects of the immune response to cancer development are thought to be mediated to some extent by the infiltration of regulatory T cells (T_regs). In the present study we evaluated the prevalence of T_reg populations in stromal and epithelial compartments of normal, post atrophic hyperplasia (PAH), prostatic intraepithelial neoplasia (PIN), and tumor lesions in men with and without prostate cancer.

Methods

Study subjects were 102 men consecutively diagnosed with localized prostate cancer undergoing radical prostatectomy and 38 men diagnosed with bladder cancer undergoing cystoprostatectomy without prostate cancer at the pathological examination. Whole mount sections from all patients were evaluated for the epithelial and stromal expression of CD4⁺ T_regs and CD8⁺ T_regs in normal, PAH, PIN, and tumor lesions. A Friedmańs test was used to investigate differences in the mean number of T_regs across histological lesions. Logistic regression was used to estimate crude and adjusted odds ratios (OR) for prostate cancer for each histological area.

Results

In men with prostate cancer, similarly high numbers of stromal CD4⁺ T_regs were identified in PAH and tumor, but CD4⁺ T_regs were less common in PIN. Greater numbers of epithelial CD4+ T_regs in normal prostatic tissue were positively associated with both Gleason score and pT‐stage. We observed a fourfold increased risk of prostate cancer in men with epithelial CD4⁺ T_regs in the normal prostatic tissue counterpart.

Conclusions

Our results may suggest a possible pathway through which PAH develops directly into prostate cancer in the presence of CD4⁺ T_regs and indicate that transformation of the anti‐tumor immune response may be initiated even before the primary tumor is established.

     

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.