Suppression of allograft rejection by regulatory B cells induced via TLR signaling.

B lymphocytes have long been recognized for their critical contributions to adaptive immunity, providing defense against pathogens through cognate antigen presentation to T cells and Ab production. More recently appreciated is that B cells are also integral in securing self-tolerance; this has led to interest in their therapeutic application to downregulate unwanted immune responses, such as transplant rejection. In this study, we found that PMA- and ionomycin-activated mouse B cells acquire regulatory properties following stimulation through TLR4/TLR9 receptors (Bregs-TLR). Bregs-TLR efficiently inhibited T cell proliferation in vitro and prevented allograft rejection. Unlike most reported Breg activities, the inhibition of alloimmune responses by Bregs-TLR relied on the expression of TGF-ß and not IL-10. In vivo, Bregs-TLR interrupted donor-specific T cell expansion and induced Tregs in a TGF-ß-dependent manner. RNA-Seq analyses corroborated the involvement of TGF-ß pathways in Breg-TLR function, identified potential gene pathways implicated in preventing graft rejection, and suggested targets to foster Breg regulation.

Transplantation in the Age of Precision Medicine: The Emerging Field of Treg Therapy.

Solid organ transplantation has experienced incredible success over the past half century in rescuing patients with end-stage organ failure and bestowing them with a high quality of life. This success in large part is the result of advances in immunosuppression, however, these regimens come with significant costs including opportunistic infections, and in some cases, direct toxicity to the newly transplanted organs. Further advances are needed. Immunosuppression and tolerance based on regulatory T cells provide an attractive alternative because of their ability to target these immunomodulatory functions specifically to the anti-allograft immune response.

Development and Implementation of a Novel Case-Based Gun Violence Prevention Training Program for First-Year Residents.

PROBLEM: Gun violence results in approximately 40,000 deaths in the United States each year, yet physicians rarely discuss gun access and firearm safety with patients. Lack of education about how to have these conversations is an important barrier, particularly among trainees. APPROACH: A 2-part training curriculum was developed for first-year residents. It included (1) a didactic presentation outlining a framework to understand types of firearm-related violence, describing institutional resources, and reviewing strategies for approaching discussions about firearms with patients, and (2) interactive case scenarios, adjusted for clinical disciplines, with standardized patients. Before and after the training, participants completed surveys on the training's relevance, efficacy, and benefit. Standardized patients provided real-time feedback to participants and completed assessments based on prespecified learning objectives. OUTCOMES: In June-August 2019, 148 first-year residents in internal medicine (n = 74), general surgery (n = 12), emergency medicine (n = 15), pediatrics (n = 22), psychiatry (n = 16), and OB/GYN (n = 9) completed the training. Most (70%, n = 104) reported having no prior exposure to gun violence prevention education. Knowledge about available resources increased among participants from 3% (n = 5) pretraining to 97% (n = 143) post-training. Awareness about relevant laws, such as Extreme Risk Protection Orders, and their appropriate use increased from 3% (n = 4) pretraining to 98% (n = 145) post-training. Comfort discussing access to guns and gun safety with patients increased from a median of 5 pretraining to 8 post-training (on a scale of 1-10, with higher scores indicating more comfort). NEXT STEPS: Delivery of a case-based gun violence prevention training program was effective and feasible in a single institution. Next steps include expanding the training to other learners (across undergraduate and graduate medical education) and institutions and assessing how the program changes practice over time.

Intrapleural transplantation of allogeneic pancreatic islets achieves glycemic control in a diabetic non-human primate.

Clinical islet transplantation has relied almost exclusively on intraportal administration of pancreatic islets, as it has been the only consistent approach to achieve robust graft function in human recipients. However, this approach suffers from significant loss of islet mass from a potent immediate blood-mediated inflammatory response (IBMIR) and a hypoxic environment. To avoid these negative aspects of the portal site, we explored an alternative approach in which allogeneic islets were transplanted into the intrapleural space of a non-human primate (NHP), treated with an immunosuppression regimen previously reported to secure routine survival and tolerance to allogeneic islets in NHP. Robust glycemic control and graft survival were achieved for the planned study period of >90 days. Our observations suggest the intrapleural space provides an attractive locale for islet transplantation due to its higher oxygen tension, ability to accommodate large transplant tissue volumes, and a lack of IBMIR-mediated islet damage. Our preliminary results reveal the promise of the intrapleural space as an alternative site for clinical islet transplantation in the treatment of type 1 diabetes.

Kidney transplantation from triple-knockout pigs expressing multiple human proteins in cynomolgus macaques.

Porcine cells devoid of three major carbohydrate xenoantigens, αGal, Neu5GC, and SDa (TKO) exhibit markedly reduced binding of human natural antibodies. Therefore, it is anticipated that TKO pigs will be better donors for human xenotransplantation. However, previous studies on TKO pigs using old world monkeys (OWMs) have been disappointing because of higher anti-TKO pig antibodies in OWMs than humans. Here, we show that long-term survival of renal xenografts from TKO pigs that express additional human transgenes (hTGs) can be achieved in cynomolgus monkeys. Kidney xenografts from TKO-hTG pigs were transplanted into eight cynomolgus recipients without pre-screening for low anti-pig antibody titers. Two recipients of TKO-hTG xenografts with low expression of human complement regulatory proteins (CRPs) (TKO-A) survived for 2 and 61 days, whereas six recipients of TKO-hTG xenografts with high CRP expression (TKO-B) survived for 15, 20, 71, 135, 265, and 316 days. Prolonged CD4+ T cell depletion and low anti-pig antibody titers, which were previously reported important for long-term survival of αGal knock-out (GTKO) xenografts, were not always required for long-term survival of TKO-hTG renal xenografts. This study indicates that OWMs such as cynomolgus monkeys can be used as a relevant model for clinical application of xenotransplantation using TKO pigs.

Properties of regulatory B cells regulating B cell targets.

Regulatory B cells (Bregs) have shown promise as anti-rejection therapy applied to organ transplantation. However, less is known about their effect on other B cell populations that are involved in chronic graft rejection. We recently uncovered that naïve B cells, stimulated by TLR ligand agonists, converted into B cells with regulatory properties (Bregs-TLR) that prevented allograft rejection. Here, we examine the granular phenotype and regulatory properties of Breg-TLR cells suppressing B cells. Cocultures of Bregs-TLR with LPS-activated B cells showed a dose-dependent suppression of targeted B cell proliferation. Adoptive transfers of Bregs-TLR induced a decline in antibody responses to antigenically disparate skin grafts. The role of Breg BCR specificity in regulation was assessed using B cell-deficient mice replenished with transgenic BCR (OB1) and TCR (OT-II) lymphocytes of matching antigenic specificity. Results indicated that proliferation of OB1 B cells, mediated through help from CD4+ OT-II cells, was suppressed by OB1 Bregs of similar specificity. Transcriptomic analyses indicated that Bregs-TLR suppression is associated with a block in targeted B cell differentiation controlled by PRDM1 (Blimp1). This work uncovered the regulatory properties of a new brand of Breg cells and provided mechanistic insights into potential applications of Breg therapy in transplantation.

Machine Perfusion of the Liver: A Review of Clinical Trials.

Although efforts have been made by transplant centers to increase the pool of available livers by extending the criteria of liver acceptance, this practice creates risks for recipients that include primary non-function of the graft, early allograft dysfunction and post-operative complications. Donor liver machine perfusion (MP) is a promising novel strategy that not only decreases cold ischemia time, but also serves as a method of assessing the viability of the graft. In this review, we summarize the data from liver machine perfusion clinical trials and discuss the various techniques available to date as well as future applications of machine perfusion. A variety of approaches have been reported including hypothermic machine perfusion (HMP) and normothermic machine perfusion (NMP); the advantages and disadvantages of each are just now beginning to be resolved. Important in this effort is developing markers of viability with lactate being the most predictive of graft functionality. The advent of machine perfusion has also permitted completely ischemia free transplantation by utilization of in situ NMP showed promising results. Animal studies that focus on defatting steatotic livers via NMP as well as groups that work on regenerating liver tissue ex vivo via MP. The broad incorporation of machine perfusion into routine clinical practice seems incredible.

Practitioner availability rather than surgical quality impacts the utilization of liver transplantation for hepatocellular carcinoma.

BACKGROUND: Liver transplantation (LT) provides better outcome than surgical resection (SR) although both are acceptable surgical options for hepatocellular carcinoma (HCC). It is unclear whether non-clinical factors drive treatment decisions. Our goal is to identify factors that may affect treatment decisions. METHODS: Patients aged 18-74 with T2 HCC undergoing either LT or SR in Surveillance, Epidemiology, and End Results Database from 2004 to 2014 were included. Healthcare resources data were analyzed to assess factors that predict utilization of LT versus SR, adjusted for demographic, clinical outcomes, and socioeconomic factors. RESULTS: 51% of patients (Total N = 2616) received LT, with a substantial state-level variation in LT rates (0.0%-66.9%). Higher LT center density [OR = 1.04 per 1% increment, P < 0.01], male gender (OR = 1.38, P = 0.02), and numbers of potential donors (OR = 1.19, P = 0.03) were positively associated with LT utilization. Conversely, higher incidence of chronic liver disease/cirrhosis (OR = 0.41 per one additional case per 100,000 populations, P = 0.001) and minority populations were negatively correlated with LT utilization. Notably, short-term surgical outcomes (in-hospital LT & SR mortality) were not associated with LT utilization. CONCLUSION: Liver transplant center density and organ availability, but not surgical outcomes, affect utilization of LT. Future studies should focus on increasing availability of resources.

Current status of porcine islet xenotransplantation.

PURPOSE OF REVIEW: Human islet transplantation has proven to be a highly effective treatment for patients with labile type 1 diabetes mellitus, which can free patients from daily glucose monitoring and insulin injections. However, the shortage of islet donors limits its' broad application. Porcine islet xenotransplantation presents a solution to the donor shortage and recent advances in genetic modification and immunosuppressive regimens provide renewed enthusiasm for the potential of this treatment. RECENT FINDINGS: Advances in genetic editing technology are leading to multigene modified porcine islet donors with alterations in expression of known xenoantigens, modifications of their complement and coagulation systems, and modifications to gain improved immunological compatibility. Recent NHP-based trials of costimulation blockade using CD154 blockade show promising improvements in islet survival, whereas results targeting CD40 are less consistent. Furthermore, trials using IL-6 receptor antagonism have yet to demonstrate improvement in glucose control and suffer from poor graft revascularization. SUMMARY: This review will detail the current status of islet xenotransplantation as a potential treatment for type I diabetes mellitus, focusing on recent advances in porcine xenogeneic islet production, assessment in nonhuman primate preclinical models, the outcome of human clinical trials and review barriers to translation of xenoislets to the clinic.

Do Social Determinants Define "Too Sick" to Transplant in Patients With End-stage Liver Disease?

BACKGROUND: Delisting for being "too sick" to be transplanted is subjective. Previous work has demonstrated that the mortality of patients delisted for "too sick" is unexpectedly low. Transplant centers use their best clinical judgment for determining "too sick," but it is unclear how social determinants influence decisions to delist for "too sick." We hypothesized that social determinants and Donor Service Area (DSA) characteristics may be associated with determination of "too sick" to transplant. METHODS: Data were obtained from the Scientific Registry of Transplant Recipients for adults listed and removed from the liver transplant waitlist from 2002 to 2017. Patients were included if delisted for "too sick." Our primary outcome was Model for End-Stage Liver Disease (MELD) score at waitlist removal for "too sick." Regression assessed the association between social determinants and MELD at removal for "too sick." RESULTS: We included 5250 delisted for "too sick" at 127 centers, in 53 DSAs, over 16 years. The mean MELD at delisting for "too sick" was 25.8 (SD ± 11.2). On adjusted analysis, social determinants including age, race, sex, and education predicted the MELD at delisting for "too sick" (P < 0.05). CONCLUSIONS: There is variation in delisting MELD for "too sick" score across DSA and time. While social determinants at the patient and system level are associated with delisting practices, the interplay of these variables warrants additional research. In addition, center outcome reports should include waitlist removal rate for "too sick" and waitlist death ratios, so waitlist management practice at individual centers can be monitored.

Regulatory B cells require antigen recognition for effective allograft tolerance induction.

Through multiple mechanisms, regulatory B cells (Breg) have been shown to play an important role in the development of allograft tolerance. However, a careful understanding of the role of antigen-specificity in Breg-mediated allograft tolerance has remained elusive. In experimental models of islet and cardiac transplantation, it has been established that Bregs can be induced in vivo by anti-CD45RB ± anti-TIM1antibody treatment, resulting in prolonged, Breg-dependent allograft tolerance. The importance of Breg antigen recognition has been suggested but not confirmed through adoptive transfer experiments, using tolerant WT C57BL/6 animals challenged with either BALB/c or C3H grafts. However, the importance of receptor-specificity has not been formally tested. Here, we utilize the novel ovalbumin-specific B cell receptor transnuclear (OBI) mice in multiple primary tolerance and adoptive transfer experiments to establish that Breg-dependent allograft tolerance relies on antigen recognition by B cells. Additionally, we identify that this Breg-dependent tolerance relies on the function of transforming growth factor-ß. Together, these experiments mark important progress toward understanding how best to improve Breg-mediated allograft tolerance.

Current state of organ transplant tolerance.

PURPOSE OF REVIEW: Immunological tolerance has long been considered the 'holy grail' of organ transplantation. Although tolerance has been an active area of research for 70 years, its clinical application has only been possible in the last two decades and widespread use remains an, as yet, unattained goal. Recent advances in the understanding of immune regulation have identified many new approaches to tolerance induction and several clinical trials are currently aimed at bringing this treatment to more patients. RECENT FINDINGS: Mixed chimerism remains the most successful approach to tolerance induction. However, many treatments, including adoptive transfer of regulatory T cells, regulatory B cells, and immune suppressive dendritic cells and myeloid derived suppressor cells have shown great promise in preclinical models. Recent clinical studies have found that both kidney and liver operational tolerance are achievable in the appropriate settings. Furthermore, combining multiple tolerance approaches has shown potential to produce durable and safer tolerance. SUMMARY: Tolerance to protect kidney and liver allografts has become a valuable therapy in the correct circumstances. Through further clinical trials and an improved understanding of immune regulatory components, tolerance is poised to have a significant impact on transplantation in the years to come.

Health Literacy Burden Is Associated With Access to Liver Transplantation.

BACKGROUND: Getting listed for liver transplantation is a complex process. Institutional health literacy may influence the ability of patients with limited educational attainment (EA) to list. As an easily accessible indicator of institutional health literacy, we measured the understandability of liver transplant center education websites and assessed whether there was any association with the percentage of low EA patients on their waitlists. METHODS: Patients on the waitlist for liver transplantation 2007-2016 were identified in Scientific Registry of Transplant Recipients. Understandability of patient education websites was assessed using the Clear Communication Index (CCI). The Centers for Disease Control and Prevention has set itself a goal CCI of 90 as being easy to understand. Low EA was defined as less than a high school education. We adjusted for center case-mix, Donor Service Area characteristics, and EA of the general population. RESULTS: Patients (84 774) were listed across 112 liver transplant centers. The median percent of waitlisted patients at each center with low EA was 11.0% (IQR, 6.6-16.8). CCI ranged from 53 to 88 and correlated with the proportion of low EA patients on the waitlist. However, CCI was not associated with the percentage of low EA in the general population. For every 1-point improvement in CCI, low EA patients increase by 0.2% (P < 0.05), translating to a 3.6% increase, or additional 3000 patients, if all centers improved their websites to CCI of 90. CONCLUSIONS: Educational websites that are easier to understand are associated with increased access to liver transplantation for patients with low EA. Lowering the health literacy burden by transplant centers may improve access to the liver transplant waitlist.

A surprising cross-species conservation in the genomic landscape of mouse and human oral cancer identifies a transcriptional signature predicting metastatic disease.

PURPOSE: Improved understanding of the molecular basis underlying oral squamous cell carcinoma (OSCC) aggressive growth has significant clinical implications. Herein, cross-species genomic comparison of carcinogen-induced murine and human OSCCs with indolent or metastatic growth yielded results with surprising translational relevance. EXPERIMENTAL DESIGN: Murine OSCC cell lines were subjected to next-generation sequencing (NGS) to define their mutational landscape, to define novel candidate cancer genes, and to assess for parallels with known drivers in human OSCC. Expression arrays identified a mouse metastasis signature, and we assessed its representation in four independent human datasets comprising 324 patients using weighted voting and gene set enrichment analysis. Kaplan-Meier analysis and multivariate Cox proportional hazards modeling were used to stratify outcomes. A quantitative real-time PCR assay based on the mouse signature coupled to a machine-learning algorithm was developed and used to stratify an independent set of 31 patients with respect to metastatic lymphadenopathy. RESULTS: NGS revealed conservation of human driver pathway mutations in mouse OSCC, including in Trp53, mitogen-activated protein kinase, phosphoinositide 3-kinase, NOTCH, JAK/STAT, and Fat1-4. Moreover, comparative analysis between The Cancer Genome Atlas and mouse samples defined AKAP9, MED12L, and MYH6 as novel putative cancer genes. Expression analysis identified a transcriptional signature predicting aggressiveness and clinical outcomes, which were validated in four independent human OSCC datasets. Finally, we harnessed the translational potential of this signature by creating a clinically feasible assay that stratified patients with OSCC with a 93.5% accuracy. CONCLUSIONS: These data demonstrate surprising cross-species genomic conservation that has translational relevance for human oral squamous cell cancer. Clin Cancer Res; 20(11); 2873-84. ©2014 AACR.

Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting.

Cancer immunoediting, the process by which the immune system controls tumour outgrowth and shapes tumour immunogenicity, is comprised of three phases: elimination, equilibrium and escape. Although many immune components that participate in this process are known, its underlying mechanisms remain poorly defined. A central tenet of cancer immunoediting is that T-cell recognition of tumour antigens drives the immunological destruction or sculpting of a developing cancer. However, our current understanding of tumour antigens comes largely from analyses of cancers that develop in immunocompetent hosts and thus may have already been edited. Little is known about the antigens expressed in nascent tumour cells, whether they are sufficient to induce protective antitumour immune responses or whether their expression is modulated by the immune system. Here, using massively parallel sequencing, we characterize expressed mutations in highly immunogenic methylcholanthrene-induced sarcomas derived from immunodeficient Rag2(-/-) mice that phenotypically resemble nascent primary tumour cells. Using class I prediction algorithms, we identify mutant spectrin-ß2 as a potential rejection antigen of the d42m1 sarcoma and validate this prediction by conventional antigen expression cloning and detection. We also demonstrate that cancer immunoediting of d42m1 occurs via a T-cell-dependent immunoselection process that promotes outgrowth of pre-existing tumour cell clones lacking highly antigenic mutant spectrin-ß2 and other potential strong antigens. These results demonstrate that the strong immunogenicity of an unedited tumour can be ascribed to expression of highly antigenic mutant proteins and show that outgrowth of tumour cells that lack these strong antigens via a T-cell-dependent immunoselection process represents one mechanism of cancer immunoediting.

Endoscopic surgical management of vidian nerve schwannoma.

Vidian nerve schwannomas are highly unusual, with only two reported cases in the world literature, and present with a constellation of symptoms that may be extremely vague. In the reported cases, open techniques have been utilized that required significant dismantling of the midfacial skeleton to access and manage these skull base lesions. Over the last 15 years, a paradigm shift in base of skull surgery has evolved with the advent of transnasal endoscopic techniques and instrumentation, improved surgical access and resectability of most skull-based pathologies, including vidian nerve lesions as reported here. We describe the varied clinical presentation and radiologic findings in two patients with such rare lesions. In addition, in one of these patients, we report on the first use of an expanded endonasal technique to access and remove vidian nerve schwannomas entirely endoscopically.