Successive emergence of two CD 8 subsets in primary CMV infection of allograft recipients

Abstract Allograft recipients with cytomegalovirus (CMV) infection develop increased proportions of circulating CD8 lymphocytes. A longitudinal study of 11 kidney and 5 liver allograft recipients with primary CMV infection but no other aetiological factor to explain graft dysfunction revealed selective imbalances in peripheral blood CD8' T cell subsets. Initially, CMV viraemia was associated with elevated CD8+bright' T cell numbers and T cell activation. Activation markers fell to normal when viral cultures became negative (before the end of the 1st month). During the 2nd-6th months, most (12/16) patients continued to have high CD8⁺ T cell counts (1050–2900 CD8⁺ cells/mm3), comprising an uncommon CD8⁺ T cell subset, as 45–73% of CD8^{+ bright} lymphocytes were CD3⁺ and TCRαβ⁺ but were not stained by anti-CD28, CD11b, CD16, CD56 and CD57 antibody. Unexpectedly, CD 8⁺ CD 57⁺ T cells, a hallmark of CMV infection, did not appear until the 2nd-6th months of primary CMV infection, and their numbers increased progressively thereafter. They became the predominant CD8⁺ T cell subset after about 6 months of infection and their persistence for several (up to 4) years was strongly correlated ( r = 0.87) with expansion of CD8⁺ cells. Persistence of CD 8 lymphocytosis was, thus, directly related to the rate of expansion of an uncommon CD 8⁺CD 57^- subset and its progressive replacement by CD 8⁺CD 57 ⁺ T cells that were chronically elicited by CMV.     

Aqueous Humor Alloreactive Cell Phenotypes, Cytokines and Chemokines in Corneal Allograft Rejection

As biopsies are not taken at the time of human corneal allograft rejection, most information on the early cellular changes in rejection is from animal models. We examined the phenotype of alloreactive cells present in the human anterior chamber during corneal graft rejection by flow cytometry and quantified aqueous humor levels of cytokines and chemokines using cytometric bead array. Aqueous and peripheral blood samples were taken from patients with graft endothelial rejection (n = 11) and from control patients undergoing cataract surgery (n = 8). CD45⁺CD4⁺, CD45⁺CD8⁺ and CD45⁺CD14⁺ cells were found in aqueous during rejection; no CD45⁺ cells were seen in control samples. Higher proportions of CD45⁺ cells found in aqueous during rejection were CD14⁺, denoting monocyte/macrophage lineage, than were CD4⁺ or CD8⁺. Large elevations were seen in aqueous levels of IL-6, MCP-1 and IP-10 during rejection compared with controls; smaller but still statistically significant increases were seen in MIP-1α and eotaxin. The role of CD14⁺ cells in allorejection is unclear as is the potential of these chemokines and their receptors as therapeutic targets. Aqueous humor samples offer a unique opportunity to analyze components of the allogeneic response in direct contact with donor tissue but without artifacts inherent in examination of tissue.     

MIP-3α/CCL20 in Renal Transplantation and Its Possible Involvement as Dendritic Cell Chemoattractant in Allograft Rejection

Graft-infiltrating dendritic cells (DC) and alloreactive T lymphocytes play a critical role in renal allograft rejection. Renal proximal tubular epithelial cells (TEC) are considered as active players in the attraction of leukocytes during renal inflammatory responses. Macrophage inflammatory protein (MIP)-3α/CCL20 is a major chemokine expressed by epithelial cells that attracts immature DC. In the present study, we present evidence that also the transplanted kidney can be a major source of MIP-3α/CCL20. Renal transplant recipients with rejection showed significantly increased excretion of urinary MIP-3α/CCL20 that correlated with transplant function. The tubular staining for MIP-3α/CCL20 in renal biopsies of patients with rejection as well as in vitro studies with primary human TEC indicated that TEC might be responsible for the increased urinary MIP-3α/CCL20. Furthermore, MIP-3α/CCL20 produced by activated TEC was highly potent in the attraction of CD1a⁺CD34⁺-derived DC precursors. These data suggest a role for MIP-3α/CCL20 in amplification of the immune response during renal allograft rejection by attraction of CCR6⁺ inflammatory cells, which may include DC, to the site of inflammation.     

Homeostatic Repopulation by CD28−CD8+ T Cells in Alemtuzumab-Depleted Kidney Transplant Recipients Treated With Reduced Immunosuppression

Alemtuzumab (CAMPATH-1H) is a depleting agent introduced recently in transplantation and often used with reduced maintenance immunosuppression. In the current study we investigated the immune response of 13 kidney allograft recipients treated with alemtuzumab followed by weaned immunosuppression with reduced dose of mycophenolate mofetil (MMF) and tacrolimus. Tacrolimus was switched to sirolimus at 6 months and MMF withdrawn at 12 months after transplantation.
We found that after alemtuzumab induction the recovery of CD8⁺ T cells was much faster than that of CD4⁺ T cells. It was complete 6 months posttransplant while CD4⁺ T cells did not fully recover even 15 months posttransplant. Repopulating CD8⁺ T cells were mainly of immunosenescent CD28⁻CD8⁺ phenotype. In a series of in vitro experiments we showed that CD28⁻CD8⁺ T cells might suppress proliferation of CD4⁺ T cells. There were three successfully treated acute rejections during the study (first at +70 day, two others +12 months) that occurred in patients with the lowest level of CD28⁻CD8⁺ T cells.
We hypothesize that expanded CD28⁻CD8⁺ T cells might compete for 'immune space' with CD4⁺ T cells suppressing their proliferation and therefore delaying CD4⁺ T-cells recovery. This delay might be associated with the clinical outcome as CD4⁺ T cells, notably CD4⁺ T effector memory cells, were shown to be associated with rejection.     

The Superiority of Hollow Fiber Membrane over Bubble Oxygenator in a Perfusion Circuit for the Evaluation of Small Caliber Endothelialized Arterial Prostheses

Abstract: A perfusion circuit was constructed from a pneumatic ventricular assist device, 2 compliance chambers, 4 small-diameter silicone tubes (ID 4 mm) simulating shear inducing vascular prostheses, and an oxygenator with a heat exchanger. A bubble oxygenator (in a BO circuit) and a hollow fiber membrane oxygenator (in an MO circuit) were studied. The circuits were perfused with 30% human serum containing culture medium for 7 days at 37^oC. The pH, Po₂, Pco₂, Na⁺, K ⁺, Ca^{2 +}, CI^-, glucose, and total protein concentrations remained the same in BO and MO circuits during the 7 days of perfusion. The differences between the values measured in the perfusion medium and in the medium maintained in the static conditions of cell culture were not significant. In the BO circuit, the amount of cholesterol and triglyceride concentrations decreased whereas the relative amounts of albumin, α1, α2, p, and γ globulins remained stable in the perfusion medium. The medium from the BO circuit did not promote the proliferation of cultured human saphenous vein endothelial cells. In the medium from the MO circuit, the cholesterol and triglyceride concentrations did not change with perfusion time; the proliferation rate and anticoagulant function of endothelial cells were maintained. The hollow fiber membrane oxygenator preserves the biological characteristics of the cell culture medium in a perfusion circuit. The MO circuit permits the performance of relevant studies on shear stress resistance and functional activity of human endothelial cells seeded onto vascular prostheses.     

New Insights into Mechanisms of Spontaneous Liver Transplant Tolerance: The Role of Foxp3-Expressing CD25+CD4+ Regulatory T Cells

Liver allografts in mice are accepted across MHC barriers without requirement for immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we investigated the role of Foxp3-expressing CD25⁺CD4⁺ regulatory T cells (Treg) in the induction of murine liver transplant tolerance. Foxp3⁺CD25⁺CD4⁺ T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-β expression and IL-4 production. Depletion of recipient CD25⁺CD4⁺ T cells using anti-CD25 mAb (250 μg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3⁺ Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4⁺ and CD8⁺ T cells in anti-CD25-mAb-treated recipients. Thus, the data suggest that Foxp3⁺CD25⁺CD4⁺Treg are involved in spontaneous acceptance of liver allografts in mice. The ratio of Treg to T effector cells appears to determine liver transplant outcome. CTLA4, IL-4, TGF-β and apoptosis of graft-infiltrating T cells are also associated with liver transplant tolerance and may contribute, at least in part, to the mechanisms of Treg-mediated immune regulation in this model.     

末端岩藻糖基化抑制剂对环孢素诱导的肾脏上皮-间充质转化的影响及其机制

目的  探讨末端岩藻糖基化抑制剂2-脱氧-D-半乳糖（2-D-gal）对环孢素（CsA）诱导的肾脏上皮-间充质转化（EMT）的影响及其机制。方法  将15只8~10周的雄性C57BL/6小鼠随机分为对照组（Ctrl组）、CsA组和CsA+2-D-gal组,每组各5只。通过蛋白质印迹法检测Ctrl组和CsA组小鼠肾脏组织岩藻糖基转移酶1（FUT1）和EMT相关蛋白E-cadherin、Vimentin、α-平滑肌肌动蛋白（α-SMA）的表达,免疫荧光法检测Ctrl组和CsA组小鼠肾脏组织末端岩藻糖的表达,Masson染色检测各组小鼠肾脏组织纤维化情况,并检测各组小鼠血尿素氮和血清肌酐水平。体外建立CsA诱导肾小管上皮HK2细胞EMT模型,分别用0、2.5、5.0和10.0 μmol/L的CsA刺激HK2细胞24 h,另将HK2细胞分为Ctrl组、2-D-gal组、CsA组和CsA+2-D-gal组,观察不同浓度CsA刺激后及各组HK2细胞形态,通过蛋白质印迹法检测不同浓度CsA刺激后及各组HK2细胞FUT1、E-cadherin、Vimentin和α-SMA的表达,免疫荧光法检测Ctrl组和CsA组HK2细胞末端岩藻糖的表达。结果  与Ctrl组比较,CsA组小鼠肾脏组织E-cadherin的蛋白相对表达量下降,FUT1、Vimentin和α-SMA蛋白相对表达量均升高（均为P < 0.05）,小鼠肾脏组织末端岩藻糖表达增多。与Ctrl组比较,CsA组和CsA+2-D-gal组小鼠的血尿素氮和血清肌酐水平均升高,与CsA组比较,CsA+2-D-gal组小鼠的血尿素氮和血清肌酐水平均降低（均为P < 0.05）。与Ctrl组比较,CsA组和CsA+2-D-gal组小鼠肾脏组织胶原纤维沉积均增多,α-SMA蛋白相对表达量均升高;与CsA组比较,CsA+2-D-gal组小鼠肾脏组织胶原纤维沉积减少,α-SMA蛋白相对表达量下降（均为P < 0.05）。随着CsA的浓度增加,HK2细胞的形态由正常的鹅卵石样逐渐变长变细,HK2细胞FUT1、Vimentin和α-SMA蛋白相对表达量升高,E-cadherin蛋白相对表达量下降,呈浓度依赖性。与Ctrl组比较,CsA组HK2细胞末端岩藻糖表达增多。CsA处理的基础上联合2-D-gal干预后,CsA+2-D-gal组的HK2细胞形态恢复到与正常HK2细胞形态相似。与CsA组比较,CsA+2-D-gal组的HK2细胞E-cadherin蛋白相对表达量升高,Vimentin和α-SMA蛋白相对表达量均下降（均为P < 0.05）。结论  CsA在体内和体外均可诱导EMT发生,并且伴有末端岩藻糖基化水平增加。2-D-gal可以通过抑制末端岩藻糖基化来抑制CsA诱导的EMT。     

Ex Vivo-Expanded Natural CD4+CD25+ Regulatory T Cells Synergize With Host T-Cell Depletion to Promote Long-Term Survival of Allografts

Foxp3⁺CD4⁺CD25⁺ natural regulatory T (nT_reg) cells have been shown in immunodeficient mice to suppress allograft rejection after adoptive cotransfer. We hypothesized that immunotherapy using ex vivo -expanded nT_reg could suppress allograft rejection in wild-type mice. Donor alloantigen (alloAg) specificity of naive splenic nT_reg was enriched in vitro by culturing with anti-CD3/CD28-coated Dynabeads plus bone marrow-derived dendritic cells (BM-DC) in the presence of interleukin (IL)-2 or IL-2 plus transforming growth factor (TGF)-β. On average, 96.2% fresh CD4⁺CD25⁺ nT_reg were intracellular Foxp3⁺. By d+20 in culture, 6.4% nT_reg were Foxp3⁺ following expansion with IL-2 alone, and 14.4% or 19.7% nT_reg were Foxp3⁺ when expanded with IL-2 plus 0.5 or 2.5 ng/mL TGF-β, respectively. In vitro , alloAg-enriched, TGF-β/IL-2-conditioned nT_reg exerted stronger donor alloAg-specific suppression than cells with IL-2 alone in mixed lymphocyte reaction (MLR) assays. In vivo , alloAg-enriched, TGF-β/IL-2-conditioned nT_reg expressed high-level Foxp3 following infusion, effectively overcame acute rejection and induced long-term survival of donor but not third-party heart allografts in peritransplant host T-cell-depleted mice. Long-term surviving allografts were noted to possess Foxp3⁺ graft-infiltrating cells of exogenous and endogenous origins. In conjunction with transient host T-cell depletion, therapeutic use of ex vivo -expanded nT_reg may be a practical means of preventing acute allograft rejection.     

Effect of Low Doses of Alpha Chlorohydrin on the Dehydrogenases and Oxidases of Rat Testis. A Histochemical Study

Effekt einer low-dose Behandlung mit α-Chlorohydrin auf die Dehydrogenase und Oxidasen im Rattenhoden — Eine histochemische Untersuchung
In dieser histochemischen Untersuchung wurden folgende Enzyme nach low-dose Behandlung mit α-Chlorohydrin (6,5 mg/kg/9 Tage) in den verschiedenen Zellarten des Rattenhodens untersucht: Isocitratdehydrogenase, Succinatdehydrogenase, Malatdehydrogenase, Glutamatdehydrogenase, Δ⁵- 3α-Hydroxysteroiddehydrogenase, NAD- und NADP-Diaphorase und Monoaminoxidase. Die Anwendung von α-Chlorohydrin führt zu einer Verminderung oder dem Verschwinden all dieser Enzyme bis auf NADP-Diaphorase und Δ⁵ -3α-HSD. Diese Veränderungen der Enzymaktivitäten im Rattenhoden zeigen deutlich, daß nach einer low-dose Behandlung mit α-Chlorohydrin lange bevor histologische Defekte auftreten, der Zitronensäurezyklus und Aminosäurestoffwechsel im Hoden geschädigt werden. Die Steroidbiosynthese bleibt jedoch unbeeinträchtigt, wie die gleichbleibenden Aktivitäten der Δ⁵ -3α-HSD und NADP-Diaphorase bewiesen.     

CD4+ T cells, without CD8+ or B lymphocytes, can reject xenogeneic skin grafts

Abstract: Previous experiments have shown that rejection of xenogeneic skin grafts by mice is particularly dependent on CD4⁺ T cells. There are two possible explantations for this finding: either 1) "help" provided by CD4⁺ T cells is essential for CD8⁺ T cell-, B cell-, or NK cell-mediated effector mechanisms of rejection, or 2) CD4⁺ cells are themselves responsible for rejection, perhaps by some nonspecific effector mechanism. To examine these two hypotheses, we transplanted pig skin onto SCID mice and then reconstituted the mice with selected subpopulations of lymphocytes. Mice that did not received CD4⁺ T cells were unable to reject their xenografts, whereas those receiving CD4⁺ cells could do so in the absence of CD8⁺ cells or B cells and even when additionally depleted of NK cells by treatment with anti-Asialo GM1 antibody. Additional experiments were performed both in vivo and vitro to confirm the absence in test mice of CD4⁺ or CD8⁺ and B lymphocytes, respectively. These results suggest that CD4⁺ T cells are not only necessary for rejection of xenogeneic skin grafts by mice, but that they can do so without CD8⁺ cells or B cells, and probably without NK cells. Since CD4⁺ cells in mice have been shown to recognize xenogeneic antigens indirectly, this suggests that a nonspecific effector mechanism may be involved in the rejection of xenografts. In these experiments allogeneic skin grafts behave quite differently as they could not be rejected by this mechanism.     

Immunohistochemical Study of Tumor-Infiltrating Lymphocytes Before and After Intravesical Bacillus Calmette-Guérin Treatment for Superficial Bladder Cancer

¹Department of Urology, Juntendo University School of Medicine, Tokyo Japan
Background This study investigated changes in the phenotypic characteristics of tumor-infiltrating lymphocytes during intravesical bacillus Calmette-Guérin (BCC) treatment using an immunohistochemical technique.
Methods A total of 16 patients with superficial bladder cancer underwent intravesical BCG treatment for therapeutic purposes. Tissue specimens were obtained from these patients before and after BCG treatment by cold cup biopsies.
Results The numbers of CD3⁺ cells, CD4⁺ cells, CD8⁺ cells, and CD19⁺ cells significantly increased after treatment compared with numbers before treatment (P <0.01). Although γ/δT cells were not observed before treatment, they appeared after treatment in 6 patients- In all these patients, the tumors disappeared or their size was reduced by more than 50%, and none of the tumors recurred. The induction of CD25⁺ cells after treatment was seen in 11 of the 16 patients.
Conclusions γ/δT cells may play an important role in the immune response of the host to the tumor in intravesical BCG treatment (although this correlation was statistically insignificant).     

Tumor-infiltrating lymphocytes derived from human renal cell carcinoma: Clonal analysis of its characteristics

Aim:   To assess the characteristics of activated tumor-infiltrating lymphocytes (TIL), we report the isolation, growth response, and functional analysis of a CD4^- CD8⁺ TIL-clone derived from human renal cell carcinoma (RCC).
Methods:   Bulk TILs were expanded from a human RCC and the lymphocytes were separated into a CD8⁺ enriched population. Subsequently, using the limiting dilution technique, a TIL clone was established and its growth response, phenotype and cytotoxic activity were analyzed.
Results:   A clone, T16-13, by day 94 numbering 1 × 10⁷ cells, was harvested and characterized as a CD4^- CD8⁺ clone. On day 144, the cytotoxic activity of this clone against the autologous tumor was relatively high (2.3 ± 0.7 LU₃₀/10⁶ cells). Meanwhile, against allogeneic renal tumors, there was no cytotoxic activity (−0.1 LU₃₀/10⁶ cells).
Conclusions:   A TIL clone possessing modest autologous tumor-specific cytotoxicity can be isolated from human RCC. The characteristics analysis of various TIL clones may provide a better understanding of an RCC tumor microenvironment and may help to establish new modalities for the treatment of patients with metastatic kidney cancer.     

Role of CD4+ T cells in the rat to mouse cardiac xenotransplantation

Abstract T cell subsets involved in rejection of xenografts were analyzed using a rat to mouse cardiac xenotransplant model. Proliferating response and interleulin-2 (IL-2) production in recipients' spleen cells were almost completely abrogated by elimination of L3T4⁺ T cells, but not by elimination of Lyt2.1⁺ T cells. Cytotoxic T lymphocyte (CTL) activities were mediated by both L3T4⁺ and Lyt2.1⁺ T cells with the help of IL-2-producing L3T4⁺ T cells. Administration of anti-L3T4 monoclonal antibody (mAb) into recipient mice resulted in a significant prolongation of graft survival (mean graft survival was 29.2 days). Moreover, anti-L3T4 mAb treatment plus thymectomy led to indefinite graft survival. Anti-rat endothelial cell (EC) antibody production in the grafted mice was remarkably suppressed by anti-L3T4 mAb treatment. In contrast, Lyt2.1 mAb treatment did not prolong the graft survival and did not suppress anti-EC antibody production. These results indicated the absolute requirement of L3T4⁺ T cells in the rejection of rat to mouse cardiac xenografts.     

CCR5 Is Required for Regulation of Alloreactive T-Cell Responses to Single Class II MHC-Mismatched Murine Cardiac Grafts

The effector CD4 T-cell response in wild-type C57BL/6 recipients of single class II MHC-disparate B6.H-2^bm12 cardiac allografts is restricted by CD4⁺CD25⁺ regulatory T cells (Tregs) resulting in long-term allograft survival. To investigate the role chemokine receptors might play in Treg function, this study tested the requirement for CCR5 on Tregs to suppress the alloimmune response in C57BL/6 recipients of B6.H-2^bm12 cardiac allografts. In contrast to the long-term survival of B6.H-2^bm12 allografts in wild-type recipients (>100 days), the allografts were acutely rejected within 25 days in CCR5^−/− recipients with intense infiltration of CD4 T cells. Numbers and duration of donor-reactive CD4 T cells producing IFN-γ and IL-4 were markedly increased in spleens of B6.CCR5^−/− versus wild-type recipients. Wild-type and B6.CCR5^−/− mice had equivalent numbers of splenic FoxP3⁺ Tregs before and following transplantation, and these Tregs were equivalently suppressive in vitro . However, diminished numbers of FoxP3⁺ Tregs infiltrated B6.H-2^bm12 allografts in B6.CCR5^−/− recipients. Adoptive transfer of wild-type, but not CCR5-deficient, CD4⁺CD25⁺ Tregs to CCR5^−/− recipients restored long-term survival of B6.H-2^bm12 cardiac grafts. Collectively, these results indicate that CCR5 expression is required for the regulatory functions of Tregs that restrict alloreactive CD4 T-cell responses to single class II MHC-mismatched cardiac allografts.     

Epidural anaesthesia blocks changes in peripheral lymphocytes subpopulation during gastrectomy for stomach cancer

To examine the effects of surgery and anaesthesia on cell-mediated and humoral immunity, we investigated changes in subpopulations of peripheral T cells and B cells during gastrectomy. Twenty-one patients with gastric cancers who underwent total or subtotal gastrectomy were randomly assigned to receive thiopental/N₂O general anaesthesia supplemented with either epidural (epidural group, n =12) or enflurane(enflurane group, n =9). The changes in peripheral lymphocyte subpopulations were detected and quantified using single and double label analysis of monoclonal antibodies against lymphocyte membrane surface markers (Leu series). Subpopulations were measured before induction, and 1 and 2 hours after skin incision. In the enflurane group, B cells (Leu12⁺), total T cells (Leu4⁺), inducer T cells (CD4⁺, Leu8⁺) and the CD4/CD8 ratio decreased and suppressor T cells (CD8⁺, Leu15⁺) increased significantly after skin incision. Whereas, the epidural group showed no change in the number of B cells and each T cell subpopulation during the study. These data suggest that epidural anaesthesia blocks the effect of stress induced by major surgery on fluctuation of peripheral lymphocyte subpopulations which may be associated with suppression of immunity.     

Monotherapy rapamycin allows an increase of CD4+ CD25bright+ FoxP3+ T cells in renal recipients

CD4⁺ CD25^bright+ FoxP3⁺ regulatory T cells (Tregs) may control donor-specific allogeneic responses in kidney transplant recipients. Recent evidence demonstrated that three phenotypical Treg-subsets, naive (CCR7⁺CD45RO⁻), central-memory (CCR7⁺CD45RO⁺) and effector-memory (CCR7⁻CD45RO⁺), are essential for the development and function of antigen-specific suppression in the lymphoid and peripheral tissues. Also, it has been appreciated that Tregs are affected by immunosuppressive agents. In clinical practice, however, the effect of a single drug remains to be determined. Therefore, we analyzed the effect of several immunosuppressive agents on the number, phenotype and function of peripheral Tregs from 46 stable kidney transplant recipients. These patients were converted to monotherapy with tacrolimus ( n  = 15), rapamycin ( n  = 17) or mycophenolate mofetil ( n  = 14). Blood was obtained at inclusion and 6 months thereafter. The number of Tregs increased significantly in patients on monotherapy with rapamycin ( P  < 0.001), which was caused by increased numbers of Tregs with a central-memory and an effector-memory phenotype (both P  < 0.05). At 6 months after conversion, however, the suppressive function of Tregs did not significantly change in co-cultures stimulated with donor-Ag. Therefore, monotherapy with rapamycin allows the signals that are needed to increase the number of functional Tregs with a memory phenotype, thereby enhancing the potential capacity to regulate donor-specific responses in the lymphoid and the peripheral tissues.     

Strategies to Induce Marked Prolongation of Secondary Skin Allograft Survival in Alloantigen-Primed Mice

Alloreactive memory T cells mediate accelerated rejection. We investigated the effect of polyclonal anti-T-cell antibody (ALS) and rapamycin (RAPA) on skin allograft survival in naïve or alloantigen-primed mice. ALS prolonged graft survival in both naïve and alloantigen-primed mice. T-cell depletion by ALS was associated with increased CD4⁺CD44^hiOX40⁺ and CD8⁺CD44^hiCD122⁺ memory T cells. Addition of RAPA to ALS extended graft survival in naïve mice, but had no effect on secondary allograft survival in alloantigen-primed mice. In adoptive transfer experiments, RAPA inhibited alloantigen-stimulated proliferation and allograft rejection by naïve T cells. In contrast, alloantigen-primed memory T cells, particularly CD4⁺CD44^hiOX40⁺ and CD8⁺CD44^hiCD122⁺ T cells, were resistant to RAPA in response to alloantigen and mediated accelerated rejection in the presence of RAPA. Resistance to RAPA by alloantigen-primed mice was overcome by the use of high-dose ALS, which achieved marked prolongation of secondary skin allograft survival (>100 days). Inhibition of CD122⁺ T cells and/or OX40/OX40L costimulation blockade, combined with low-dose ALS and RAPA, was also effective. These results demonstrate that tolerance may be achieved in allosensitized individuals by T-cell depletion- and RAPA-based strategies employing high-dose ALS or targeting CD122⁺CD8⁺ T cells and/or the OX40/OX40L costimulatory pathway.     

Albumin-induced epithelial mesenchymal transformation

Background: Progressive chronic kidney disease is often associated with albuminuria and renal fibrosis linked to the accumulation of myofibroblasts producing extracellular matrix. Renal myofibroblasts are derived from a number of cells including tubular epithelial cells (TECs) through epithelial mesenchymal transformation (EMT). This study explores the hypothesis that exposure of TECs to albumin induces EMT. Methods: Normal rat TECs (NRK52E) were exposed in culture to de-lipidated bovine serum albumin (dBSA; 10 mg/ml) for 2, 4 and 6 days. Binding/uptake of fluoresceined albumin by PTCs was evaluated. Transformation into myofibroblasts was assessed by light and electron microscopy, immunofluorescence and Western blotting for α-smooth muscle actin (α-SMA), E-cadherin and transforming growth factor-β1 (TGF-β1). We also investigated the expression of fibroblast-specific protein-1 (FSP-1) and collagens I, III and IV. TGF-β1 biological activity, mRNA and protein were measured. A neutralising anti-TGF-β1 antibody was used to analyse the role of TGF-β1 in albumin-induced EMT. Results: Exposure of TECs to dBSA led to binding/uptake of albumin as well as fibroblastic morphological changes. Incubation of TECs with dBSA caused a reduction of TEC marker E-cadherin (ANOVA p = 0.0002) and de novo expression of fibroblast markers α-SMA and FSP-1 (ANOVA p = 0.0001) in a time-dependent manner. It also increased expression and activity of TGF-β1. Neutralisation of TGF-β1 significantly reduced EMT (p < 0.01). Conclusion: This study demonstrates that in vitro, albumin induces the transformation of TECs into cells with myofibroblast characteristics; a process that may be TGF-β1 dependent.