Late Antibody-Mediated Rejection in a Large Prospective Cross-Sectional Study of Kidney Allograft Recipients--Preliminary Results of the Screening Phase of the BORTEJECT Trial.

There is limited data on the rate of late antibody-mediated rejection (ABMR) in unselected transplant cohorts. Here, we investigated the prevalence and characteristics of ABMR in a large cohort of long-term kidney allograft recipients. Patients were screened in the context of a randomized controlled trial (BORTEJECT study; ClinicalTrials.gov: NCT01873157) designed to investigate the impact of bortezomib on the course of late ABMR. The study (initiation in October 2013) includes a cross-sectional ABMR screening (key inclusion criterion: functioning graft at ≥ 180 days) to identify 44 recipients eligible for inclusion in the intervention trial. Patients were screened for donor-specific antibodies (DSA) applying solid phase technology and DSA+ recipients underwent protocol biopsies. Through November 2014, ABMR screening (after a median of 6.5 years post-transplantation) had been completed for 714 recipients. One hundred one patients (14%) had DSA above a threshold of 1,000 mean fluorescence intensity (MFI). Forty-four of 78 DSA+ recipients (6% of the overall cohort) subjected to biopsy were diagnosed with C4d-positive (n = 17) or -negative (n = 27) ABMR and 40 consented to participate in the intervention trial. DSA+ABMR+ and DSA+ABMR- patients differed significantly with respect to the MFI of the highest level DSA (P < 0.001), whereby ABMR or C4d positivity were moderately predicted by MFI values (area under the receiver operating characteristic curve: 0.75 and 0.84, respectively). In conclusion, the results of this cross-sectional analysis suggest a ≥ 6% prevalence of late ABMR. We demonstrate that the more frequent finding of circulating DSA may not necessarily associate with ABMR diagnosis, especially in patients with low antibody levels.

Characterization of the transcriptome in isolated and transplanted mouse pancreatic islets: associations with engraftment and dysfunction.

The transplantation of pancreatic islets is an option for therapeutic management of hypoglycemia unawareness in select patients with type 1 diabetes mellitus. Characteristics of the transcriptome of freshly isolated islets, islet allografts, and islet isograft are reported in the literature. However, no single experiment has undertaken a comparison of the islet allograft to isograft. Potential implications of the latter are the use in diagnosis of rejection and to discover the molecular pathways in islet allograft dysfunction after transplant. Here, the mouse model of islet transplant is used to characterize the transcriptome of freshly isolated islets and compare islet graft in an isogeneic vs. allogeneic host using an Affymetrix GeneChip® Array assay. A set of islet associated transcripts (IAT) was developed, and subsequently shown to have high level of expression in islet allografts and isografts harvested either five- or ten-days after transplant. Furthermore, specific analysis of transcriptome differences between islet isografts and pre-rejection allografts (ten-day), reveal a series of islet rejection associated transcripts (IRAT). Nearly half of IRAT show overlap with previously described pathogenesis based transcript sets identified in the setting of mouse kidney allograft rejection. The novel transcripts identified to be associated with islet rejection include those involved in chemotaxis or lymphocyte function. Although use of biopsy based monitoring of humans islet transplants remains difficult at the present time, this study provides proof of principle for a transcriptome based technique for islet graft rejection monitoring and describes the transcripts associated with islet graft dysfunction.

Expression of CTL associated transcripts precedes the development of tubulitis in T-cell mediated kidney graft rejection.

The usual phenotype of clinical kidney allograft rejection is infiltration by lymphocytes and macrophages and evolution of histologic Banff lesions, particularly tubulitis, which indicate parenchymal injury. Using Affymetrix microarrays, we evaluated the relationship between the evolution of pathologic lesions and the transcriptome. We studied CBA/J into C57Bl/6 mouse kidney allografts in which one host kidney is left in place to permit observation of lesion development. Histology was dominated by early infiltration by mononuclear cells from day 3 and slower evolution of tubulitis after day 7. We defined a set of cytotoxic T lymphocyte-associated transcripts (CATs) on the basis of expression in purified cytotoxic T lymphocytes (CTL) and in a mixed lymphocyte culture, and absence in normal kidney. CATs were detectable by day 3 and highly expressed by day 5 in rejecting kidneys, with a median signal 14% of that in CTL, compared to 4% in isografts and normal kidneys, and persisted through day 42. Lack of mature B cells had little effect on CAT expression, confirming that CATs reflect T-cell-mediated rejection. Expression of CATs was established before diagnostic lesions and remained remarkably consistent through day 42 despite massive alterations in the pathology, and probably reflects T cells recruited to the graft.

Lesions of T-cell-mediated kidney allograft rejection in mice do not require perforin or granzymes A and B.

Organ allograft rejection is strongly associated with the presence of alloreactive cytotoxic T cells but the role of cytotoxicity in the pathologic lesions is unclear. Previous studies showed that the principal lesions of kidney rejection - interstitial infiltration, tubulitis, and endothelial arteritis - are T-cell-dependent and antibody-independent. We studied the role of cytotoxic granule components perforin and granzymes A and B in the evolution of the T-cell-mediated lesions of mouse kidney transplant rejection. By real-time RT-PCR, allografts rejecting in wild-type hosts at days 5, 7, 21, and 42 showed massively elevated and persistent expression of perforin and granzymes A and B, but evolution of tubulitis and arteritis did not correlate with increasing granzyme or perforin expression. Allografts transplanted into hosts with disrupted genes for perforin or granzymes A and B showed no change in tubulitis, arteritis, or MHC induction. Thus the development of the histologic lesions diagnostic of T-cell-mediated kidney transplant rejection are associated with but not mediated by perforin or granzyme A or B. Together with previous graft survival studies, these results indicate that the granule-associated cytotoxic mechanisms of T cells are not the effectors of T-cell-mediated allograft rejection.

Heterogeneity in the evolution and mechanisms of the lesions of kidney allograft rejection in mice.

The natural history and pathogenesis of the pathologic lesions that define rejection of kidney transplants have not been well characterized. We studied the evolution of the pathology of rejection in mouse kidney allografts, using four strain combinations across full major histocompatibility complex (MHC) plus nonMHC disparities, to permit more general conclusions. Interstitial infiltrate, MHC induction, and venulitis appeared by day 5, peaked at day 7-10, then stabilized or regressed by day 21. In contrast, tubulitis, arteritis, and glomerulitis were absent or mild at days 5 and 7, but progressed through day 21, indicating separate regulation and homeostatic control of these lesions. Edema, hemorrhage, and necrosis also increased through day 21. All lesions were T-dependent, failing to develop in T-cell-deficient hosts. Allografts into immunoglobulin-deficient hosts manifested typical infiltration, MHC induction, and tubulitis at days 7 and 21, indicating that these lesions are alloantibody-independent. However at day 21 kidneys rejecting in immunoglobulin-deficient hosts showed decreased edema, arteritis, venulitis, and necrosis. Thus the three groups of lesions are: T-cell-mediated interstitial infiltration, MHC induction, and venulitis, which develops rapidly then stabilizes; slower but progressive T-cell-mediated tubulitis and arteritis; and late antibody-mediated endothelial injury, which contributes to late edema, arteritis, and venulitis.

Differential usage of class II transactivator promoters PI and PIV during inflammation and injury in kidney.

Expression of class II transactivator (CIITA), the transcriptional regulator that controls all class II expression, is controlled in cell lines in vitro by three promoters: the dendritic cell promoter PI, the B cell promoter PIII, and the interferon-gamma (IFN-gamma)-inducible promoter, PIV. The authors examined the promoter usage in vivo in mouse kidney in the basal state and in response to IFN-gamma, endotoxin, allostimulation, and renal injury. Genetically modified mice were used to examine the dependency of each promoter on IFN-gamma and on the transcription factor interferon regulatory factor 1 (IRF-1). Usage of distinct CIITA promoters was monitored by real-time reverse transcriptase polymerase chain reaction (RT-PCR) using the unique sequences in the 5' end of the transcript from each promoter. Kidneys in both control mice and IFN-gamma knockouts expressed chiefly PI- and PIV-related products. Administration of recombinant IFN-gamma activated only promoter PIV. Endotoxin or allogeneic stimulation elevated the PIV-related mRNA, dependent on IFN-gamma and on IRF-1. Ischemic renal injury, however, increased the PI- and PIV-driven mRNA expression in wild-type but also in IFN-gamma-deficient mice. Thus the in vivo control of CIITA promoters in kidney is similar to that observed in vitro (i.e., basal-state usage of PI and IFN-gamma-dependent usage of PIV during inflammation), but it also shows additional levels of control: IFN-gamma-independent basal activity of PIV and IFN-gamma-independent induction of PIV during tissue injury.

IFN-gamma is an absolute requirement for spontaneous acceptance of liver allografts.

Experimental liver allografts undergo spontaneous acceptance despite undergoing rejection during the first few weeks post transplant. We explored the role of interferon-gamma (IFN-gamma) in the spontaneous acceptance of mouse liver allografts. Strain of mouse (CBA) liver allografts transplanted into normal BALB/c mice developed histologic changes typical of rejection that spontaneously regressed, permitting long-term survival of these allografts similar to that of syngeneic grafts. In contrast, CBA liver allografts in IFN-gamma-deficient hosts manifested not only infiltration but also hemorrhage and necrosis, with no survival beyond 14 days. Despite differences in survival, local expression of cytotoxic T-cell genes in the transplant was not increased in IFN-gamma-deficient hosts, but livers in interferon-gamma-deficient mice (GKO) hosts displayed much less induction of major histocompatibility complex (MHC) class I and II expression. To determine whether the difference in survival was secondary to the direct effects of IFN-gamma on the liver, we transplanted livers from IFN-gamma-receptor-deficient mice into normal hosts. Liver allografts lacking IFN-gamma receptors also developed hemorrhage and necrosis with minimal induction of MHC expression. Thus IFN-gamma mediates a direct effect on rejecting liver allografts that reduces hemorrhage and necrosis, induces MHC expression, and is absolutely required for spontaneous acceptance.

Transcription factor IRF-1 in kidney transplants mediates resistance to graft necrosis during rejection.

In many circumstances kidney transplants remain viable despite extensive inflammation, permitting rejection episodes to be reversed. The mechanisms by which the kidney resists host effector mechanisms are not known. In mouse kidney transplants, resistance requires interferon-gamma (IFN-gamma), which acts on the graft to protect the graft from necrosis during the first days of rejection as well as inducing major histocompatibility complex (MHC) expression. Because some effects of IFN-gamma are mediated by transcription factor IRF-1, the role of IRF-1 in the donor tissue early phases of rejection of mouse kidney allografts was studied. H-2(b) kidneys were transplanted from mice with wild-type IRF-1 genes (WT) or mice with disrupted IRF-1 genes (IRF-1KO) into CBA (H-2(k)) recipients. At day 5 and day 7, IRF-1KO and WT kidneys were functioning despite typical rejection pathology: interstitial infiltration and tubulitis. However, function deteriorated rapidly in rejecting IRF-1KO allografts, associated with widespread epithelial necrosis, peritubular capillary congestion, glomerulitis, and fibrin thrombi in small veins by day 7. At day 21, WT kidneys were viable despite severe tubulitis and arteritis, whereas IRF-1KO kidneys showed massive necrosis of the epithelium despite patent large vessels. Compared with WT kidneys, rejecting IRF-1KO kidneys showed less induction of donor MHC yet had similar mRNA levels of perforin, granzyme B, and Fas ligand and evoked host alloantibody responses. Thus in rejecting kidney transplants, IRF-1 in the graft mediates MHC induction, but it also mediates resistance to necrosis, an effect that could be crucial to permit success in interventions against rejection.

Telomere shortening in kidneys with age.

The histology and function of the kidney deteriorates with age and age-related diseases, but the mechanisms involved in renal aging are not known. In vitro studies suggest that telomere shortening is important in replicative senescence, and is accelerated by stresses that increase replication. This study explored the relationship between age and telomere length in surgical samples from 24 human kidneys, which were either histologically normal (17) or displayed histologic abnormalities (7). Telomere loss was assessed by two independent methods: Southern blotting of terminal restriction fragments (TRF) and slot blotting using telomere-specific probes. The results of these methods correlated with each other. The mean TRF length determined by Southern blotting in cortex was about 12 kb pairs (kbp) in infancy and was shorter in older kidneys. The slope of the regression line was about 0.029 kbp (0.24%, P = 0.023) per year. Telomere DNA loss in cortex by the slot blot method was 0.25% per year (P = 0.011). By both methods, the telomere loss in medulla was not significant and was less than in cortex. Comparisons of TRF length from 20 paired samples from cortex and medulla showed that TRF was greater in cortex than medulla, with the differences being greater in young kidneys and lessening with age due to telomere loss in cortex. These findings indicate that telomeres shorten in an age-dependent manner in the kidney, either due to developmental factors or aging, particularly in renal cortex.