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Transplantation is a life-saving medical intervention that unfortunately is constrained by scarcity of available organs. An ideal system for allocating organs should seek to achieve the greatest good for the greatest number of people. It also must be fair and not disadvantage certain populations. However, policies aimed at reducing disparities also must be balanced with considerations of utility (graft outcomes), cost, efficiency, and any adverse effects on organ utilization. Here, we discuss the ethical challenges of creating a fair and equitable organ allocation system, focusing on the principles governing deceased donor kidney transplant waitlists around the world. The kidney organ allocation systems in the United States, Australia, and Hong Kong are used as illustrations.

Human Leukocyte Antigen (HLA) diversity is the key driver of alloimmune responses. Ideally, patients would receive an allograft that is fully matched at the allelic level. However, the extensive polymorphism in the HLA loci renders this impractical. Thus, there is growing interest in determining whether HLA mismatches at the eplet/epitope level better reflects the true disparity between a donor-recipient pair, with the goal of predicting permissible mismatches versus those that should be avoided because they will elicit a strong alloimmune response. Here, we will discuss the available algorithms used to predict immunogenicity/antigenicity of mismatches and prognosticate graft outcomes.

Induction of longstanding immunologic tolerance is essential for survival of transplanted organs and tissues. Despite recent advances in immunosuppression protocols, allograft damage inflicted by antibody specific for donor organs continues to represent a major obstacle to graft survival. Here we report that activation of regulatory CD8 T cells (CD8 Treg) that recognize the Qa-1 class Ib major histocompatibility complex (MHC), a mouse homolog of human leukocyte antigen-E (HLA-E), inhibits antibody-mediated immune rejection of heart allografts. We analyzed this response using a mouse model that harbors a point mutation in the class Ib MHC molecule Qa-1, which disrupts Qa-1 binding to the T cell receptor (TCR)-CD8 complex and impairs the CD8 Treg response. Despite administration of cytotoxic T lymphocyte antigen 4 (CTLA-4) immunoglobulin (Ig), Qa-1 mutant mice developed robust donor-specific antibody responses and accelerated heart graft rejection. We show that these allo-antibody responses reflect diminished Qa-1-restricted CD8 Treg-mediated suppression of host follicular helper T cell-dependent antibody production. These findings underscore the critical contribution of this Qa-1/HLA-E-dependent regulatory pathway to maintenance of transplanted organs and suggest therapeutic approaches to ameliorate allograft rejection.

Adaptive immunity can target a nearly infinite range of antigens, yet it is tempered by tolerogenic mechanisms that limit autoimmunity. Such immunological tolerance, however, creates a gap in adaptive immunity against microbes decorated with self-like antigens as a form of molecular mimicry. Our results demonstrate that the innate immune lectin galectin-7 (Gal-7) binds a variety of distinct microbes, all of which share features of blood group-like antigens. Gal-7 binding to each blood group expressing microbe, including strains of

T cells play a pivotal role in orchestrating immune responses directed against a foreign (allogeneic) graft. For T cells to become fully activated, the T-cell receptor (TCR) must interact with the major histocompatibility complex (MHC) plus peptide complex on antigen-presenting cells (APCs), followed by a second "positive" costimulatory signal. In the absence of this second signal, T cells become anergic or undergo deletion. By blocking positive costimulatory signaling, T-cell allo-responses can be aborted, thus preventing graft rejection and promoting long-term allograft survival and possibly tolerance (Alegre ML, Najafian N, Curr Mol Med 6:843-857, 2006; Li XC, Rothstein DM, Sayegh MH, Immunol Rev 229:271-293, 2009). In addition, costimulatory molecules can provide negative "coinhibitory" signals that inhibit T-cell activation and terminate immune responses; strategies to promote these pathways can also lead to graft tolerance (Boenisch O, Sayegh MH, Najafian N, Curr Opin Organ Transplant 13:373-378, 2008). However, T-cell costimulation involves an incredibly complex array of interactions that may act simultaneously or at different times in the immune response and whose relative importance varies depending on the different T-cell subsets and activation status. In transplantation, the presence of foreign alloantigen incites not only destructive T effector cells but also protective regulatory T cells, the balance of which ultimately determines the fate of the allograft (Lechler RI, Garden OA, Turka LA, Nat Rev Immunol 3:147-158, 2003). Since the processes of alloantigen-specific rejection and regulation both require activation of T cells, costimulatory interactions may have opposing or synergistic roles depending on the cell being targeted. Such complexities present both challenges and opportunities in targeting T-cell costimulatory pathways for therapeutic purposes. In this chapter, we summarize our current knowledge of the various costimulatory pathways in transplantation and review the current state and challenges of harnessing these pathways to promote graft tolerance (summarized in Table 10.1).

Background: Calculated panel reactive antibody (cPRA) scoring is used to assess whether platelet refractoriness is mediated by human leukocyte antigen (HLA) antibodies in the recipient. cPRA testing uses a national sample of US kidney donors to estimate the population frequency of HLA antigens, which may be different than HLA frequencies within local platelet inventories. We aimed to determine the impact on patient cPRA scores of using HLA frequencies derived from typing local platelet donations rather than national HLA frequencies. Methods: We built an open-source web service to calculate cPRA scores based on national frequencies or custom-derived frequencies. We calculated cPRA scores for every hematopoietic stem cell transplantation (HSCT) patient at our institution based on the United Network for Organ Sharing (UNOS) frequencies and local frequencies. We compared frequencies and correlations between the calculators, segmented by gender. Finally, we put all scores into three buckets (mild, moderate, and high sensitizations) and looked at intergroup movement. Results: 2531 patients that underwent HSCT at our institution had at least 1 antibody and were included in the analysis. Overall, the difference in medians between each group's UNOS cPRA and local cPRA was statistically significant, but highly correlated (UNOS vs. local total: 0.249 and 0.243, ρ = 0.994; UNOS vs. local female: 0.474 and 0.463, ρ = 0.987, UNOS vs. local male: 0.165 and 0.141, ρ = 0.996; P < 0.001 for all comparisons). The median difference between UNOS and cPRA scores for all patients was low (male: 0.014, interquartile range [IQR]: 0.004–0.029; female: 0.0013, IQR: 0.003–0.028). Placement of patients into three groups revealed little intergroup movement, with 2.96% (75/2531) of patients differentially classified. Conclusions: cPRA scores using local frequencies were modestly but significantly different than those obtained using national HLA frequencies. We released our software as open source, so other groups can calculate cPRA scores from national or custom-derived frequencies. Further investigation is needed to determine whether a local-HLA frequency approach can improve outcomes in patients who are immune-refractory to platelets.

For over thirty years, antibody (mAb)-based therapies have been a standard component of transplant immunosuppression, and yet much remains to be learned in order for us to truly harness their therapeutic capabilities. Current mAbs used in transplant directly target and destroy graft-destructive immune cells, interrupt cytokine and costimulation-dependent T and B cell activation, and prevent down-stream complement activation. Areas covered: This review summarizes our current approaches to using antibody-based therapies to prevent and treat allograft rejection. It also provides examples of promising novel mAb therapies, and discusses the potential for future mAb development in transplantation. Expert opinion: The broad capability of antibodies, in parallel with our growing ability to synthetically modulate them, offers exciting opportunities to develop better biologic therapeutics. In order to do so, we must further our understanding about the basic biology underlying allograft rejection, and gain better appreciation of how characteristics of therapeutic antibodies affect their efficacy.

B cells play a central role in antibody-mediated rejection and certain autoimmune diseases. However, B cell-targeted therapy such as anti-CD20 B cell-depleting antibody (aCD20) has yielded mixed results in improving outcomes. In this study, we investigated whether an accelerated B cell reconstitution leading to aCD20 depletion resistance could account for these discrepancies. Using a transplantation model, we found that antigen-independent inflammation, likely through toll-like receptor (TLR) signaling, was sufficient to mitigate B cell depletion. Secondary lymphoid organs had a quicker recovery of B cells when compared to peripheral blood. Inflammation altered the pharmacokinetics (PK) and pharmacodynamics (PD) of aCD20 therapy by shortening drug half-life and accelerating the reconstitution of the peripheral B cell pool by bone marrow-derived B cell precursors. IVIG (intravenous immunoglobulin) coadministration also shortened aCD20 drug half-life and led to accelerated B cell recovery. Repeated aCD20 dosing restored B cell depletion and delayed allograft rejection, especially B cell-dependent, antibody-independent allograft rejection. These data demonstrate the importance of further clinical studies of the PK/PD of monoclonal antibody treatment in inflammatory conditions. The data also highlight the disconnect between B cell depletion on peripheral blood compared to secondary lymphoid organs, the deleterious effect of IVIG when given with aCD20 and the relevance of redosing of aCD20 for effective B cell depletion in alloimmunity.

T cells play a pivotal role in orchestrating immune responses directed against a foreign (allogeneic) graft. For T cells to become fully activated, the T-cell receptor (TCR) must interact with the major histocompatibility complex (MHC) plus peptide complex on antigen-presenting cells (APCs), followed by a second “positive” costimulatory signal. In the absence of this second signal, T cells become anergic or undergo deletion. By blocking positive costimulatory signaling, T-cell allo-responses can be aborted, thus preventing graft rejection and promoting long-term allograft survival and possibly tolerance (Alegre ML, Najafian N, Curr Mol Med 6:843–857, 2006; Li XC, Rothstein DM, Sayegh MH, Immunol Rev 229:271–293, 2009). In addition, costimulatory molecules can provide negative “coinhibitory” signals that inhibit T-cell activation and terminate immune responses; strategies to promote these pathways can also lead to graft tolerance (Boenisch O, Sayegh MH, Najafian N, Curr Opin Organ Transplant 13:373–378, 2008). However, T-cell costimulation involves an incredibly complex array of interactions that may act simultaneously or at different times in the immune response and whose relative importance varies depending on the different T-cell subsets and activation status. In transplantation, the presence of foreign alloantigen incites not only destructive T effector cells but also protective regulatory T cells, the balance of which ultimately determines the fate of the allograft (Lechler RI, Garden OA, Turka LA, Nat Rev Immunol 3:147–158, 2003). Since the processes of alloantigen-specific rejection and regulation both require activation of T cells, costimulatory interactions may have opposing or synergistic roles depending on the cell being targeted. Such complexities present both challenges and opportunities in targeting T-cell costimulatory pathways for therapeutic purposes. In this chapter, we summarize our current knowledge of the various costimulatory pathways in transplantation and review the current state and challenges of harnessing these pathways to promote graft tolerance (summarized in Table 10.1).

Supplemental Digital Content is available in the text., Background. Many kidney transplant centers in the United States report both HLA class I and II antibodies detected by sensitive solid-phase assays (SPAs) to United Network for Organ Sharing as unacceptable antigens, significantly reducing the compatible donor organ pool and prolonging waiting time for highly sensitized patients. However, the clinical relevance of all detected donor-specific antibodies (DSAs) by SPA is not unequivocal, because fluorescence intensity does not always accurately reflect antibody pathogenicity. Our center does not exclude patients from transplantation based on DSA class II. Methods. We performed a retrospective analysis in 179 deceased-donor kidney transplant recipients with solely DSA class II before transplant and patients without DSA and compared graft survival, rejection, and clinical outcomes. Patient survival was also compared with matched controls on the waiting list. Results. Patients transplanted with DSA class II showed a clear survival benefit compared with matched patients who remained on dialysis or were waitlisted on dialysis/transplanted at 5 years (100%, 34%, and 73%, respectively). After a mean follow-up of 5.5 years, there was no significant difference in death-censored graft survival between transplanted patients without DSA and those with preformed DSA class II (adjusted HR 1.10; 95% confidence interval, 0.41–2.97), although the incidence of rejection was higher in recipients with DSA class II (adjusted HR 5.84; 95% confidence interval, 2.58–13.23; P < 0.001). Serum creatinine levels at 1, 3, and 5 years posttransplant did not differ between groups. No predictors of rejection were found, although patients who received basiliximab induction therapy had higher incidence of rejection (100%) compared with those who received antithymocyte globulin (52%). Conclusions. We conclude that for highly sensitized patients, deceased-donor kidney transplantation with DSA class II yields a survival benefit over prolonged waiting time on dialysis. Instead of listing DSA class II as unacceptable antigens, an individual approach with further immunologic risk assessment is recommended.

Purpose: The programmed death-1/programmed death ligand (PD-1/PD-L) pathway in T cell activation has been shown to play an important role in tumor evasion from host immunity. The predictive value of PD-L1 and PDL2 expression in colorectal cancer (CRC) remains still under discussion. We analyzed whether negative signaling of infiltrating PD-1-positive T cells through PD-L1 and PD-L2 within the tumor could promote further tumor progression through downregulation of anti-tumor immunity. Methods: We investigated PD-L1 and PD-L2 expression in tumors from patients with CRC and analyzed its prognostic significance with respect to outcome analysis. Results: T cell infiltration was observed in 90.5% of the tumors, with 58% of the patients demonstrating PD-1- positive T cells in their tumors. Patients who developed PD-1-positive T cell infiltration showed increased PD-L1- expression within their tumors than PD-1-T cell negative individuals. Presence of tumor infiltrating PD-1-positive T cells was more pronounced in advanced stage cancers than in early cancers. Ligand expression (PD-L1/PD-L2) in the tumors combined with dense PD-1-positive T cell infiltration was associated with poor prognosis. Multivariate analysis demonstrated that PD-L expression in the tumors was an independent prognostic factor in CRC. Conclusion: The presented results from primary tumors and CRC patient outcome analysis suggest that negative signaling of infiltrating PD-1-positive T cells through PD-L1 expression within the tumor may promote further tumor progression through downregulation of anti-tumor immunity. Co-expression of PD-1 on CD4/Foxp3- positive T cells was found indicating T regulatory cell-mediated mechanisms by which tumor cells can evade immune recognition and destruction. This study demonstrates the importance of strategies inhibiting negative PD-1/ PD-L1 signaling in CRC.

Background: A number of studies have demonstrated the role of CX3CR1 in regulating the migration of monocytes into peripheral tissue and their transformation into DC. No data are yet available on the importance of chemokine pathways in regulating homeostasis of dendritic cell (DC) in heart transplants. Recently, we showed that recipients of heart allografts from CXC3R1-/- donors show longer survival. To dissect the role of CX3CR1 signaling in the homeostasis of heart dDC, we have developed and tested a novel in vivo imaging tool in CX3CR1GFP/+ DC (B6 background) heart graft into BALB/c recipient model. Results: Majority of GFP positive cells located in the middle of cardiac myocyte and their shapes were stretching and still roundish in the early phase of post transplant (3 and 24hours). However, images from 72hours at cardiac graft showed many of GFP positive cells move to vessel areas. In addition, significant immunological events such as GFP positive cell exiting from cardiac myocyte and changes of GFP positive cell motility and shape were detected. GFP positive cells were detected in the vasculature and lymph nodes. Only 1 GFP positive cell was observed in three lymph nodes (2 mesenteric, 1 inguinal) (72hours). Conclusions: Current data will define the function of dDC in regulating alloimmune responses in vivo and provide information to develop novel strategies designed to achieve durable tolerance and prevent chronic rejection.

we here present a miniature suction-assisted endoscope suitable for imaging cellular events in the beating heart graft of the mouse and we demonstrated the applicability of our imaging method for a wide range of cardiovascular research (1-3). Over the past decade, intravital optical microscopy (IVM) has emerged as a powerful tool in animal research. By monitoring the complex behavior of cells, it will possible to answer many of outstanding questions in the field. Despite their earlier success with openheart vasculature imaging (4), This article is protected by copyright. All rights reserved.

Background A number of studies have demonstrated the role of CX3CR1 in regulating the migration of monocytes into peripheral tissue and their transformation into dendritic cell (DC). No data are yet available on the importance of chemokine pathways in regulating homeostasis of DC in heart transplants. Recently, we showed that recipients of heart allografts from CX3CR1−/− donors show longer survival. To assess the trafficking of dDC, we have developed and tested a novel in vivo imaging tool in CX3CR1GFP/+ DC (B6 background) heart graft into BALB/c recipient model. Results Majority of GFP+ cells were noted in the middle of cardiac myocyte. However few hours post transplant, they experienced morphological changes including stretching their extensions (3 and 24 h). However, images from 72 h at cardiac graft showed many of GFP+ cells moved to vessel areas. GFP+ cells were detected in near vessel wall. Only one GFP+ cell was observed in three lymph nodes (two mesenteric and one inguinal) (72 h). Conclusion Our data indicate that immediately post transplant dDC undergo morphological changes and traffic out of the organs via systemic circulation. While, we still noted presence of dDC in the transplanted organs, their trafficking to lymphoid tissue remains to be fully explored.

Ligands of the B7 family provide both positive and negative costimulatory signals to the CD28 family of receptors on T lymphocytes, the balance of which determines the immune response. B7-H3 is a member of the B7 family whose function in T-cell activation has been the subject of some controversy: in autoimmunity and tumor immunity, it has been described as both costimulatory and coinhibitory, while in transplantation, B7-H3 signaling is thought to contribute to graft rejection. However, we now demonstrate results to the contrary. Signaling through a putative B7-H3 receptor prolonged allograft survival in a fully MHC-mismatched cardiac model and promoted a shift toward a Th2 milieu; conversely, B7-H3 blockade, achieved by use of a blocking antibody, resulted in accelerated rejection, an effect associated with enhanced IFN-γ production. Finally, graft prolongation achieved by CTLA4 Ig was shortened both by B7-H3 blockade and the absence of recipient B7-H3. These findings suggest a coinhibitory role for B7-H3. However, experience with other CD28/B7 family members suggests that immune redundancy plays a crucial role in determining the functions of various pathways. Given the abundance of conflicting data, it is plausible that, under differing conditions, B7-H3 may have both positive and negative costimulatory functions.

T cell Ig domain and mucin domain protein 1 (TIM-1) is a costimulatory molecule that regulates immune responses by modulating CD4+ T cell effector differentiation. However, the function of TIM-1 on other immune cell populations is unknown. Here, we show that in vivo in mice, TIM-1 is predominantly expressed on B rather than T cells. Importantly, TIM-1 was expressed by a large majority of IL-10-expressing regulatory B cells in all major B cell subpopulations, including transitional, marginal zone, and follicular B cells, as well as the B cell population characterized as CD1d(hi)CD5+. A low-affinity TIM-1-specific antibody that normally promotes tolerance in mice, actually accelerated (T cell-mediated) immune responsiveness in the absence of B cells. TIM-1+ B cells were highly enriched for IL-4 and IL-10 expression, promoted Th2 responses, and could directly transfer allograft tolerance. Both cytokine expression and number of TIM-1+ regulatory B cells (Bregs) were induced by TIM-1-specific antibody, and this was dependent on IL-4 signaling. Thus, TIM-1 is an inclusive marker for IL-10+ Bregs that can be induced by TIM-1 ligation. These findings suggest that TIM-1 may be a novel therapeutic target for modulating the immune response and provide insight into the signals involved in the generation and induction of Bregs.

The balance between effector T cells and regulatory T cells (Tregs) is central to transplant tolerance. Therefore, the impact of immunosuppressive therapies on their fate is a major determinant of long-term allograft outcome. The biologic agents are new molecules that show promise as more selective therapies with less adverse effects. Because they target mostly surface markers or costimulatory pathways of lymphocytes, it is of critical importance to evaluate their effect on both effector T cells and Tregs. The present review provides a brief summary of Tregs physiology and then discusses actual knowledge on the impact of the biologic agents on the frequency of Tregs as well as their function in vitro and in vivo in humans.

Aim Mandatory HLA-DPB and DQA typing has recently been instituted for deceased organ donors. While these loci are not routinely used for organ allocation or listed as unacceptable antigens, if they are in the future, consistent typings will be important. We sought to determine the current state of compliance with the mandatory typing as well as to analyze repeat typings for discrepant results. Methods We examined 162,598 HLA typings submitted to UNOS on deceased donors from 10/1/87 through 12/31/17. Data was obtained with permission from UNOS as a SAS “STAR” file and analyzed with JMP® (SAS Institute). Results Prior to January, 2016, just fewer than ten percent of the 146,896 donors were typed for DPB, DPA, DQA while typing results were submitted for DPB and DQA for virtually all donors subsequently and in 76% DPA was also submitted. In the era prior to 1/1/16, less than 3% of typings were repeated. This increased to 30–36% in the current era (Table 1). The total number of discrepancies for DPB typings was 152 out of 5,495 typings that were repeated (2.8%). The most common discrepancies in DPB typing were between DPB*104:01 and 03:01 (N = 47), DPB*04:02 and 105:01(N = 29), and DPB*04:02 with 04:01 (N = 13). A number of other discrepancies were observed involving DPB*03:01, 04:02, 104:01, 105:01 and 04:01, which accounted for 71% of the DPB discrepancies. Download : Download high-res image (223KB) Download : Download full-size image Conclusions HLA-DPB and DQA typing of deceased donors is now mandatory. There has been excellent compliance with submission of typing results. Over three quarters of typings also include DPA results. The large proportion of repeat typings has permitted an analysis of both the rate of discrepancies and which antigens account for most of them. If these loci are to be considered for allocation, further analysis of the nature and reasons for these discrepancies will be needed to reduce the error rate.

Aim Currently, there are two different approaches to calculating cPRA. The first, used in USA since 2009, is the expectation maximization (EM) algorithm. The second, utilized in Europe/Canada, is to simply observe the percentage of donors with a particular antigen (“count method”). Recently, the UNOS/OPTN Histocompatibility Committee has been re-evaluating the optimal method for determining cPRA. This has been driven by the need to consider incorporating DP (and other) locus antibodies. The benefit of the EM method, based on haplotype frequencies, is to estimate population frequencies that would include unobserved, rare phenotypes. However, because DP is not in linkage disequilibrium, it may no longer be feasible to use. Here, we evaluate the use of the count method in comparison with the EM approach for determining HLA antigen frequencies in the U.S. donor population. Methods Using the UNOS/OPTN data, we evaluated HLA typings from 39,568 deceased donors from 1/2015–1/2018. We then compared the observed antigen frequencies (count method) with those derived from the UNet cPRA calculator (EM method). Results See table. Download : Download high-res image (81KB) Download : Download full-size image Conclusions There was high concordance between the two methods of calculating cPRA in antigen frequencies within the A, B, and DR loci. Instances where there were large discrepancies were primarily due to parent antigens that were subsequently split; the UNet cPRA was based upon a pool of donors from 2007–9. This suggests, at the very least, that updates to the cPRA calculator are needed. Since DPB is not in linkage disequilibrium with the other loci, thereby negating our ability to use the EM algorithm, we suggest a new updated cPRA calculator be based upon the count method of calculating antigen frequencies.

Aim Single antigen bead (SAB) assay is used to test the presence of antibodies against HLA antigens including HLA-DQ. Each SAB DQ bead is comprised of an alpha and a beta chain representative of an HLA-DQ antigen. A positive result represents reactivity to either or both chains. The aim of this report is to evaluate the frequency of HLA-DQ alpha antibodies in transplant candidates known to have Class II antibody. Methods Class II SAB data (One Lambda Inc.) was collected from 2346 transplant candidates at Brigham and Women’s Hospital from 6/9/2012 to 1/15/2014. The reactivity to HLA-DQ alpha was considered positive (MFI >3000) if all the beads containing a specific HLA-DQ alpha were reactive but the beads with corresponding HLA-DQ beta chain were negative. For example, the sera which were considered positive for HLA DQA1*03:02, had positive reaction with two beads containing DQA1*03:02, [DQA1*03:02, DQB1*03:02] and [DQA1*03:02, DQB1*03:03], but negative reaction with five beads positive for either DQB1*03:02 or DQB1*03:03 combined with DQA1 chains other than DQA1*03:02. Results Of the 29 HLA-DQ assay beads, 12 different HLA-DQ alpha chains were combined with different HLA-DQ beta chains. Sixteen (16/2346, 0.69%) sera reacted with beads specific for DQA1*03:02. These sera reacted with the alpha chain in combination with either DQB1*03:02 or DQB1*03:03. A similar pattern could be observed with sera (17/2346, 0.72%) that had specific reactivity to DQA1*02:01. Interestingly, none of the sera appeared to react selectively with DQA1*01:01, DQA1*01:02, DQA1*03:01, or DQA1*05:01. Although reactions with beads coated with DQA1*01:03, DQA1*03:03, DQA1*04:01, DQA1*05:03, DQA1*05:05, or DQA1*06:01 were present, they could not be attributed to DQA1 as the number of reactions or beads were not sufficient to rule out reactivity to DQB1. Out of the 33 patients who showed reactivity to either DQA1*03:02 or DQA1*02:01, only three (9.1%) had reactivity exclusively to the HLA-DQ alpha chain with no reactivity to any other HLA class II antigen. Conclusions The frequency of antibodies which appear to have HLA-DQ alpha specificity in transplant candidates is low (1.4%). Our results imply that a large number of sensitized patients will have to be studied in order to determine the clinical significance of antibodies to DQAlpha.

B7 ligands deliver both costimulatory and coinhibitory signals to the CD28 family of receptors on T lymphocytes, the balance between which determines the ultimate immune response. Although B7-H4, a recently discovered member of the B7 family, is known to negatively regulate T cell immunity in autoimmunity and cancer, its role in solid organ allograft rejection and tolerance has not been established. Targeting the B7-H4 molecule by a blocking antibody or use of B7-H4(-/-) mice as recipients of fully MHC-mismatched cardiac allografts did not affect graft survival. However, B7-H4 blockade resulted in accelerated allograft rejection in CD28-deficient recipients. B7-1/B7-2-double-deficient recipients are truly independent of CD28/CTLA-4:B7 signals and usually accept MHC-mismatched heart allografts. Blockade of B7-H4 in these mice also precipitated rejection, demonstrating regulatory function of this molecule independent of an intact CD28/CTLA-4:B7 costimulatory pathway. Accelerated allograft rejection was always accompanied by increased frequencies of alloreactive IFN-γ-, IL-4- and Granzyme B-producing splenocytes. Finally, intact recipient, but not donor, B7-H4 is essential for prolongation of allograft survival by blocking CD28/CTLA4:B7 pathway using CTLA4-Ig. These data are the first to provide evidence of the regulatory effects of B7-H4 in alloimmune responses in a murine model of solid organ transplantation.

Current immunosuppressive regimens suppress alloimmunity by nonspecifically targeting T-cell proliferation, differentiation, and activation. In doing so, they have been effective in dramatically reducing rates of acute rejection and improving short-term allograft survival. However, this is often at the expense of overimmunosuppression. Furthermore, chronic rejection remains a significant problem. CD4+CD25+FoxP3+ regulatory T cells (Treg) act to counterbalance effector mechanisms in immune homeostasis. Their function has been shown to be critical in autoimmune disease, transplantation, and allergy and inflammation. In this article, we will explore the current knowledge of Treg immunobiology in experimental models, as well as in human organ transplantation. The impact of current immunosuppressive agents on Tregs will be reviewed, and future promising targets for Treg-based therapies will be explored.

Aim DNA library preparation for next generation sequencing (NGS) consists of several hundred material transfer and wash steps, which are time consuming, labor intensive, and increase the risk of repetitive strain injury. Robotic liquid handlers can automate and standardize library preparation, but few HLA laboratories have experience with high-complexity robotic platforms. We sought to evaluate the use of robotics for library preparation in conjunction with a NGS based HLA typing assay. Methods A Beckman Coulter Biomek NXp Span-8 robotic liquid handler was acquired along with the vendor supplied robotics method for the Illumina TruSight HLA v2 sequencing panel. The robotics method was first optimized with a series of runs using water, 50% glycerol, 85% ethanol, and AMPure beads. Next, DNA and reagents were run to identify sample viscosity and reagent issues. After each run, issues were communicated to the vendor for method improvement. The finalized method was run on 78 DNA samples over seven runs to assess the quality of normalization, pooling, sequencing, and reproducibility of results. Results Robotic issues were encountered with pipette calibration, labware definitions, proper labware selection, and efficiency. To add complexity, these issues had subtle compounding effects on the overall success of the method, which required that each be addressed individually and in detail. Eleven versions of the method were subsequently produced and tested to ensure robustness and consistency. The finalized method produced consistent high quality sequencing metrics on the NGS typing platform which agreed with the known HLA typing results. Conclusions Detailed documentation of issues by lab staff and clear communication with the vendor allowed for a rapid and iterative improvement of the robotics method. The lessons learned during the implementation process formed a roadmap that allowed for successful implementation of a robotics platform for NGS based HLA typing.

Aim Chimerism testing is a valuable tool for determining the proportion of donor vs. recipient DNA in a sample. The commonly used polymerase chain reaction amplifications of short tandem repeats assay (Promega, Madison, WI) produces results for 21 loci, but only 9 loci are used in manual calculations. We sought to improve the tedious and time consuming process of manually calculating a patient’s percent chimerism by developing an automated chimerism calculator for rapid and accurate chimerism calculations based off of all 21 loci. Methods The calculator was developed using Microsoft Excel Macros and is able to report the percent donor chimerism +/− the margin of error. To determine whether precision is improved by using 21 loci, the results of a 21 loci analysis and a 9 loci analysis were compared in 111 patient samples. Additionally, each of the 21 loci was evaluated for its average deviation from the mean. Finally, 298 patient samples were analyzed by the program in order to determine if precision is equal at all chimerism percentages. Results When the same loci are analyzed, the chimerism calculator results are identical to the manual calculations, but completed in 10% of the time. The analysis of patient samples using 21 and 9 loci yielded, on average, chimerism percentages within 2.0% of each other. The average margin of error using 9 loci for analysis is 2.41% compared to 0.8% using all 21 loci. Among the 21 loci, the average deviation from the mean was found to range from 8.9% and 5.9%. Finally, we found that the average margin of error for patients between 90 and 100 percent donor chimerism is 0.19% while patients between 30 and 40 percent donor chimerism have 10.1% margin of error. Conclusions The chimerism calculator allows for routine use of all 21 STR loci with significantly reduced analysis time, 30% less margin of error, and an informative statistical analysis. In various laboratory settings, similar methods of computer assisted chimerism analysis can be easily implemented.

Aim Accurate classification of serologically-distinct antigens (Ag) is critical in organ allocation, and nowadays it is possible to define anti-HLA antibody specificity to the allelic level. We examined class I antibodies (Ab) for allelic specificities that may be serologically distinct and evaluated its impact on crossmatch (XM) results and organ allocation. Methods 6726 sera were tested for anti-HLA Ab using LABScreen Single Ag assay (One Lambda), in which 17 class I HLA Ag are represented by more than one allele. Discordance in MFI values between the two allelic beads was examined as a categorical variable to mimic clinical practice, and as a continuous variable (MFI value) to better reflect biological plausibility. To evaluate the significance of an allele-specific Ab on XM results, we examined an unbiased cohort of deceased donor-candidate testing (125,828 CDC XMs between 2014 and 2017). In our lab, all candidates within a blood group have a CDC XM performed, regardless of whether they have donor-specific antibody (DSA). This uniquely allows us to examine whether a candidate with an allele-specific DSA, who has already been excluded from the match run, would indeed have a positive XM against a donor who has a different allele. Statistical analyses were performed using JMP12Pro (SAS Institute). Results 1. The incidence of discordance between the two allelic beads varied depending on whether MFI values were considered as continuous vs categorical variables. 2. The presence of allele-specific Ab for rare alleles was out of proportion to their frequencies in the population. 3. The presence of Ab against only the rare allele was a poor predictor of a positive CDC XM, with a positive predictive value of 0–7% compared with 52.5% if the candidate had DSA against an A or B locus Ag. Conclusions 9 out of 17 Class I HLA antigens had potential allele-specific reactivity, meaning that candidates with antibody selectivity against rare alleles may be unnecessarily excluded from many organ offers based on definition of unacceptable antigens by serological equivalence. Conversely, for centers relying on virtual-XM, prematurely defining alleles as new serological splits could unfortunately allow transplants for which the recipient has DSA. Further testing is needed to rigorously confirm these allele-specific reactivity patterns.

The pivotal role of costimulatory pathways in regulating T-cell activation versus tolerance has stimulated tremendous interest in their manipulation for therapeutic purposes. Of these, the CD28-B7 pathway is arguably the most important and best studied. Therapeutic targets of CD28 are currently used in the treatment of melanoma, autoimmune diseases and in transplantation.In this review, we summarize our current knowledge of CD28 and cytotoxic T-lymphocyte antigen-4 (CTLA-4) signaling, and review the current state and challenges of harnessing them to promote transplant tolerance.Despite the success of belatacept, a first-in-class CTLA-4 fusion protein now clinically used in transplantation, it is apparent that we have only scratched the surface in understanding the complexities of how costimulatory pathways modulate the immune system. Our initial assumption that positive costimulators activate effector T cells and prevent tolerance, while negative costimulators inhibit effector T cells and promote tolerance, is clearly an oversimplified view. Indeed, belatacept is not only capable of blocking deleterious CD28-B7 interactions that promote effector T-cell responses but can also have undesired effects on tolerogenic regulatory T-cell populations.

Summary Background and objectives Acute rejection remains a problem in renal transplantation. This study sought to determine the utility of a noninvasive cytokine assay in screening of acute rejection. Design, setting, participants, & measurements In this observational cross-sectional study, 64 patients from two centers were recruited upon admission for allograft biopsy to investigate acute graft dysfunction. Blood was collected before biopsy and assayed for a panel of 21 cytokines secreted by PBMCs. Patients were classified as acute rejectors or nonrejectors according to a classification rule derived from an initial set of 32 patients (training cohort) and subsequently validated in the remaining patients (validation cohort). Results Although six cytokines (IL-1β, IL-6, TNF-α, IL-4, GM-CSF, and monocyte chemoattractant protein-1) distinguished acute rejectors in the training cohort, logistic regression modeling identified a single cytokine, IL-6, as the best predictor. In the validation cohort, IL-6 was consistently the most accurate cytokine (area under the receiver-operating characteristic curve, 0.85; P =0.006), whereas the application of a prespecified cutoff level, as determined from the training cohort, resulted in a sensitivity and specificity of 92% and 63%, respectively. Secondary analyses revealed a strong association between IL-6 levels and acute rejection after multivariate adjustment for clinical characteristics ( P Conclusions In this pilot study, the measurement of a single cytokine can exclude acute rejection with a sensitivity of 92% in renal transplant recipients presenting with acute graft dysfunction. Prospective studies are needed to determine the utility of this simple assay, particularly for low-risk or remote patients.

The proinflammatory cytokine IL-6 plays an important role in controlling T-cell differentiation, especially the development of Th17 and regulatory T cells. To determine the function of IL-6 in regulating allograft rejection and tolerance, BALB/c cardiac grafts were transplanted into wild-type or IL-6-deficient C57BL/6 mice. We observed that production of IL-6 and IFN-γ was upregulated during allograft rejection in untreated wild-type mice. In IL-6-deficient mice, IFN-γ production was greater than that observed in wild-type controls, suggesting that IL-6 production affects Th1/Th2 balance during allograft rejection. CD28-B7 blockade by CTLA4-Ig inhibited IFN-γ production in C57BL/6 recipients, but had no effect on the production of IL-6. Although wild-type C57BL/6 recipients treated with CTLA4-Ig rejected fully MHC-mismatched BALB/c heart transplants, treatment of IL-6-deficient mice with CTLA4-Ig resulted in graft acceptance. Allograft acceptance appeared to result from the combined effect of costimulatory molecule blockade and IL-6-deficiency, which limited the differentiation of effector cells and promoted the migration of regulatory T cells into the grafts. These data suggest that the blockade of IL-6, or its signaling pathway, when combined with strategies that inhibit Th1 responses, has a synergistic effect on the promotion of allograft acceptance. Thus, targeting the effects of IL-6 production may represent an important part of costimulation blockade-based strategies to promote allograft acceptance and tolerance.

Fetomaternal tolerance has been shown to depend both on regulatory T cells (Tregs) and negative signals from the PD1–PDL1 costimulatory pathway. More recently, IL-17–producing T cells (Th17) have been recognized as a barrier in inducing tolerance in transplantation. In this study, we investigate the mechanisms of PDL1-mediated regulation of fetomaternal tolerance using an alloantigen-specific CD4+ TCR transgenic mouse model system (ABM-tg mouse). PDL1 blockade led to an increase in embryo resorption and a reduction in litter size. This was associated with a decrease in Tregs, leading to a lower Treg/effector T cell ratio. Moreover, PDL1 blockade inhibited Ag-specific alloreactive T cell apoptosis and induced apoptosis of Tregs and a shift toward higher frequency of Th17 cells, breaking fetomaternal tolerance. These Th17 cells arose predominantly from CD4+Foxp3− cells, rather than from conversion of Tregs. Locally in the placenta, similar decrease in regulatory and apoptotic markers was observed by real-time PCR. Neutralization of IL-17 abrogated the anti-PDL1 effect on fetal survival rate and restored Treg numbers. Finally, the adoptive transfer of Tregs was also able to improve fetal survival in the setting of PDL1 blockade. This is to our knowledge the first report using an alloantigen-specific model that establishes a link between PDL1, Th17 cells, and fetomaternal tolerance.

The PD1:PDL1 pathway is an essential negative costimulatory pathway that plays a key role in regulating the alloimune response. PDL1 is expressed not only on antigen-presenting cells (APCs) but also cardiac endothelium. In this study, we investigated the importance of PDL1 expression on donor cardiac allograft in acquired transplantation tolerance in a fully MHC-mismatched model. We generated PDL1 chimeric mice on B6 background that expressed PDL1 on either hematopoietic cells or nonhematopoietic cells of the heart. Sham animals were used as controls. These hearts were then transplanted into BALB/c recipients and treated with CTLA4-Ig to induce tolerance. Cardiac endothelium showed significant expression of PDL1, which was upregulated upon transplantation. While the absence of PDL1 on hematopoietic cells of the heart resulted in delayed rejection and prevented long-term tolerance in most but not all recipients, we observed an accelerated and early graft rejection of all donor allografts that lacked PDL1 on the endothelium. Moreover, PDL1-deficient endothelium hearts had significant higher frequency of IFN-γ-producing alloreactive cells as well as higher frequency of CD8(+) effector T cells. These findings demonstrate that PDL1 expression mainly on donor endothelium is functionally important in a fully allogeneic mismatched model for the induction of cardiac allograft tolerance.

Injured epithelial cells of the proximal tubule upregulate the glycoprotein kidney injury molecule 1 (KIM-1), suggesting its potential as a biomarker of incipient kidney allograft injury. It is unknown whether KIM-1 expression changes in kidney allografts with delayed graft function (DGF), which often follows ischemia-reperfusion injury. Here, we prospectively measured KIM-1 RNA and protein expression in preperfusion biopsies of 30 living- and 85 deceased-donor kidneys and correlated the results with histologic and clinical outcomes after transplantation. We detected KIM-1 expression in 62% of deceased-donor kidneys and only 13% of living-donor kidneys (P < 0.0001). The level of KIM-1 expression before reperfusion correlated inversely with renal function at the time of procurement and correlated directly with the degree of interstitial fibrosis. Surprising, however, we did not detect a significant correlation between KIM-1 staining intensity and the occurrence of DGF. Our findings are consistent with a role for KIM-1 as an early indicator of tubular injury but do not support tissue KIM-1 measurement before transplantation to identify kidneys at risk for DGF.

High mobility group box 1 (HMGB-1) has recently received attention as a late mediator of lipopolysaccharide-induced shock, and is thought to function as a mediator in such a disorder as multi-organ failure (MOF). In Japan, we have access to an immobilized polymyxin B fiber column using a direct hemoperfusion (PMX-DHP) for patients with septic shock to improve hemodynamics and organ dysfunction. In this study, we looked at HMGB-1 levels in each category based on the sequential organ failure assessment (SOFA) scores to further dissect its importance in specific aspects of organ failure in patients undergoing PMX-DHP.Sixty patients with septic shock (40 survivors and 20 non-survivors). We analyzed HMGB-1 and IL-6 levels before and after PMX-DHP and defined organ failure as two or more SOFA points.There was a significant positive correlation between SOFA score and HMGB-1 level (P0.05). The HMGB-1 level before PMX-DHP significantly increased as the number of organ failures increased (P0.01: comparing 2 versus 5 organ failures). IL-6 levels decreased after PMX-DHP (P0.05 compared with before PMX-DHP), but HMGB-1 levels remained unchanged. HMGB-1 levels of survivors with organ failure in liver decreased after PMX-DHP, but those of non-survivors significantly increased 24h after PMX-DHP compared with before PMX-DHP (P0.01). In non-survivors with organ failure in liver, HMGB-1 levels were significantly higher than among survivors 24h after PMX-DHP (P0.01).Our results indicate that HMGB-1 is a useful prognostic biomarker in sepsis-induced organ failure in patients undergoing PMX-DHP.