T cell exhaustion is associated with antigen abundance and promotes transplant acceptance

Exhaustion of T cells limits their ability to clear chronic infections or eradicate tumors. Here, in the context of transplant, we investigated whether T cell exhaustion occurs and has a role in determining transplant outcome. A peptide/MHC tetramer‐based approach was used to track exhausted CD8⁺ T cells in a male‐to‐female skin transplant model. Transplant of large whole‐tail skins, but not small tail skins (0.8 cm × 0.8 cm), led to exhaustion of anti‐male tetramer⁺ CD8⁺ T cells and subsequently the acceptance of skin grafts. To study CD4⁺ T cell exhaustion, we used the TCR‐transgenic B6 TEa cells that recognize a major transplant antigen I‐Eα from Balb/c mice. TEa cells were adoptively transferred either into B6 recipients that received Balb/c donor skins or into CB6F1 mice that contained an excessive amount of I‐Eα antigen. Adoptively transferred TEa cells in skin‐graft recipients were not exhausted. By contrast, virtually all adoptively transferred TEa cells were exhausted in CB6F1 mice. Those exhausted TEa cells lost ability to reject Balb/c skins upon further transfer into lymphopenic B6.Rag1^?/? mice. Hence, T cell exhaustion develops in the presence of abundant antigen and promotes transplant acceptance. These findings are essential for better understanding the nature of transplant tolerance.     

Persistent cytomegalovirus infection is associated with increased expression of TGF-beta1, PDGF-AA and ICAM-1 and arterial intimal thickening in kidney allografts.

BACKGROUND: Cytomegalovirus (CMV) is a suggested risk factor for the development of chronic allograft nephropathy. Transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) are important molecules in this process. We analysed the impact of persistent CMV infection in kidney allografts on the expression of growth factors, adhesion molecules and inflammation markers. METHODS: In a population of 172 renal transplant recipients, CMV was diagnosed in 82 patients by pp65 antigenaemia test and viral cultures. Biopsies taken after CMV infection were available from 48 of the 82 patients for the demonstration of CMV antigens by immunohistochemistry and in situ DNA hybridization. Biopsy material for further analyses was available from 16 CMV patients. Five patients with no previous CMV infection were used as controls. Biopsy histology was scored according to Banff 97 classification. RESULTS: In 11 out of 16 patients, persistent CMV antigens and/or DNA were demonstrated in the biopsy >2 months after the last positive finding in blood or urine. Increased expression of TGF-beta1 was recorded in tubuli and in arterial endothelium in biopsies with a positive CMV finding compared with controls. Also, the expression of PDGF-AA was increased in tubuli and somewhat in arterial endothelium in CMV-positive biopsies. The expression of intercellular adhesion molecule-1 (ICAM-1) was increased significantly in peritubular capillary endothelium. Vascular intimal thickening was increased in the biopsies with persistent CMV infection. CONCLUSIONS: Persistent CMV infection in kidney allografts was associated with increased vascular changes and increased expression of TGF-beta1, PDGF-AA and ICAM-1.     

Less expression of prohibitin is associated with increased caspase-3 expression and cell apoptosis in renal interstitial fibrosis rats

Aims: Prohibitin (PHB), a ubiquitous protein, is involved in a variety of molecular functions. Renal interstitial fibrosis (RIF) is a hallmark of common progressive chronic diseases that lead to renal failure. This study was performed to investigate whether PHB was associated with Caspase‐3 expression/cell apoptosis in RIF rats. Methods: Twenty‐four male Wistar rats were randomly divided into two groups: sham operation group (SHO) and model group subjected to unilateral ureteral obstruction (GU), n = 12, respectively. The model was established by left ureteral ligation. Renal tissues were collected at 14 days and 28 days after surgery. RIF index, cell apoptosis index, protein expression of PHB, transforming growth factor‐βl (TGF‐β1), collagen‐IV (Col‐IV), fibronectin (FN) or Caspase‐3 in renal interstitium, and mRNA expression of PHB in renal tissue were detected. Results: Compared with that in the SHO group, the PHB expression (mRNA and protein) was significantly reduced (P < 0.01). Protein expressions of TGF‐β1, Col‐IV, FN and Caspase‐3, and RIF index or cell apoptosis index in GU group were markedly elevated compared with those in SHO group (all P < 0.01). The protein expression of PHB had a negative correlation with the protein expression of TGF‐β1, Col‐IV, FN or Caspase‐3, and RIF index or cell apoptosis index (each P < 0.01). Conclusions: Less expression of PHB is associated with increased Caspase‐3 expression/cell apoptosis in RIF rats. However, further research is needed to determine the effect of PHB on Caspase‐3 expression/cell apoptosis and to determine the potential of PHB as a therapeutic target.     

Dynamic changes of B‐cell compartments in kidney transplantation: lack of transitional B cells is associated with allograft rejection

B cells play an important role in the immune responses which affect the outcomes of kidney allografts. Dynamic changes of B‐cell compartments in clinical kidney transplantation are still poorly understood. B‐cell subsets were prospectively monitored using flow cytometry for 1 year in 98 kidney transplant recipients. Data were correlated with immunosuppression and clinical outcomes. An increase in the total population of B lymphocytes was observed during the first week after transplantation. The level of IgM^highCD38^highCD24^high transitional B cells reduced significantly up until the third month, with partial repopulation in the first year. Lower numbers of transitional B cells in the third month were associated with higher risk of graft rejection. IgM⁺IgD⁺CD27^? naive B cells did not change within follow‐up. IgM⁺CD27⁺ nonswitched memory B cells and IgM^?CD27⁺ switched memory B cells increased on post‐operative day 7. IgM^?CD38^highCD27^high plasmablasts showed similar kinetics during the first post‐transplant year, similar to transitional B cells. In conclusion, sensitized kidney transplant recipients as well as those with either acute or chronic rejection within the first post‐transplant year exhibited lower levels of transitional B cells. Therefore, these data further support the hypothesis that transitional B cells have a protective role in kidney transplantation.     

Persistent cytomegalovirus infection in kidney allografts is associated with inferior graft function and survival

The long-term effects of cytomegalovirus (CMV) infections on kidney allografts are unknown. We examined the impact of persistent intragraft CMV infection on long-term kidney allograft function and survival. CMV was diagnosed in 82/172 renal transplant recipients by antigenemia test and viral cultures. Biopsies from 48 of 82 patients taken after CMV infection and from 15 patients with no previous CMV infection detected were available for the immunohistochemical demonstration of CMV antigens and DNA hybridization in situ. Five-year follow-up data from these 63 patients were analysed. In 17 patients, CMV antigens and/or DNA persisted in the biopsy >2 months after the last positive finding in blood or urine. Patients with persistent intragraft CMV had reduced graft survival (P = 0.041) and Cox regression analysis showed persistent CMV as a risk factor for reduced graft survival (RR: 3.5). Recipients with persistent intragraft CMV had reduced creatinine clearance 1 and 2 years after transplantation (P = 0.007) and in multivariate logistic regression analyses including several potential pre- and posttransplant risk factors, persistent CMV was an independent risk factor for lower clearance at 1 and 2 years (OR: 4.4 and 4.9). Our novel findings show that persistent intragraft CMV infection was associated with reduced kidney allograft function and survival.     

Sympathetic activity is increased in polycystic kidney disease and is associated with hypertension.

Hypertension is common in patients with polycystic kidney disease (PKD). This study addresses the hypothesis that sympathetic activity is enhanced in hypertensive PKD patients, not only when renal function is impaired but also when renal function is still normal. Muscle sympathetic nerve activity (MSNA, peroneal nerve), plasma renin activity (PRA), heart rate, and BP were studied in PKD patients with normal and with impaired renal function and in matched controls. In hypertensive patients with normal renal function, MSNA and mean arterial pressure (MAP) were higher than in normotensive patients (23 +/- 5 versus 15 +/- 7 bursts/min; 110 +/- 10 versus 90 +/- 3 mmHg; P < 0.05), whereas PRA and heart rate did not differ. In PKD with chronic renal failure (CRF) (creatinine clearance rate, 39 +/- 19 ml/min), MAP, MSNA and PRA were higher than in controls (resp, 116 +/- 7 versus 89 +/- 9 mmHg; 34 +/- 14 versus 19 +/- 9 bursts/min; 405 [20 to 1640] versus 120 [40 to 730] fmol/L per sec; all P < 0.05). Heart rate in PKD CRF did not differ from controls. MSNA correlated with MAP (r = 0.42; P = 0.01) and age with MSNA (r = 0.45; P < 0.01). Regression line of age and MSNA in patients was steeper than that in controls. This study indicates that MSNA is increased in hypertensive PKD patients regardless of renal function. The data support the idea that sympathetic hyperactivity contributes to the pathogenesis of hypertension in PKD.     

Higher mycophenolate dosage is associated with an increased risk of squamous cell carcinoma in kidney transplant recipients

BackgroundKidney transplant recipients are at increased risk of keratinocyte cancers, namely squamous cell and basal cell carcinomas (SCCs and BCCs). This is primarily due to the high levels of immunosuppression that are required to prevent allograft rejection. Different immunosuppressive medications confer different risks, and the effect of mycophenolate mofetil on SCC and BCC risk is unclear. We explored the relationship between mycophenolate dose prescribed over the entire transplant period and the risk of SCC and BCC.MethodsKidney transplant recipients from Queensland, Australia, were recruited between 2012 and 2014 and followed until mid-2016. During this time transplant recipients underwent regular skin examinations to diagnose incident SCCs and BCCs. Immunosuppressive medication regimens were obtained from hospital records, and the average mycophenolate dose/day over the entire transplantation period was calculated for each patient. Doses were divided into three ranked groups, and adjusted relative risks (RR_adj) of developing SCC and BCC tumours were calculated using negative binomial regression with the lowest dosage group as reference. Recipients who had used azathioprine previously were excluded; further sub-group analysis was performed for other immunosuppressant medications.ResultsThere were 134 kidney transplant recipients included in the study. The average age was 55, 31% were female and 69% were male. At the highest median mycophenolate dose of 1818 mg/day the SCC risk doubled (RR_adj 2.22, 95% CI 1.03–4.77) when compared to the reference group of 1038 mg/day. An increased risk persisted after accounting for ever-use of ciclosporin, ever-use of tacrolimus, and when excluding mammalian target of rapamycin users. This increased risk was mainly carried by kidney transplant recipients immunosuppressed for five or more years (RR_adj = 11.05 95% CI 2.50–48.81). In contrast, there was no significant association between BCC incidence and therapy with the highest compared with the lowest mycophenolate dosage (RR_adj = 1.27 95% CI 0.56–2.87).ConclusionHigher mycophenolate dosage is associated with increased SCCs in kidney transplant recipients, particularly those immunosuppressed for more than five years. The increased SCC risk persists after accounting for usage of other immunosuppressant medications.     

Hepatic expression of IL-15 mRNA is associated with liver graft acceptance

BACKGROUND: Acute allograft rejection continues to be a major cause of morbidity following organ transplantation. The aim of this study was to investigate the local expression of a range of immunomodulatory molecules which may be mediating rejection of, or tolerance to, liver allografts. METHODS: RNA was extracted from 31 protocol liver biopsies taken 7-10 days post-transplantation, reverse transcribed and screened by a sensitive RT-PCR for a wide range of cytokines and other immunomodulatory molecules. The mRNA profile of each biopsy was subsequently related to the histological and clinical status of the graft. Samples of RNA isolated from activated leukocytes and T cell clones, and from normal liver, were used as controls to compare to the 'immunological snapshot' obtained from the biopsies. RESULTS: Presence of tumour necrosis factor-alpha, fas ligand, granzyme B and perforin mRNA in most of the liver biopsies reflected the occurrence of cell-mediated immune reactions. However, the expression of only one cytokine, interleukin-15 (IL-15), was significantly more frequent in allografts that showed no histological or biochemical signs of rejection during the early post-transplant period. Using an in vitro model it was demonstrated that recombinant IL-15 expands tenfold the number of CD3(+)CD56(+) (natural T; NT) cells from peripheral blood mononuclear cells cultures. Conditioning with IL-15 also increased cytotoxic activity of lymphocytes against leukaemic target cells. CONCLUSIONS: Although considerable evidence for cell-mediated immunity was shown for all liver allografts, the only clinical association was for IL-15 mRNA expression and graft acceptance. An in vitro model suggested that IL-15 may be enhancing the numbers and the activity of local regulatory cells, in particular resident NT cells in the liver, which may have a role in killing activated lymphocytes such as graft-reactive host T cells.     

Plasma cadmium is associated with increased risk of long-term kidney graft failure

1. Download : Download high-res image (302KB)
2. Download : Download full-size image

     

Membranoproliferative glomerulonephritis associated with low-grade B cell lymphoma presenting in the kidney

Low-grade B cell lymphoma of mucosa-associated tissue type (MALToma) rarely may involve the kidney. Membranoproliferative glomerulonephritis (MPGN) is an uncommon complication of B cell lymphoma and may be related to cryoglobulin and/or immunoglobulin synthesis by a secretory B cell clone. We report 2 patients with the novel renal biopsy findings of coexistent MALToma and MPGN. Both subjects presented with nephrotic proteinuria and renal insufficiency. One patient had a serum M protein (IgG K) but neither individual had any other clinical or serologic evidence of systemic disease, including hematolymphoid malignancy, autoimmune disease, cryoglobulinemia, or hepatitis C viral infection. Both renal biopsies demonstrated MPGN type I with immunoglobulin deposits that in 1 case showed light chain restriction (IgM K). Electron microscopy disclosed corresponding glomerular electron dense deposits in subendothelial locations. Both biopsies also contained atypical interstitial lymphoid infiltrates comprising marginal zone (centro-cyte-like) cells that infiltrated tubules and showed extra-capsular extension. Immunostains demonstrated a predominantly B cell population that lacked expression of CD5 and cycline D1, and gene rearrangement studies confirmed the presence of a monoclonal B cell population in both cases. These findings indicate that low-grade B cell lymphoma in the kidney may be an unexpected finding in patients with nephrotic syndrome related to MPGN. Immunophenotypic and gene rearrangement studies are important ancillary tools for the evaluation of atypical lymphoid infiltrates in kidney biopsies.     

Reduction of Foxp3-expressing regulatory T cell infiltrates during the progression of renal allograft rejection in a mouse model

BACKGROUND: Regulatory T (Treg) cells are the immune suppressors in the maintenance of immune homeostasis and tolerance to self and non-self antigens, and may have therapeutic potential in the treatment of transplant rejection in patients. However, Treg cell development and action are poorly understood in transplantation. In this study, the association of CD4(+)Foxp3(+) infiltrates within renal allograft tissue with graft survival was investigated in a mouse model. METHODS: Kidney donors from C57BL/6J mice (H-2(b)) were transplanted to bilaterally nephrectomized Balb/c recipient mice (H-2(d)). Treg cells were examined with FACS and immunohistochemical staining. RESULTS: Here we showed that without any immunosuppressive regimen, kidney allografts were mostly rejected from 20 to 60 days after transplantation. During the progression of allograft rejection Foxp3(+) Treg phenotype infiltrates were significantly diminished, while intragraft expression of TGF-beta1, IL-6, IL-17 and IL-23 was up-regulated. The regulatory function of CD4(+)CD25(+) infiltrates was confirmed by their suppressive activity in mixed lymphocyte reaction. Further in vitro studies indicated that primary renal tubular epithelial cell (TEC) cultures produced high levels of IL-6 in response to allogeneic lymphocyte or IL-17 stimulation, and neutralization of IL-6 increased CD4(+)CD25(+)Foxp3(+) cells in co-cultures with TEC. CONCLUSION: Diminution of Foxp3(+) Treg infiltrates associates with renal allograft rejection, and neutralization of IL-6 activity enhances Foxp3(+) Treg cell differentiation. Our findings suggest that increase in intragraft IL-6 may down-regulate infiltrating Foxp3(+) Treg cells.     

Epstein-Barr virus infection is associated with endothelial Bcl-2 expression in transplant liver allografts

INTRODUCTION: In liver transplant recipients with Epstein-Barr virus (EBV) disease, we reported a low rate of acute rejection after stopping or markedly lowering immunosuppression. This observation led to the hypothesis that EBV, as a means of viral persistence, induces expression of antiapoptotic factors and these factors, in turn, confer protection to the transplanted organ. Bcl-2, an antiapoptotic factor induced by EBV in various host cells, is not normally expressed in the liver. We questioned whether bcl-2 is expressed in the transplanted liver and whether its expression is modified by EBV. MATERIALS AND METHODS: Retrospective liver biopsy specimen from liver transplant patients diagnosed with EBV (n=12) were examined for the presence of bcl-2 by immunohistochemistry and compared with EBV (-) transplant (n=15), and nontransplant (n=13) livers. RESULTS: The most significant finding was the presence of endothelial bcl-2 expression in the majority of EBV (+) transplant samples examined (67%) and its relative absence in the other two groups (P<0.005). There was also bcl-2 expression in the hepatocytes and lymphocytes of the majority of transplant liver samples, irrespective of EBV status. DISCUSSION: We have identified a strong association between EBV infection and endothelial bcl-2 expression in transplant livers. We also found that transplantation, in itself, was associated with bcl-2 expression in the hepatocytes and lymphocytes of liver allografts.     

Reduced expression of α-tocopherol-associated protein is associated with tumor cell proliferation and the-increased risk of prostate cancer recurrence

Aim： To examine the impact and prognostic significance of α-tocopherol associated protein （TAP） expression in a series of prostate cancer patients. Methods： Tissues from 87 patients underwent radical prostatectomy were examined for TAP expression by immunohistochemistry. The relationships of the staining results, the clinic pathological characteristics and the recurrence times were analyzed. Results： Compared with the adjacent areas of normal and benign glands, immunoreactivity of TAP was reduced in areas of prostate cancer. A lower TAP-positive cell number per mm^2 of the largest cancer area （defined as TAP-PN） was associated with higher clinical stage （r = -0.248, P = 0.0322）. Inverse associations were found among the TAP-PN and positive lymph nodes （r = -0.231, P = 0.0325）, preoperative prostatespecific antigen （PSA） levels （r = -0.423, P = 0.0043）, tumor size （r = -0.315, P = 0.0210） and elevated tumor cell proliferation, which was indicated by the staining of Ki-67 （r = -0.308, P = 0.0026）. TAP-PN was a significant predictor of recurrence univariately （P = 0.0006）, as well as multivariately, adjusted for known markers including preoperative PSA, clinical stage, Gleason score, surgical margin, extra-prostatic extension, seminal vesicle invasion and lymph node metastasis （P = 0.0012）. Conclusion： Reduced expression of TAP was associated with the cell proliferation status of prostate cancer, adverse pathological parameters and the increased risk of recurrence.     

Progressive histological damage in renal allografts is associated with expression of innate and adaptive immunity genes

The degree of progressive chronic histological damage is associated with long-term renal allograft survival. In order to identify promising molecular targets for timely intervention, we examined renal allograft protocol and indication biopsies from 120 low-risk pediatric and adolescent recipients by whole-genome microarray expression profiling. In data-driven analysis, we found a highly regulated pattern of adaptive and innate immune gene expression that correlated with established or ongoing histological chronic injury, and also with development of future chronic histological damage, even in histologically pristine kidneys. Hence, histologically unrecognized immunological injury at a molecular level sets the stage for the development of chronic tissue injury, while the same molecular response is accentuated during established and worsening chronic allograft damage. Irrespective of the hypothesized immune or nonimmune trigger for chronic allograft injury, a highly orchestrated regulation of innate and adaptive immune responses was found in the graft at the molecular level. This occurred months before histologic lesions appear, and quantitatively below the diagnostic threshold of classic T-cell or antibody-mediated rejection. Thus, measurement of specific immune gene expression in protocol biopsies may be warranted to predict the development of subsequent chronic injury in histologically quiescent grafts and as a means to titrate immunosuppressive therapy.     

High expression of granzyme B in conventional CD4+ T cells is associated with increased relapses after allogeneic stem cells transplantation in patients with hematological malignancies

Granzyme B is known to be a serine protease contained in granules of cytotoxic T cells. We have previously reported an influence of granzyme B expression in T regulatory cells (Tregs) on the risk of acute graft versus host disease (GVHD) onset. However, it is still unknown if conventional T cells (Tcon) use the granzyme B pathway as a mechanism of alloimmunity. We hypothesized that granzyme B in Tcon may affect recurrence within the first 6 months after allogeneic transplantation (allo-HSCT). A total of 65 patients with different hematological malignancies were included in this study. Blood samples were collected on day +30 after allo-HSCT. The percentage of granzyme B positive conventional T cells in patients who developed relapse in the first 6 months after allo-HSCT was 11.3 (4.5–35.3) compared to the others in continuous complete remission—1.3 (3.65–9.7), р = 0.011. The risk of relapse after allo-HSCT was in 3.9 times higher in patients with an increased percentage of granzyme B positive conventional T cells. The findings demonstrated that the percentage of granzyme B positive conventional T cells on day +30 after allo-HSCT could be a predictable marker of relapse within the first 6 months after allo-HSCT.     

Reduction of the vitamin D hormonal system in kidney disease is associated with increased renal inflammation

To examine any potential role for 1,25-dihydroxyvitamin D (1,25(OH)2D) in inflammation associated with chronic kidney disease we measured vitamin D metabolites, markers of inflammation and gene expression in 174 patients with a variety of kidney diseases. Urinary MCP-1 protein and renal macrophage infiltration were each significantly but inversely correlated with serum 1,25(OH)2D levels. Logistic regression analysis with urinary MCP-1 as binary outcome showed that a 10-unit increase in serum 1,25(OH)2D or 25OHD resulted in lower renal inflammation. Analysis of 111 renal biopsies found that renal injury was not associated with a compensatory increase in mRNA for the vitamin D-activating enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), its catabolic counterpart 24-hydroxylase, or the vitamin D receptor. There was, however, a significant association between tissue MCP-1 and CYP27B1. Patients with acute renal inflammation had a significant increase in urinary and tissue MCP-1, macrophage infiltration, and macrophage and renal epithelial CYP27B1 expression but significantly lower levels of serum 1,25(OH)2D in comparison to patients with chronic ischemic disease despite similar levels of renal damage. In vitro, 1,25(OH)2D attenuated TNFalpha-induced MCP-1 expression by human proximal tubule cells. Our study indicates that renal inflammation is associated with decreased serum vitamin D metabolites and involves activation of the paracrine/autocrine vitamin D system.