MHC I-associated peptides preferentially derive from transcripts bearing miRNA response elements

MHC I–associated peptides (MIPs) play an essential role in normal homeostasis and diverse pathologic conditions. MIPs derive mainly from defective ribosomal products (DRiPs), a subset of nascent proteins that fail to achieve a proper conformation and the physical nature of which remains elusive. In the present study, we used high-throughput proteomic and transcriptomic methods to unravel the structure and biogenesis of MIPs presented by HLA-A and HLA-B molecules on human EBV-infected B lymphocytes from 4 patients. We found that although HLA-different subjects present distinctive MIPs derived from different proteins, these MIPs originate from proteins that are functionally interconnected and implicated in similar biologic pathways. Secondly, the MIP repertoire of human B cells showed no bias toward conserved versus polymorphic genomic sequences, were derived preferentially from abundant transcripts, and conveyed to the cell surface a cell-type–specific signature. Finally, we discovered that MIPs derive preferentially from transcripts bearing miRNA response elements. Furthermore, whereas MIPs of HLA-disparate subjects are coded by different sets of transcripts, these transcripts are regulated by mostly similar miRNAs. Our data support an emerging model in which the generation of MIPs by a transcript depends on its abundance and DRiP rate, which is regulated to a large extent by miRNAs (read more).

Infusion of High-Dose Intravenous Immunoglobulin Fails to Lower the Strength of Human Leukocyte Antigen Antibodies in Highly Sensitized Patients.

BACKGROUND: Human leukocyte antigen (HLA) sensitization presents a major obstacle for patients awaiting renal transplantation. HLA antibody reduction and favorable transplantation rates have been reported after treatment with high-dose intravenous immunoglobulin (IVIg). METHODS: We enrolled 27 patients whose median flow cytometric calculated panel reactive antibody (CPRA) was 100% and mean wait-list time exceeded 4 years in a protocol whereby high-dose IVIg was administered, HLA antibody profiles of sera obtained before and after treatment were characterized, and cross-match tests were performed with all blood group identical kidney offers. RESULTS: Whereas 12.8% of a similarly sensitized historic control cohort underwent transplantation in the course of a year, 41% of the IVIg-treated group underwent transplantation during the study period. Surprisingly, HLA antibody profiles, measured by CPRA, showed no significant change in response to IVIg treatment. In fact, retrospective cross-match testing using pretreatment sera of those receiving deceased-donor allografts showed that all patients would have been eligible for transplantation with their respective donors before IVIg infusions. CONCLUSIONS: This study does not corroborate previous reports of CPRA reduction leading to increased deceased-donor transplantation rates in broadly sensitized patients undergoing desensitization with high-dose IVIg. The increased rate of transplantation relative to historic controls is not related to improved cross-match eligibility and likely resulted from frequent crossmatching using a cytotoxic strength threshold, improved medical readiness for transplantation, and newly recognized options for live-donor transplantation, all of which could have been achieved without IVIg treatment (read more)

Blood donors implicated in transfusion-related acute lung injury with patient-specific HLA antibodies are more broadly sensitized to HLA antigens compared to other blood donors

BACKGROUND: The prevalence of HLA antibodies in randomly surveyed blood donors was compared to the prevalence of antibody in donors who were associated with transfusion-related acute lung injury (TRALI) cases reported to Canadian Blood Services (CBS).

STUDY DESIGN AND METHODS: Current operating procedure mandates that the CBS TRALI Medical Review Group (TMRG) refer possible TRALI cases to the (CBS) Platelet Immunology Laboratory for investigation. Donor samples from these TRALI cases were screened for HLA antibodies. In parallel, a survey was conducted to screen serum samples from blood donors who were not associated in TRALI cases. A comparison analysis of HLA antibody profiles in the two groups of donors was performed.

RESULTS: We studied 121 TRALI-associated donors (TDs) who were recalled in a total of 44 cases reported to CBS and classified by TMRG. We also studied 149 survey donors (SDs) who were deferred for donation for varied reasons and consented to participate in a survey for HLA antibody screening. Twenty-two percent of SDs and 50.4% of TDs tested positive for HLA antibodies. In addition, TDs who were implicated in TRALI demonstrated broader sensitization and higher level of quantitative HLA antibody compared to nonimplicated TDs and SDs.

CONCLUSION: Patient-specific Class I and II HLA antibodies are directly related to the risk of TRALI. Moreover, it supports the concept that HLA antibody strength is directly related to the risk of TRALI when the HLA antibody is patient specific; however, no clear cutoff as defined by mean fluorescence intensity is evident (read more).

Controversies in kidney paired donation.

Kidney paired donation represented 10% of living kidney donation in the United States in 2011. National registries around the world and several separate registries in the United States arrange paired donations, although with significant variations in their practices. Concerns about ethical considerations, clinical advisability, and the quantitative effectiveness of these approaches in paired donation result in these variations. For instance, although donor travel can be burdensome and might discourage paired donation, it was nearly universal until convincing analysis showed that living donor kidneys can sustain many hours of cold ischemia time without adverse consequences. Opinions also differ about whether the last donor in a chain of paired donation transplants initiated by a nondirected donor should donate immediately to someone on the deceased donor wait-list (a domino or closed chain) or should be asked to wait some length of time and donate to start another sequence of paired donations later (an open chain); some argue that asking the donor to donate later may be coercive, and others focus on balancing the probability that the waiting donor withdraws versus the number of additional transplants if the chain can be continued. Other controversies in paired donation include simultaneous versus nonsimultaneous donor operations, whether to enroll compatible pairs, and interactions with desensitization protocols. Efforts to expand public awareness of and participation in paired donation are needed to generate more transplant opportunities (read more)

Immune self-reactivity triggered by drug-modified HLA-peptide repertoire

Human leukocyte antigens (HLAs) are highly polymorphic proteins that initiate immunity by presenting pathogen-derived peptides to T cells. HLA polymorphisms mostly map to the antigen-binding cleft, thereby diversifying the repertoire of self-derived and pathogen-derived peptide antigens selected by different HLA allotypes. A growing number of immunologically based drug reactions, including abacavir hypersensitivity syndrome (AHS) and carbamazepine-induced Stevens–Johnson syndrome (SJS), are associated with specific HLA alleles. However, little is known about the underlying mechanisms of these associations, including AHS, a prototypical HLA-associated drug reaction occurring exclusively in individuals with the common histocompatibility allele HLA-B*57:01, and with a relative risk of more than 1,000 (refs 6, 7). We show that unmodified abacavir binds non-covalently to HLA-B*57:01, lying across the bottom of the antigen-binding cleft and reaching into the F-pocket, where a carboxy-terminal tryptophan typically anchors peptides bound to HLA-B*57:01. Abacavir binds with exquisite specificity to HLA-B*57:01, changing the shape and chemistry of the antigen-binding cleft, thereby altering the repertoire of endogenous peptides that can bind HLA-B*57:01. In this way, abacavir guides the selection of new endogenous peptides, inducing a marked alteration in ‘immunological self’. The resultant peptide-centric ‘altered self’ activates abacavir-specific T-cells, thereby driving polyclonal CD8 T-cell activation and a systemic reaction manifesting as AHS. We also show that carbamazepine, a widely used anti-epileptic drug associated with hypersensitivity reactions in HLA-B*15:02 individuals, binds to this allotype, producing alterations in the repertoire of presented self peptides. Our findings simultaneously highlight the importance of HLA polymorphism in the evolution of pharmacogenomics and provide a general mechanism for some of the growing number of HLA-linked hypersensitivities that involve small-molecule drugs (read more).

Posttransplant MIC-A Antibodies and Long-Term Graft Outcomes in a Multicenter Cohort of 779 Kidney Transplant Recipients

Background: The impact of major histocompatibility class I chain-related A (MICA) antibodies on renal graft outcomes is unclear. The goal of this work was to assess the impact of posttransplant MICA antibodies, assayed at 1 year, with two commercially available kits, on long-term renal graft outcomes.
Methods: We retrospectively tested sera from 779 kidney transplant recipients with two single-antigen flow bead assays 1 year after transplantation. Samples were considered positive for MICA if they were positive in both tests or positive for MICA specificities that were present in one kit only. The main outcome was 4-year death-censored graft survival.
Results: The prevalence of MICA antibodies was 5.4% at 1 year. MICA+ patients were more frequently human leukocyte antigen (HLA) sensitized and regrafted. Four-year death-censored graft survival was not different between MICA+ and MICA− patients (97% vs. 94%, P=0.28). By Cox multivariate analysis, independent risk factors for graft loss were as follows: number of HLA DR mismatches, acute rejection within the first year posttransplantation, 1-year serum creatinine, and the presence of HLA antibodies at 1 year, but not the presence of MICA antibodies.
Conclusions: These data do not support an independent pathogenic role for MICA in long-term renal graft injury and question the interest of posttransplant monitoring of MICA antibodies with single-antigen flow bead assays currently available (read more).

Plasma bilirubin and late graft failure in renal transplant recipients

Exogenous bilirubin has been shown to protect against oxidative stress in ischemia-reperfusion injury. Oxidative stress has been implicated in the pathophysiology of chronic transplant dysfunction leading to late graft failure after renal transplantation. We prospectively investigated whether high endogenous bilirubin is protective against development of late graft failure in renal transplant recipients (RTR). Baseline data were collected between August 2001 and July 2003 in nonicteric outpatient RTR with a functioning graft for >1 year. At baseline, bilirubin and liver enzymes were measured using routine assays on a Merck Mega analyzer. Graft failure was prospectively recorded until May 19 2009. During follow-up for 7.1[6.2–7.2] years, 55 RTR developed graft failure. We found that circulating levels of bilirubin are inversely associated with late graft failure in RTR (HR = 0.29 [95% CI: 0.16–0.52], P < 0.001). This association was independent of potential confounders, including creatinine clearance, urinary protein excretion, calcineurin inhibitors, and gender (HR = 0.31 [95% CI: 0.15–0.62] P = 0.001). Our findings are consistent with a protective effect of increased endogenous bilirubin against development of late graft failure in RTR. If our findings are confirmed by other studies, intervention with endogenous or exogenous bilirubin may be of interest for long-term preservation of renal function in RTR (read more).

Donor B Cells in Transplants Augment Clonal Expansion and Survival of Pathogenic CD4+ T Cells That Mediate Autoimmune-like Chronic Graft-versus-Host Disease

We reported that both donor CD4⁺ T and B cells in transplants were required for induction of an autoimmune-like chronic graft-versus-host disease (cGVHD) in a murine model of DBA/2 donor to BALB/c recipient, but mechanisms whereby donor B cells augment cGVHD pathogenesis remain unknown. In this study, we report that, although donor B cells have little impact on acute GVHD severity, they play an important role in augmenting the persistence of tissue damage in the acute and chronic GVHD overlapping target organs (i.e., skin and lung); they also markedly augment damage in a prototypical cGVHD target organ, the salivary gland. During cGVHD pathogenesis, donor B cells are activated by donor CD4⁺ T cells to upregulate MHC II and costimulatory molecules. Acting as efficient APCs, donor B cells augment donor CD4⁺ T clonal expansion, autoreactivity, IL-7Rα expression, and survival. These qualitative changes markedly augment donor CD4⁺ T cells’ capacity in mediating autoimmune-like cGVHD, so that they mediate disease in the absence of donor B cells in secondary recipients. Therefore, a major mechanism whereby donor B cells augment cGVHD is through augmenting the clonal expansion, differentiation, and survival of pathogenic CD4⁺ T cells (read more).

New approaches for detecting complement-fixing antibodies.

PURPOSE OF REVIEW: Complement-fixing human leukocyte antigen (HLA) antibodies are a contraindication to solid organ transplant. Newer solid phase assays for HLA antibody definition have created treatment quandaries because many more antibodies are detected by these methods. It is unclear which of the antibodies identified are clinically relevant as all IgG-binding antibodies are detected whether they can fix complement or not. RECENT FINDINGS: Two methods have been developed to assess complement-fixing capability in the solid phase assays: C4d and C1q. These assays, especially the sensitive C1q method, have been reported to more closely correlate with renal and cardiac graft dysfunction, rejection, and graft failure than antibodies detected only by the traditional IgG method. Additionally, the C1q method can be used to predict and monitor desensitization status pre and posttransplant in patients being treated with intravenous immunoglobulin. SUMMARY: The availability of these complement-fixing assays provides new tools for making treatment decisions by discriminating antibodies with known clinical relevance and to assess the clinical relevance of binding antibodies that cannot fix complement. The assays also provide a means of assessing when to transplant a patient undergoing desensitization or when to discontinue augmented immunosuppression for resolving antibody-mediated rejection (read more).

A GPS for finding the route to transplantation for the sensitized patient.

PURPOSE OF REVIEW: To identify factors that affect the choice of route to renal transplantation for the sensitized patient. The evolution of protocols for transplanting sensitized patients has been desensitization (DES), paired donation, and most recently, paired donation combined with DES. Use of these protocols has revealed various factors that influence which route is the most likely to work for a given patient. RECENT FINDINGS: The data indicate that patient blood type and HLA sensitization have the dominant influence on what route is best for a patient but numerous other factors, particularly the number, HLA type, and ABO type of donors a patient brings to a program will also affect the likelihood of transplantation. The distribution of these factors among patients transplanted or unable to find a compatible donor can be used to calculate the probability of transplantation via paired donation. SUMMARY: Kidney paired donation with or without DES provides benefits that cannot be achieved with DES alone. However, DES may provide the fastest route to transplantation (read more)

The role of human leukocyte antigen matching in the development of multiethnic "haplobank" of induced pluripotent stem cell lines.

Among the tools of regenerative medicine, induced pluripotent stem cells (iPSCs) are interesting because the donor genotype can be selected. The construction of banks of iPSC cell lines selected from human leukocyte antigen (HLA) homozygous donors has been proposed to be an effective way to match a maximal number of patients receiving cell therapy from iPSC lines. However, what effort would be required to constitute such a bank for a worldwide application has remained unexplored. We developed a probabilistic model to compute the number of donors to screen for constituting banks of best-chosen iPSC lines with homozygous HLA haplotypes (haplobanks) in four ancestry backgrounds. We estimated what percentage of the patients would be provided with single HLA haplotype matched cell lines. Genetic diversity leads to different outcomes for the four sets in all terms. A bank comprising iPSC lines representing the 20 most frequent haplotypes in each population would request quite different number of donors to screen, between 26,000 for European Americans and 110,000 for African Americans. It would also match different fractions of the recipient population, namely, more than 50% of the European Americans and 22% of African Americans. Conversely, a bank comprising the 100 iPSC lines with the most frequent HLA in each population would leave out only 22% of the European Americans, but 37% of the Asians, 48% of the Hispanics, and 55% of the African Americans. The constitution of a haplobank of iPSC lines is achievable through a large-scale concerted worldwide collaboration (read more).

Identification and expansion of highly suppressive CD8+FoxP3+ regulatory T cells after experimental allogeneic bone marrow transplantation

FoxP3⁺ confers suppressive properties and is confined to regulatory T cells (T_reg) that potently inhibit autoreactive immune responses. In the transplant setting, natural CD4⁺ T_reg are critical in controlling alloreactivity and the establishment of tolerance. We now identify an important CD8⁺ population of FoxP3⁺ T_reg that convert from CD8⁺ conventional donor T cells after allogeneic but not syngeneic bone marrow transplantation. These CD8⁺ T_reg undergo conversion in the mesenteric lymph nodes under the influence of recipient dendritic cells and TGF-β. Importantly, this population is as important for protection from GVHD as the well-studied natural CD4⁺FoxP3⁺ population and is more potent in exerting class I–restricted and antigen-specific suppression in vitro and in vivo. Critically, CD8⁺FoxP3⁺ T_reg are exquisitely sensitive to inhibition by cyclosporine but can be massively and specifically expanded in vivo to prevent GVHD by coadministering rapamycin and IL-2 antibody complexes. CD8⁺FoxP3⁺ T_reg thus represent a new regulatory population with considerable potential to preferentially subvert MHC class I–restricted T-cell responses after bone marrow transplantation (read more).

Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock-out pigs

Background:  Anti-Galα1,3Galβ-R natural antibodies are responsible for hyperacute rejection in pig-to-primate xenotransplantation. Although the generation of pigs lacking the α1,3galactosyltransferase (GalT) has overcome hyperacute rejection, antibody-mediated rejection is still a problem. It is possible that other enzymes synthesize antigens similar to Galα1,3Gal epitopes that are recognized by xenoreactive antibodies. The glycosphingolipid isoglobotrihexosylceramide (iGb₃) represents such a candidate expressing an alternative Galα1,3Gal epitope. The present work determined whether the terminal Galα1,3Gal disaccharide is completely absent in Immerge pigs lacking the GalT using several different highly sensitive methods.

Methods:  The expression of Galα1,3Gal was evaluated using a panel of antibodies and lectins by flow cytometry and fluorescent microscopy; GalT activity was detected by an enzymatic assay; and ion trap mass spectroscopy of neutral cellular membranes extracted from aortic endothelial was used for the detection of sugar structures. Finally, the presence of iGb₃ synthase mRNA was tested by RT-PCR in pig thymus, spleen, lymph node, kidney, lung, and liver tissue samples.

Results:  Aortic endothelial cells derived from GalT knockout pigs expressed neither Galα1,3Gal nor iGb₃ on their surface, and GalT enzymatic activity was also absent. Lectin staining showed an increase in the blood group H-type sugar structures present in GalT knockout cells as compared to wild-type pig aortic endothelial cells (PAEC). Mass spectroscopic analysis did not reveal Galα1,3Gal in membranes of GalT knockout PAEC; iGb₃ was also totally absent, whereas a fucosylated form of iGb₃ was detected at low levels in both pig aortic endothelial cell extracts. Isoglobotrihexosylceramide 3 synthase mRNA was expressed in all pig tissues tested whether derived from wild-type or GalT knockout animals.

Conclusions:  These results confirm unequivocally the absence of terminal Galα1,3Gal disaccharides in GalT knockout endothelial cells. Future work will have to focus on other mechanisms responsible for xenograft rejection, in particular non-Galα1,3Gal antibodies and cellular responses (read more).

Complete Steroid Avoidance Is Effective and Safe in Children With Renal Transplants: A Multicenter Randomized Trial with Three-Year Follow-Up

To determine whether steroid avoidance in pediatric kidney transplantation is safe and efficacious, a randomized, multicenter trial was performed in 12 pediatric kidney transplant centers. One hundred thirty children receiving primary kidney transplants were randomized to steroid-free (SF) or steroid-based (SB) immunosuppression, with concomitant tacrolimus, mycophenolate and standard dose daclizumab (SB group) or extended dose daclizumab (SF group). Follow-up was 3 years posttransplant. Standardized height Z-score change after 3 years follow-up was –0.99 ± 2.20 in SF versus –0.93 ± 1.11 in SB; p = 0.825. In subgroup analysis, recipients under 5 years of age showed improved linear growth with SF compared to SB treatment (change in standardized height Z-score at 3 years –0.43 ± 1.15 vs. –1.07 ± 1.14; p = 0.019). There were no differences in the rates of biopsy-proven acute rejection at 3 years after transplantation (16.7% in SF vs. 17.1% in SB; p = 0.94). Patient survival was 100% in both arms; graft survival was 95% in the SF and 90% in the SB arms (p = 0.30) at 3 years follow-up. Over the 3 year follow-up period, the SF group showed lower systolic BP (p = 0.017) and lower cholesterol levels (p = 0.034). In conclusion, complete steroid avoidance is safe and effective in unsensitized children receiving primary kidney transplants (read more).

Amphiregulin Stimulates Liver Regeneration After Small-for-Size Mouse Liver Transplantation

This study investigated whether amphiregulin (AR), a ligand of the epidermal growth factor receptor (EGFR), improves liver regeneration after small-for-size liver transplantation. Livers of male C57BL/6 mice were reduced to ∼50% and ∼30% of original sizes and transplanted. After transplantation, AR and AR mRNA increased in 50% but not in 30% grafts. 5-Bromodeoxyuridine (BrdU) labeling, proliferating cell nuclear antigen (PCNA) expression and mitotic index increased substantially in 50% but not 30% grafts. Hyperbilirubinemia and hypoalbuminemia occurred and survival decreased after transplantation of 30% but not 50% grafts. AR neutralizing antibody blunted regeneration in 50% grafts whereas AR injection (5 μg/mouse, iv) stimulated liver regeneration, improved liver function and increased survival after transplantation of 30% grafts. Phosphorylation of EGFR and its downstream signaling molecules Akt, mTOR, p70S6K, ERK and JNK increased markedly in 50% but not 30% grafts. AR stimulated EGFR phosphorylation and its downstream signaling pathways. EGFR inhibitor PD153035 suppressed regeneration of 50% grafts and largely abrogated stimulation of regeneration of 30% grafts by AR. AR also increased cyclin D1 and cyclin E expression in 30% grafts. Together, liver regeneration is suppressed in small-for-size grafts, as least in part, due to decreased AR formation. AR supplementation could be a promising therapy to stimulate regeneration of partial liver grafts (read more).

Pretransplant IFN-γ ELISPOT assay as a potential screening test to select immunosuppression protocols for patients receiving basiliximab induction therapy.

The use of basiliximab induction therapy has increased in standard immunological risk patients. The objective of this study was to identify whether pretransplant donor-reactive interferon-γ enzyme-linked immunosorbent spot (ELISPOT) assay results were associated with post-transplant clinical outcomes in patients receiving basiliximab induction therapy and whether this could be helpful for choosing an efficacious immunosuppressive regimen. In 154 living donor renal transplant recipients who received basiliximab induction therapy without desensitization, we determined pretransplant ELISPOT frequencies and correlated the results with clinical outcomes based on the use of calcineurin inhibitors (tacrolimus [TAC] or cyclosporine [CSA]). The ELISPOT (+) patients had higher rate of post-transplant biopsy-proven acute rejection (AR) than ELISPOT (-) patients (P = 0.001) regardless of immunosuppressive regimen. In the logistic and multivariate regression analysis, ELISPOT was the only significant correlate of AR (P = 0.002), and the patients with increased ELISPOT results and CSA therapy were associated with AR. Our results suggest that the pretransplant ELISPOT (+) may assess the risk of poor post-transplant outcomes in patients with basiliximab induction (read more)

The immune-modulator AS101 reduces anti-HLA antibodies in sera of sensitized patients: A structural approach.

BACKGROUND: Significant efforts are dedicated to identification of agents that eliminate anti-HLA antibodies (Ab) from sera of transplant candidates. Antibody titer following in vitro incubation of sera with desensitizing agents has shown to reflect the probability that a patient would benefit from clinical de-sensitization protocols. AS101 is a non-toxic, synthetic, organic tellurium compound. The aim of this research was to assess the ability ofAS101 to reduce anti-HLA Abs and to identify patients likely to benefit from this effect.
METHODS: Sera of sensitized patients awaiting transplant were incubated in the presence of AS101. Measured mean fluorescence intensity (MFI) represents reactivity of anti HLA Abs in the serum, as detected by the Luminex platform. The repertoire of HLA antigen epitopes was recognized using HLA Matchmaker software.
RESULTS: AS101 Incubation caused a significant Ab titer decrease in approximately two thirds of the samples. The median Class I and II MFI decrease among the responding samples was 16.7% and 14%, respectively (p<0.05). HLA Matchmaker analysis of the patients' class I epitope sequences revealed apparent amino-acid differences between the patterns of the responding and non-responding patients.
CONCLUSION: In vitro incubation of sera in the presence of AS101 causes a decrease in the anti-HLA Ab's reactivity in several patient samples. Sera most likely to demonstrate this effect are characterized by a moderate MFI level and a distinct antibody reactivity pattern specific for defined HLA antigen epitopes. These results support further investigation of AS101 as a potential agent for desensitization of humoral reactivity prior to transplantation (read more).

Effect of high-dose intravenous immunoglobulin on anti-HLA antibodies in sensitized kidney transplant candidates

Limited data exist on the effect of intravenous immunoglobulin (IVIg) on anti-HLA antibodies as determined by solid-phase assays. We reviewed our experience treating sensitized wait-listed kidney transplant recipients with IVIg as a method for desensitization and report our results utilizing Luminex single antigen (LSA) bead assay to quantify antibody reactivity (MFI). Fifteen patients with a cPRA > 40% received 2 g/kg IVIg per month for four months or until transplanted. LSA testing was performed before and after IVIg. Median MFI for anti-class I antibodies fell in 11 (73%) and increased in 4 (27%) patients after IVIg. Similar significant changes in MFI for anti-class II antibodies were observed in 10 patients (66%). Administration of IVIg was associated with a modest decrease in reactivity to both class I and II HLA antigens (median MFI change 493 and 1110, respectively; p < 0.0001) but did not significantly alter mean cPRA (85% before IVIg vs. 80% after IVIg; p = 0.1). Our data suggest a smaller effect of IVIg on HLA antibody reactivity than previously described, leading us to question how best to measure the efficacy of a desensitization protocol in current practice (read more).

Using Donor-Specific Antibodies to Monitor the Need for Immunosuppression

Background: Experience with tolerance protocols has shown that none is perfect and that each escape from tolerance must be identified early to prevent graft failure. In addition, some test is needed for patients who are weaned off immunosuppression (IS) to forewarn of weaning failure. The usual measures of function—such as serum creatinine levels—are not sensitive enough to detect rejection in a timely manner.
Methods: A study was carried out on 72 patients who received living-donor kidney transplants with clonal deletion protocol (total lymphoid irradiation or bortezomib), and followed with reduced doses of maintenance IS. Every month or every 2 months, a test was performed for donor-specific antibodies (DSA) using Luminex mixed and/or single antigen beads.
Results: After transplantation, DSA developed in 17% of the patients at 6 months, 41% at 1 year, and 57% at 2 years, with 95% confidence limits of 10%, 28%; 30%, 55%; and 44%, 71%, respectively. Fifty-three percent of patients weaned IS to less than 10 mg prednisone daily experienced DSA within 3 months. Furthermore, prednisone dose (per 2.5 mg) and years after transplantation were inversely associated with DSA production (risk ratio 0.92 [95% confidence limits: 0.85, 0.99], and 0.70 [0.49, 1.00]).
Conclusions: DSA monitoring is highly effective for detecting escape from tolerance and reemergence of the immune response in weaned patients. DSA appearance was inversely proportional to the level of maintenance drugs in the weaning process. Measurement of DSA on a monthly basis is adequate for detection of the change in immune reactivity (read more).

Allocation of Vascularized Composite Allografts: What Is It?

To date, there are more than 80 reports of vascularized composite allografts (VCAs; a.k.a. composite tissue allotransplantation). However, the classification of this type of transplantation for oversight purposes has not been clarified. From a biological and regulatory perspective, we propose that VCA retrieval and transplantation is akin to organ transplantation and should be incorporated into the organ oversight structure. As VCA becomes more of a clinical reality, the need for a methodical approach to allocation and access to more donors will develop. Such an allocation system will likely incorporate parameters that deviate from those used for organ transplantation. To develop an effective and balanced system, the use of existing regulatory agencies that oversee solid organs should provide the maximum benefit to the patients and the society (read more).

CD47 functions as a molecular switch for erythrocyte phagocytosis

CD47 on erythrocytes inhibits phagocytosis through interaction with the inhibitory immunoreceptor SIRPα expressed by macrophages. Thus, the CD47-SIRPα interaction constitutes a negative signal for erythrocyte phagocytosis. However, we report here that CD47 does not only function as a "do not eat me" signal for uptake but can also act as an "eat me" signal. In particular, a subset of old erythrocytes present in whole blood was shown to bind and to be phagocytosed via CD47-SIRPα interactions. Furthermore, we provide evidence that experimental aging of erythrocytes induces a conformational change in CD47 that switches the molecule from an inhibitory signal into an activating one. Preincubation of experimentally aged erythrocytes with human serum before the binding assay was required for this activation. We also demonstrate that aged erythrocytes have the capacity to bind the CD47-binding partner thrombospondin-1 (TSP-1) and that treatment of aged erythrocytes with a TSP-1–derived peptide enabled their phagocytosis by human red pulp macrophages. Finally, CD47 on erythrocytes that had been stored for prolonged time was shown to undergo a conformational change and bind TSP-1. These findings reveal a more complex role for CD47-SIRPα interactions in erythrocyte phagocytosis, with CD47 acting as a molecular switch for controlling erythrocyte phagocytosis (read more and editorial).

Antibody Mediated Rejection Associated With Complement Factor H-Related Protein 3/1 Deficiency Successfully Treated With Eculizumab

Antibody mediated rejection (AMR) activates the classical complement pathway and can be detrimental to graft survival. AMR can be accompanied by thrombotic microangiopathy (TMA). Eculizumab, a monoclonal C5 antibody prevents induction of the terminal complement cascade (TCC) and has recently emerged as a therapeutic option for AMR. We present a highly sensitized 13-year-old female with end-stage kidney disease secondary to spina bifida-associated reflux nephropathy, who developed severe steroid-, ATG- and plasmapheresis-resistant AMR with TMA 1 week post second kidney transplant despite previous desensitization therapy with immunoglobulin infusions. Eculizumab rescue therapy resulted in a dramatic improvement in biochemical (C3; creatinine) and hematological (platelets) parameters within 6 days. The patient was proven to be deficient in complement Factor H-related protein 3/1 (CFHR3/1), a plasma protein that regulates the complement cascade at the level of C5 conversion and has been involved in the pathogenesis of atypical hemolytic uremic syndrome caused by CFH autoantibodies (DEAP-HUS). CFHR1 deficiency may have worsened the severe clinical progression of AMR and possibly contributed to the development of donor-specific antibodies. Thus, screening for CFHR3/1 deficiency should be considered in patients with severe AMR associated with TMA (read more).

Bortezomib for Acute Antibody-Mediated Rejection in Liver Transplantation

Antibody-mediated rejection (AMR) is an uncommon, but challenging type of rejection after solid organ transplantation. We review three cases of AMR in ABO-compatible liver transplant recipients. These cases were characterized by severe acute rejection resistant to steroids and antithymocyte globulin, histologic evidence of plasma cell infiltrates, C4d positivity and high serum anti-HLA donor-specific antibodies. All three patients were treated with bortezomib, a proteasome inhibitor effective in depleting plasma cells. After treatment, all patients had improved or normal liver function tests, resolution of C4d deposition and significant decline in their HLA donor-specific antibodies (read more).

Randomized Phase 2b Trial of Tofacitinib (CP-690,550) in De Novo Kidney Transplant Patients: Efficacy, Renal Function and Safety at 1 Year

In this Phase 2b study, 331 low-to-moderate risk de novo kidney transplant patients (approximately 60% deceased donors) were randomized to a more intensive (MI) or less intensive (LI) regimen of tofacitinib (CP-690, 550), an oral Janus kinase inhibitor or cyclosporine (CsA). All patients received basiliximab induction, mycophenolic acid and corticosteroids. Primary endpoints were: incidence of biopsy-proven acute rejection (BPAR) with a serum creatinine increase of ≥0.3 mg/dL and ≥20% (clinical BPAR) at Month 6 and measured GFR at Month 12. Similar 6-month incidences of clinical BPAR (11%, 7% and 9%) were observed for MI, LI and CsA. Measured GFRs were higher (p < 0.01) at Month 12 for MI and LI versus CsA (65 mL/min, 65 mL/min vs. 54 mL/min). Fewer (p < 0.05) patients in MI or LI developed chronic allograft nephropathy at Month 12 compared with CsA (25%, 24% vs. 48%). Serious infections developed in 45%, 37% and 25% of patients in MI, LI and CsA, respectively. Anemia, neutropenia and posttransplant lymphoproliferative disorder occurred more frequently in MI and LI compared with CsA. Tofacitinib was equivalent to CsA in preventing acute rejection, was associated with improved renal function and less chronic allograft histological injury, but had side-effects at the doses evaluated (read more).

Lymphocytic Bronchiolitis After Lung Transplantation Is Associated With Daily Changes in Air Pollution

Acute rejection represents a major problem after organ transplantation, being a recognized risk for chronic rejection and mortality. Recently, it became clear that lymphocytic bronchiolitis (LB, B-grade acute rejection) is more important than previously thought, as it predisposes to chronic rejection. We aimed to verify whether daily fluctuations of air pollution, measured as particulate matter (PM) are related to histologically proven A-grade rejection and/or LB and bronchoalveolar lavage (BAL) fluid cellularity after lung transplantation. We fitted a mixed model to examine the association between daily variations in PM₁₀ and A-grade rejection/LB on 1276 bronchoscopic biopsies (397 patients, 416 transplantations) taken between 2001 and 2011. A difference of 10 μg/m³ in PM₁₀ 3 days before diagnosis of LB was associated with an OR of 1.15 (95% CI 1.04–1.27; p = 0.0044) but not with A-grade rejection (OR = 1.05; 95% CI 0.95–1.15; p = 0.32). Variations in PM₁₀ at lag day 3 correlated with neutrophils (p = 0.013), lymphocytes (p = 0.0031) and total cell count (p = 0.024) in BAL. Importantly, we only found an effect of PM10 on LB in patients not taking azithromycin. LB predisposed to chronic rejection (p < 0.0001). The risk for LB after lung transplantation increased with temporal changes in particulate air pollution, and this was associated with BAL neutrophilia and lymphocytosis. Azithromycin was protective against this PM effect (read more).

Compound heterozygosity of HLA-DRB3*01:01 and HLA-DRB4*01:01 as a potential predictor of fetal neonatal alloimmune thrombocytopenia

BACKGROUND: Fetal neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder in the fetus or neonate caused by maternal alloantibodies directed against fetal platelet (PLT) antigens inherited from the father. The immune-dominant antigen leading to severe FNAIT is the human PLT antigen (HPA)-1, whose polymorphism constitutes an epitope for human leukocyte antigens (HLAs), usually DRB3*0101 leading to an immune response.

STUDY DESIGN AND METHODS: In this study our aims were to find whether other allele variants of the β subunit of the HLA-DR family specifically focused on the HLA residues that bind Position 33 of the HPA-1 integrin contribute to FNAIT development and affect response to treatment and whether coexistence of both anti-HPA-1a and anti-HLA Class I specific against the father's antigens leads to a more severe thrombocytopenia in the newborn. We examine the genotype of 23 mothers to newborns with FNAIT compared to a control group.

RESULTS: Our results suggested that, when HPA-1 incompatibility with the husband is found, the presence of two HLA alleles (DRB3*01:01 and DRB4*01:01) in the mother increases the risk and severity of FNAIT and reduces the success of a preventive immunoglobulin G treatment. We provide a structural model for the molecular basis of the rational effects of the different HLA alleles. In addition, we found that the presence of both anti-HPA-1 and anti-HLAs did not aggravate FNAIT in comparison to mothers harboring only anti-HPA-1.

CONCLUSION: Overall, we suggest that a specific genotyping of the mother in relation to HLA-DRB as well as HPA-1 can serve as an antenatal diagnostic tool, particularly in siblings of women who gave birth to neonates with FNAIT (read more).

HLA-B27 Homodimers and Free H Chains Are Stronger Ligands for Leukocyte Ig-like Receptor B2 than Classical HLA Class I

Possession of HLA-B27 (B27) strongly predisposes to the development of spondyloarthritis. B27 forms classical heterotrimeric complexes with β₂-microglobulin (β2m) and peptide and (β2m free) free H chain (FHC) forms including B27 dimers (termed B27₂) at the cell surface. In this study, we characterize the interaction of HLA-B27 with LILR, leukocyte Ig-like receptor (LILR)B1 and LILRB2 immune receptors biophysically, biochemically, and by FACS staining. LILRB1 bound to B27 heterotrimers with a K_D of 5.3 ± 1.5 μM but did not bind B27 FHC. LILRB2 bound to B27₂ and B27 FHC and B27 heterotrimers with K_Ds of 2.5, 2.6, and 22 ± 6 μM, respectively. Domain exchange experiments showed that B27₂ bound to the two membrane distal Ig-like domains of LILRB2. In FACS staining experiments, B27 dimer protein and tetramers stained LILRB2 transfectants five times more strongly than B27 heterotrimers. Moreover, LILRB2Fc bound to dimeric and other B27 FHC forms on B27-expressing cell lines more strongly than other HLA-class 1 FHCs. B27-transfected cells expressing B27 dimers and FHC inhibited IL-2 production by LILRB2-expressing reporter cells to a greater extent than control HLA class I transfectants. B27 heterotrimers complexed with the L6M variant of the GAG KK10 epitope bound with a similar affinity to complexes with the wild-type KK10 epitope (with K_Ds of 15.0 ± 0.8 and 16.0 ± 2.0 μM, respectively). Disulfide-dependent B27 H chain dimers and multimers are stronger ligands for LILRB2 than HLA class I heterotrimers and H chains. The stronger interaction of B27 dimers and FHC forms with LILRB2 compared with other HLA class I could play a role in spondyloarthritis pathogenesis (read more).

A CD3+ count-based thymoglobulin induction regimen permits delayed introduction of calcineurin inhibitors in kidney transplantation

Withholding calcineurin inhibitors (CNIs) can be considered when graft function is inadequate following kidney transplantation (KT). Thymoglobulin (rATG) can be used to prevent acute rejection while CNIs are being withheld. Here, we report our results of a novel CNI-sparing induction protocol, which utilizes a CD3+ cell count-based rATG treatment regimen when delayed graft function (DGF) develops in the immediate postoperative period. In a cohort of 153 consecutive deceased-donor KT recipients, all received a single intraoperative dose of basiliximab; 84 subsequently developed DGF and therefore received rATG (rATG+ group), while 69 demonstrated immediate graft function and received CNIs (rATG− group). In the rATG+ group, mean duration of therapy was 8.5 ± 6.0 d, permitting CNI initiation to be delayed until postoperative day 10.3 ± 6.2. Cumulative dose of rATG was only 5.1 ± 4.5 mg/kg while targeting CD3+ counts of ≤30 cells/mm³. CD3+ counts were reduced to a mean of 16.7 ± 17.0 cells/mm³ during therapy. At one yr, patient and graft survival rates were 97.6% and 92.9%, respectively, while the frequency of infections and malignancies were not significantly increased compared to the rATG− group. A unique induction regimen successfully delayed CNI initiation by using modest doses of rATG to deplete CD3+ cells, while yielding excellent long-term graft outcome without increased risk of infection or malignancy (read more).

Human leukocyte antigen mismatches associated with increased risk of rejection, graft failure, and death independent of initial immunosuppression in renal transplant recipients

Human leukocyte antigen (HLA) mismatches have been shown to adversely affect renal allograft outcomes and remain an important component of the allocation of deceased donor (DD) kidneys. The ongoing importance of HLA mismatches on transplant outcomes in the era of more potent immunosuppression remains debatable. Using Australia and New Zealand Dialysis and Transplant Registry, live and DD renal transplant recipients between 1998 and 2009 were examined. The association between the number of HLA mismatches and HLA-loci mismatches and outcomes were examined. Of the 8036 renal transplant recipients, 59% had between 2 and 4 HLA mismatches. Compared with 0 HLA mismatch, increasing HLA mismatches were associated with a higher risk of graft failure and patient death in the adjusted models. HLA mismatches were associated with an incremental risk of rejection although the relative risk was higher for live donor kidney transplants. Increasing HLA-AB and HLA-DR mismatches were associated with a greater risk of acute rejection, graft failure, death-censored graft failure, and/or death. There was no consistent association between initial immunosuppressive regimen and outcomes. Our results corroborate and extend the previous registry analyses demonstrating that HLA mismatches are associated with poorer transplant outcomes independent of immunosuppression and transplant era (read more).