Role Of Transplantation Research Articles

Effect of Immunosuppressive Therapy After Liver Transplantation and the Relationship Between Changes in the Gut Microbiome and Graft Rejection

Solid organ transplantation, particularly liver transplantation, necessitates the use of immunosuppressive therapy to prevent organ rejection. However, these agents profoundly affect the gut microbiota, whose altered state can significantly impact transplant outcomes. This review synthesizes current knowledge on the interactions between immunosuppressants and the gut microbiota and explores the mechanisms by which these interactions affect graft survival and rejection. Immunosuppressive drugs, including cyclosporine, tacrolimus, sirolimus, mycophenolate mofetil, prednisone, and azathioprine, are known to cause dysbiosis by decreasing microbial diversity and favoring pathogenic bacteria and fungi. This disruption can lead to increased susceptibility to infections, chronic inflammation, impaired drug metabolism, and direct effects on graft rejection. The review also discusses the potential of modifying the gut microbiota to improve transplant outcomes through dietary interventions, probiotics, prebiotics, and fecal microbiota transplantation (FMT). These interventions aim to restore a healthy microbial balance, enhance immune tolerance, and reduce the incidence of graft rejection. The paper highlights the need for integrated approaches that consider the microbiome’s role in transplantation and outlines future directions for research and clinical practice, aiming to optimize the management of transplant recipients. By understanding and manipulating the gut microbiome, new therapeutic strategies could revolutionize transplant medicine, reducing complications and improving the quality of life for recipients.

Artificial intelligence-driven automated lung sizing from chest radiographs

Lung size measurements play an important role in transplantation, as optimal donor-recipient size matching is necessary to ensure the best possible outcome. Although several strategies for size matching are currently used, all have limitations, and none has proven superior. In this pilot study, we leveraged deep learning and computer vision to develop an automated system for generating standardized lung size measurements using portable chest radiographs to improve accuracy, reduce variability, and streamline donor/recipient matching. We developed a 2-step framework involving lung mask extraction from chest radiographs followed by feature point detection to generate 6 distinct lung height and width measurements, which we validated against measurements reported by 2 radiologists (T.A. and W.B.G.) for 50 lung transplant recipients. Our system demonstrated <2.5% error (<7.0 mm) with robust interrater and intrarater agreement compared with an expert radiologist review. This is especially promising given that the radiographs used in this study were purposely chosen to include images with technical challenges such as consolidations, effusions, and patient rotation. Although validation in a larger cohort is necessary, this study highlights artificial intelligence’s potential to both provide reproducible lung size assessment in real patients and enable studies on the effect of lung size matching on transplant outcomes in large data sets.

Energy Metabolism and Metformin: Effects on Ischemia-Reperfusion Injury in Kidney Transplantation.

Metformin (MTF) is the only biguanide included in the World Health Organization's list of essential medicines; representing a widespread drug in the management of diabetes mellitus. With its accessibility and affordability being one of its biggest assets, it has become the target of interest for many trying to find alternative treatments for varied pathologies. Over time, an increasing body of evidence has shown additional roles of MTF, with unexpected interactions of benefit in other diseases. Metformin (MTF) holds significant promise in mitigating ischemia-reperfusion injury (IRI), particularly in the realm of organ transplantation. As acceptance criteria for organ transplants expand, IRI during the preservation phase remain a major concern within the transplant community, prompting a keen interest in MTF's effects. Emerging evidence suggests that administering MTF during reperfusion may activate the reperfusion injury salvage kinase (RISK) pathway. This pathway is pivotal in alleviating IRI in transplant recipients, potentially leading to improved outcomes such as reduced rates of organ rejection. This review aims to contextualize MTF historically, explore its current uses, pharmacokinetics, and pharmacodynamics, and link these aspects to the pathophysiology of IRI to illuminate its potential future role in transplantation. A comprehensive survey of the current literature highlights MTF's potential to recondition and protect against IRI by attenuating free radical damage, activating AMP-activated protein kinase to preserve cellular energy and promote repair, as well as directly reducing inflammation and enhancing microcirculation.

Normothermic liver perfusion derived extracellular vesicles have concentration-dependent immunoregulatory properties.

Extracellular vesicles (EVs) are major contributors to immunological responses following solid organ transplantation. Donor derived EVs are best known for their role in transplant rejection through transferring donor major histocompatibility complex proteins to recipient antigen presenting cells, a phenomenon known as ‛cross-decoration'. In contrast, donor liver-derived EVs are associated with organ tolerance in small animal models. Therefore, the cellular source of EVs and their cargo could influence their downstream immunological effects. To investigate the immunological effects of EVs released by the liver in a physiological and transplant-relevant model, we isolated EVs being produced during normothermic ex vivo liver perfusion (NEVLP), a novel method of liver storage prior to transplantation. We found EVs were produced by the liver during NEVLP, and these EVs contained multiple anti-inflammatory miRNA species. In terms of function, liver-derived EVs were able to cross-decorate allogeneic cells and suppress the immune response in allogeneic mixed lymphocyte reactions in a concentration-dependent fashion. In terms of cytokine response, the addition of 1×109 EVs to the mixed lymphocyte reactions significantly decreased the production of the inflammatory cytokines TNF-α, IL-10 and IFN-γ. In conclusion, we determined physiologically produced liver-derived EVs are immunologically regulatory, which has implications for their role and potential modification in solid organ transplantation.

Uncommon HLA Alleles Observed in a Population of Istanbul Province

Objective: New polymorphisms are formed in human leucocyte antigen (HLA) genes with point mutations, gene conversions, and duplication, and the diversity continues to increase. Various new HLA alleles have significant roles in transplantation, and epidemiologic and population studies. The aim of our study was to determine the status of HLA alleles in the Turkish population, which is uncommon, well-defined, and non-defined in the world population according to the international ImMunoGeneTics information system® (IMGT) database. Methods: We performed HLA-A, -B, -C, -DQB1, and DRB1 loci at the four-field resolution level, using Sanger- sequence-based typing (SBT) for 5592 healthy, unrelated bone marrow donor volunteers from Istanbul Province. The uncommon alleles were also confirmed using high-throughput next-generation sequencing (NGS). Results: Uncommon alleles were determined at five loci as follows: HLA-A*01:155, 02:66, 02:90, 02:110, 02:343, 03:82, 24:28, 24:146, 24:276, 24:356, 31:23,33:33, 68:38; HLA-B *07:240, 18:19, 35:193, 40:303, 51:69, 51:169; HLA-C*04:39, 06:40, 07:93, 12:149, 15:73; HLA-DRB1*11:149, 13:14:02 and HLA-DQB1*03:27. All alleles were arranged according to the common and well‐documented (CWD) 3.0.0 catalog. Conclusion: This is the first study to show uncommon alleles in our population. These reported data increase the knowledge of HLA polymorphisms in the Turkish population and provide a basis for further studies in population genetics. This information may also be useful in determining whether a matched, unrelated donor is unlikely to be found so that a mismatch strategy, an extended family search, or alternate therapy, can be pursued, thus saving time and cost for patients.

Revitalizing Urologists' Role in Transplantation as a Novel Strategy to Address Surgeon Shortage: Results from a Cross-Sectional Survey.

Urologists represent functional alternatives for transplant surgeons, but their involvement is minimal. Evaluating urologists' interests in transplant and identifying associated factors may help to determine whether recruitment of more urological providers is a viable strategy to address transplant surgeon shortages in the United States. We emailed a 10-question survey to individuals pursuing urology in the United States and collected demographic data, education and training backgrounds, and preferences for proposed integrated residency programs and abbreviated transplant fellowships. We stratified respondents based on transplant interest (yes/no); we made comparisons by using t-tests for continuous variables and Fisher exact tests for categorical variables. We used multivariable logistic regression to identify factors associated with interest in transplant surgery. Of 104 respondents, 98 were included in the final analysis, with 47% indicating a current or prior interest in transplantation. Male respondents were 3.7 times more likely than female respondents to be interested (odds ratio = 4.675; 95% CI, 1.411-15.495; P = .012). Participants aged <30 years were 93% less likely than older participants to be interested in transplantation (odds ratio = 0.071; 95% CI, 0.006-0.779; P = .03). International medical graduates reported higher enthusiasm for transplantation compared with US-trained counterparts (89% vs 42%), with a trend toward significance (P = .06). Nearly all (93%, 43/46) who expressed interest endorsed having an integrated training pathway. Only 70% (32/46) supported an abbreviated fellowship (<24 mo). Lifestyle concerns and insufficient exposure during residency were the most frequently cited reasons for lack of interest. Compared with male and older urology trainees, female and younger urology trainees were less inclined to pursue transplant surgery. Nonetheless, urologists represent an untapped pool of transplant surgeons. Proposing an integrated training program for urologists and increasing exposure to transplantation during urology residency represent potential strategies to decrease transplant surgeon shortages.

HLA-B*51 Frequency in Transplant Patients and Donors.

HLA molecules play a crucial role in transplantation. The best treatment modality in patients with end-stage renal disease is renal transplant. HLA mismatches between patients and donors can prolong time for renal transplant therapy, reduce graft survival, and increase mortality. HLA region is the most polymorphic genetic region and is essential for antigen presentation. The main target of the recipient's immune system is HLA molecules on the surface of donor cells. HLA-B*51 is associated with Behcet disease, a rare multisystemic disease characterized by autoimmunity and inflammatory processes. In transplant recipients, inflammation and vasculitis are immunologic mechanisms that are responsible for damage of graft tissue. In this retrospective study, we aimed to investigate the frequency of HLA-B*51 in patients diagnosed with end-stage renal disease and in controls and to investigate correlations with rejection episodes. Patients who applied to Baskent University Adana Dr. Turgut Noyan Research and Medical Center (between 2010 and 2022) with end-stage renal disease (n = 1732) and a control group (n = 5277) received HLA typing for class I (HLA-A, HLA-B). Sequence-specific primers or sequencespecific oligonucleotides were used. Among patients diagnosed with end-stage renal disease, 321 had kidney transplant. Frequency of HLA-B*51 was 25.92% in patients and 25.22% in controls. No significant differences were found between patients and controls in the frequency of HLA-B*51. Among kidney transplant recipients with HLA-B*51 (n = 72), 38.89% had rejection episodes and 61.11% had no rejection. No significant association was found between HLA-B*51 allele positivity and rejection. No significant relationship was shown between patients diagnosed with end-stage renal disease and HLA-B*51 positivity. Previous studies support frequency of the HLA-B*51 allele in the control group. Although Behçet disease is known to cause renal vasculitis, HLA-B*51 positivity alone was not associated with vasculitis or inflammation.

Renal Cell Carcinoma in End-Stage Kidney Disease and the Role of Transplantation.

Kidney transplant patients have a higher risk of renal cell carcinoma (RCC) compared to non-transplanted end-stage kidney disease (ESKD) patients. This increased risk has largely been associated with the use of immunosuppression; however, recent genetic research highlights the significance of tissue specificity in cancer driver genes. The implication of tissue specificity becomes more obscure when addressing transplant patients, as two distinct metabolic environments are present within one individual. The oncogenic potential of donor renal tissue is largely unknown but assumed to pose minimal risk to the kidney transplant recipient (KTR). Our review challenges this notion by examining how donor and recipient microenvironments impact a transplant recipient's associated risk of renal cell carcinoma. In doing so, we attempt to encapsulate how ESKD-RCC and KTR-RCC differ in their incidence, pathogenesis, outcome, and approach to management.

Exploring Perturbations in Peripheral B Cell Memory Subpopulations Early after Kidney Transplantation Using Unsupervised Machine Learning.

B cells have a significant role in transplantation. We examined the distribution of memory subpopulations (MBCs) and naïve B cell (NBCs) phenotypes in patients soon after kidney transplantation. Unsupervised machine learning cluster analysis is used to determine the association between the cellular phenotypes and renal function. MBC subpopulations and NBCs from 47 stable renal transplant recipients were characterized by flow cytometry just before (T0) and 6 months after (T6) transplantation. T0 and T6 measurements were compared, and clusters of patients with similar cellular phenotypic profiles at T6 were identified. Two clusters, clusters 1 and 2, were formed, and the glomerular filtration rate was estimated (eGFR) for these clusters. A significant increase in NBC frequency was observed between T0 and T6, with no statistically significant differences in the MBC subpopulations. Cluster 1 was characterized by a predominance of the NBC phenotype with a lower frequency of MBCs, whereas cluster 2 was characterized by a high frequency of MBCs and a lower frequency of NBCs. With regard to eGFR, cluster 1 showed a higher value compared to cluster 2. Transplanted kidney patients can be stratified into clusters based on the combination of heterogeneity of MBC phenotype, NBCs and eGFR using unsupervised machine learning.

Does ABO Blood Type Implicate Susceptibility to Respiratory Abnormalities? A Controlled Cross-sectional Study among Indigenous University Students in Rivers State, Nigeria

Background: The blood typing is a genetically defined immune system characteristic that has a well-known role in transplantation and chemotherapy. However, it is unclear what role it could serve in diagnosing or predicting respiratory pathologies. The study aims at investigating the prevalence of respiratory disorder among ABO blood type. This study investigated the prevalence of respiratory disorder among ABO blood types.&#x0D; Methods: Using a quantitative survey research design, this cross-sectional study comprises of 102 young University students (64 females and 36 males) within the age of 17-28 years, extracted from different local Government Area in Rivers State. A semi-structured questionnaire was used to gather the social demograpgic characteristics of respondent’s. Anthropometric investigations and respiratory function test was done.&#x0D; Results: The prevalent blood types A, B, O, and AB was 19.6%, 16.7%, 56.9%, and 6.9% respectively. BMI showed that 2.9%, 39.2%, 38.2% and 19.6% was underweight, normal weight, overweight and obese respectively. Respiratory problems were distributed unevenly among blood types, with risk of obstructive pulmonary dysfunction having the highest frequency of occurrence.&#x0D; Conclusion: The susceptibility to respiratory abnormalities is not associated with any blood type, the risk of obstructive pulmonary disease is higher in blood type O, which is largely influenced by the predominancy.

Clinical Analysis of Pediatric T-Cell Acute Lymphoblastic Leukemia Using the MRD-Oriented Strategy System.

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) has historically been associated with a poor prognosis. However, prognostic indicators and methods of treatment used for T-ALL remain controversial. A total of 136 children newly diagnosed with T-ALL between 2005 and 2018 were consecutively enrolled in this study. We assessed the effect of different prognostic factors, such as clinical characteristics, minimal residual disease (MRD), and the role of transplantation in postremission treatment, as the outcomes. Compared with B-ALL patients, patients with T-ALL are generally older, more likely to be male and have a higher white blood cell count. The complete remission (CR) rate was 95.6%, while the 5-year overall survival (OS), event-free survival (EFS), and cumulative incidence of relapse (CIR) were 74.3 ± 3.7%, 71.3 ± 3.9%, and 24.4 ± 3.8%, respectively. In the multivariate analysis, day 33 MRD ≥0.1% and hyperleukocytosis were associated with a significantly worse prognosis in the whole group. Transplantation resulted in a significant survival advantage, compared with chemotherapy, for high-risk (HR) patients (5-year CIR: 15.6 ± 10.2% vs. 55.6 ± 11.7%, P=.029). The prognosis of children with T-ALL was poor, and the MRD on day 33 was found to be an important predictive factor of clinical outcome at our center.

The role of transplantation in Hodgkin lymphoma.

Despite the success of frontline anthracycline-based chemotherapy for classical Hodgkin Lymphoma (cHL), approximately 15% of patients do not achieve an adequate response and require further therapy. For transplant-eligible patients, additional treatment followed by high-dose chemotherapy and autologous hematopoietic stem cell transplantation (autoHCT) provides a durable response in 50% of patients. The most refractory patients, including those requiring multiple lines of therapy to achieve a response or those relapsing after an autoHCT, may achieve long-term survival with allogeneic hematopoietic stem cell transplant (alloHCT). Contemporary salvage regimens used as a bridge to transplant have expanded to include not only non-cross resistant chemotherapy, but also brentuximab vedotin (BV) and checkpoint inhibitors (CPI). As the management of relapsed/refractory (R/R) cHL evolves with the introduction of novel agents, so too does the role of transplantation. The paradigm of chemosensitivity as a predictor for autoHCT efficacy is being challenged by favorable post- autoHCT outcomes in heavily pre-treated CPI-exposed patients. Contemporary supportive care measures, validated comorbidity assessments, and an increased donor pool with haploidentical donors have broadened the application of transplantation to an increasingly older and diverse patient population. Despite the introduction of increasingly effective treatment options for R/R cHL, transplantation continues to play an important role in the management of these patients. In this review, we explore the impact of salvage therapy on autoHCT, conditioning regimens, maintenance therapy and the diminishing role of alloHCT for patients with cHL.

Intensive Therapy for NPM1 Mutant AML Patients: Negative Impact of FLT3-ITDhighbut Not FLT3-ITDlow and FLT3-TKD and Role of Transplant in Patients over 60 Years of Age

Introduction: Nucleophosmin 1 (NPM1) mutations without an FMS-like tyrosine kinase-3 ITD (ITD) mutation are associated with a favorable prognosis in younger (age <60 years) patients (pts) with acute myeloid leukemia (AML). However, the prognostic impact of NPM1 mutations with concurrent low ITD allelic ratio (ITDlow) is unclear. Furthermore, the benefits of hematopoietic stem cell transplant (SCT) in NPM1+ AML in pts >60 is not known. In this retrospective study, we investigated the impact of FLT3 mutational status in pts with NPM1+ AML and the role of SCT in patients >60 years with ITD negative (ITDneg). Methods: We included patients from January 2013 to August 19, 2021 from 7 centers with NPM1+ AML who received induction chemotherapy with an anthracycline and cytarabine. Data was collected by chart review and mutational status was determined by next generation sequencing at each institution according to local guidelines. ITDhigh was defined as an allelic ratio (AR) of ≥0.5; AR <0.5 was considered ITDlow. The log-rank test was used to compare overall survival between groups. Results: A total of 345 pts met the inclusion criteria. The mean age was 58 years (range 18-79) and 56% were female. Normal karyotype was seen in 80%. ITD was detected in 142 of the 345 (41%); a FLT3-TKD mutation was detected in 48 (14%) pts. Common co-mutations included DNMT3A (42%), TET2 (23%), IDH2 (19%), IDH1 (19%), and RAS (19%). The complete response (CR) rates for NPM1+/FLT3neg pts was 79.8%; the CR rates for the NPM1/ITDpos pts treated with IC+TKI, IC alone, and NPM1+/ITDneg/TKD+ were 73.3%, 74.1%, and 80%, respectively. In the entire ITD+ population, AR was known in 42.3% (N=60) of pts. The 2-year overall survival (OS) for the NPM1+/ITDhigh (N=29) was 48.6% (90% CI: 35.0%, 67.5%) and the 2-year duration of response (DOR) was 56.1% (90% CI: 38.7%, 81.5%), while the NPM1+/ITDlow group (N=31) had a 2-year OS of 66.4% (90% CI: 53.5%, 82.4%) and 2-year DOR of 62.0% (90% CI: 46.6%, 82.3%). The comparison of OS in these two groups by log-rank was not significant (p=0.31). In the ITDlow group, pts who had SCT (N=15) had a 2-year OS of 71.8% (90% CI: 52.6%, 94.5%) and a 2-year DOR of 76.2% (90% CI: 58.7%, 98.8%); those treated with chemotherapy alone (N=14) had a 2-year OS of 62.9% (90% CI: 44.4%, 88.9%) and 2-year DOR of 50.8% (90% CI: 28.7%, 90.0%). OS was not significantly different (p=0.35). Comparing survival between FLT3 groups with a Cox PH model, FLT3-ITDhigh was significantly associated with inferior OS vs. FLT3neg(HR = 2.30, p=0.005). However, FLT3-ITDlow and FLT3-TKD+ were not associated with inferior OS vs. FLT3neg (HR=1.56, p=0.17 and HR=1.65, p=0.08, respectively). In pts >60 and NPM1+/ITDneg (N=104), the mean age was 67 years (range 60-78), median WBC count at baseline 7.7 (range 0-127), and 48.1% of pts were female. The CR rate was 75.0% (N=78). A total of 32 pts underwent SCT and 40 pts received a median of 3 cycles of consolidation. The 2-year OS and DOR in the SCT group was 71.1% (90% CI: 58.8%, 85.8%) and 53.2% (90% CI: 40.1%, 70.7%), respectively as compared to 77.3% (90% CI: 66.4%, 89.9%) and 51.4% (90% CI: 39.1%, 67.6%), respectively in the non-SCT group. Albeit with small numbers SCT did not improve OS in this age group (p=0.2) In the entire cohort, the five most common co-mutations other than FLT3 were evaluated for influence on survival. Using the log-rank test, only DNMT3A and TET2 were associated with inferior OS (P=0.026 and 0.039, respectively), while IDH1, IDH2, and RAS were not significantly associated with survival (p-values of 0.43, 0.6, and 0.69, respectively). Conclusion: In our multicenter cohort of pts with NPM1+ AML, OS was significantly inferior in pts with ITDhigh v. FLT3neg, but not in ITDlow and TKD. Co-mutations in DNMT3A and TET2 adversely affected overall survival. Our data did not find a significant difference in OS in pts >60 with NPM1+/FLT3-ITDneg who were consolidated with chemotherapy versus SCT, though larger follow up studies are needed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Long-Term Outcome of a Prospective Randomized Trial Comparing Continuous Lenalidomide/Dexamethasone with Lenalidomide/Dexamethasone Induction, MEL140 with Autologous Blood Stem Cell Transplantation and Single Agent Lenalidomide Maintenance in Patients of Age 60-75 Years with Newly Diagnosed Multiple Myeloma

Background: Lenalidomide/dexamethasone has been approved as a standard treatment in multiple myeloma and is applied preferentially in older patients with comorbidities. The addition of daratumumab to this therapy is the best treatment option today for patients who are not transplant eligible. Intensity-reduced high-dose melphalan (MEL140) with autologous transplantation also is an effective treatment option for older patients who are transplant eligible but may not tolerate full dose high-dose melphalan (MEL200). A combination of MEL140 with lenalidomide/dexamethasone potentially could produce a better outcome compared with lenalidomide/dexamethasone alone. Methods: In this trial (DSMM XIII; EudraCT-No: 2008-004083-39) patients of age 60-75 years with newly diagnosed multiple myeloma who had symptomatic and measurable disease were randomly assigned to either (A1) 3 cycles of lenalidomide (25 mg po d1-21/28d) with low-dose dexamethasone (40 mg po d1, d8, d15, d22/28d), followed by stem cell mobilization and then further lenalidomide/dexamethasone cycles until progression or (A2) 3 cycles of lenalidomide/dexamethasone as induction, followed by stem cell mobilization, intensity-reduced high-dose melphalan 140 mg/m2 (MEL140) (single or double) with autologous blood stem cell transplantation and lenalidomide maintenance with 10 mg daily until progression. At randomization, patients were stratified according to age (60-70 years vs 71-75 years) and ISS stage (I, II vs III). The primary endpoint was progression-free survival, and secondary endpoints included safety, response rates and overall survival. Here, we report the long-term outcome. Results: 348 patients were included into the study and 339 patients could be analysed per intention-to-treat (ITT). Patient characteristics were well balanced between treatment arms. For all patients, median age was 68 years (range 60 - 75), 29% were older than 70 years, 36.5% had ISS stage I, 35.6% ISS stage II, and 27.9% ISS stage III. After a median follow-up of 68 months, the median progression-free survival (PFS) was 40 months for A1 (169 patients) and 33 months for A2 (170 patients) (HR 1.16; p=0.19) in the ITT analysis. The median overall survival (OS) was 87 months for A1 and 96 months for A2 (HR 1.04; p=0.81). A prespecified per-protocol (PP) analysis was defined to include patients who received at least 6 months of study treatment (also including patients with progressive disease or death during this time period). In this PP analysis the median PFS was 42 months for A1 (139 patients) and 36 months for A2 (131 patients) (HR 1.15; p=0.27); the median OS was 89 months for A1 and 96 months for A2 (HR 1.01; p=0.96). In arm A2, 115 patients (68%) received one or two transplants. For transplanted patients, median PFS was 40 months and median OS was 114 months. Second primary malignancies (SPMs) were observed in 24 patients in arm A1 (skin 12, solid tumor 11, hematologic 1) and in 20 patients in arm A2 (skin 5, solid tumor 11, hematologic 4). Conclusion: In the intention-to-treat population, the combination of MEL140 with lenalidomide/dexamethasone induction and lenalidomide single agent maintenance was not superior to continuous lenalidomide/dexamethasone treatment. Subgroup analyses with a focus on the role of transplant will be presented at the meeting. Sponsor: Gesellschaft für Medizinische Innovation in der Hämatologie/Onkologie - GMIHO

Co-expression of HLA-E and HLA-G in Porcine Endothelial Cells Attenuates Human Natural Killer Cell-Mediated Cytotoxicity

INTRODUCTION: Pig-to-human xenotransplantation (XTx) is a promising solution to the organ shortage. Genetically-modified (GM) pigs lacking major xenoantigens have reduced hyperacute rejection and prolonged xenograft survival. Despite these advancements, acute rejection remains a barrier to clinical XTx. Natural killer cells (NK) play a crucial role in transplantation. In the context of XTx, suppressing human NK activity by expressing HLA class I molecules in pig organs may protect xenografts from immune attack. Therefore, we aimed to co-express HLA class I molecules in GM pig endothelial cell line null for 5 key xenoantigens (5GKO) and to evaluate human NK response to 5GKO+HLA class I+ cells. METHODS: DNA sequences encoding HLA-E and HLA-G were designed, synthesized, and cloned into expression vectors. Three GM porcine cell lines (5GKO.HLA-E, 5GKO.HLA-G, 5GKO.HLA-E+G) were generated by transfection. IL-2–activated human peripheral blood mononuclear cells (PBMCs) from 5 donors were co-cultured with GM porcine cell lines to initiate NK cell–mediated immune responses. NK activation (CD107a expression) was evaluated by flow cytometry. Three experimental replicates were averaged and analyzed by one-way ANOVA and a post hoc Tukey test. RESULTS: We successfully co-expressed HLA-G, HLA-E, and HLA-E+G in 5GKO cells. Co-expression of HLA-E+G in 5GKO cells significantly reduced human NK activation compared with 5GKO, 5GKO.HLA-E, and 5GKO.HLA-G. CONCLUSION: Co-expression of HLA-E and HLA-G in porcine cells significantly reduces NK cell–mediated cytotoxicity and shows promise for incorporation into GM pigs for pig-to-human xenotransplantation.

Soluble CD83 improves and accelerates wound healing by the induction of pro-resolving macrophages.

To facilitate the recovery process of chronic and hard-to-heal wounds novel pro-resolving treatment options are urgently needed. We investigated the pro-regenerative properties of soluble CD83 (sCD83) on cutaneous wound healing, where sCD83 accelerated wound healing not only after systemic but also after topical application, which is of high therapeutic interest. Cytokine profile analyses revealed an initial upregulation of inflammatory mediators such as TNFα and IL-1β, followed by a switch towards pro-resolving factors, including YM-1 and IL-10, both expressed by tissue repair macrophages. These cells are known to mediate resolution of inflammation and stimulate wound healing processes by secretion of growth factors such as epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF), which promote vascularization as well as fibroblast and keratinocyte differentiation. In conclusion, we have found strong wound healing capacities of sCD83 beyond the previously described role in transplantation and autoimmunity. This makes sCD83 a promising candidate for the treatment of chronic- and hard-to-heal wounds.

415.6: Exploring Cell-Specific Mechanisms of Ischemia/Reperfusion Injury Mediated Reactivation of Cytomegalovirus From Latency in the Orthotopic Mouse Lung Transplantation

Purpose: Cytomegalovirus (CMV) is the most common opportunistic infection in lung transplant recipients, with the highest risk seen in CMV seronegative recipients (R-) of seropositive organs (D+). This study aims to explore key molecular pathways responsible for transplant-mediated CMV reactivation by using single-cell RNA sequencing (scRNA-seq) in a mouse model of syngeneic D+/R- lung transplantation. Methods: Left lungs from naïve (D-) mice or MCMV latently infected (D+) mice were transplanted to syngeneic R- recipients, while the contralateral right lungs served as controls. Lung tissues from the transplants at day 2 post-transplant (POD) or controls were harvested for viral RNA analysis and single cell isolation. CD31+ and CD45+ enriched single cell suspensions were processed using 10X Genomics, followed by sequencing. The sequenced data were processed using Cell Ranger pipeline and Seurat. Additional transplants were treated with small molecule Myd88 inhibitor to determine whether innate immunity plays a role in transplant induced CMV reactivation. Results: MCMV IE-1 mRNA transcripts were significantly upregulated at POD 2, but were not detectable in the controls, indicating that transcriptional activation is induced in the early phase of transplant and ischemia/reperfusion (I/R) injury associated with the transplant procedures is an important contributing factor. In addition, viral DNA amplification was detected in the salivary glands of D+/R- recipients at POD14, confirming that transplant of a D+ graft results in viral reactivation and dissemination. These data support the mouse lung transplant model as a viable approach to study mechanisms of CMV reactivation. Data from scRNAseq analysis showed distinct lung and immune cell clusters in lung transplants and controls. There were minimal differences in transcriptome landscapes between D- and D+ lungs prior to transplant (controls). In contrast, the differences were prominent between lungs from D-/R- transplant vs D+/R- lung grafts in the myeloid populations [e.g. neutrophils, macrophages (Macs)] and endothelial cells. Gene expression of Macs showed more differences between transplant lungs and control lungs than any endothelial cells or neutrophils. Pathway analysis of differentially expressed genes for myeloid cell types showed several significant immune response pathways including MIF regulation of innate immunity and communication between innate and adaptive immune cells. Expression of Myd88 and c-fos were increased in the lung grafts regardless of latent MCMV infection. Furthermore, our preliminary study using a Myd88 inhibitor showed a trend of CMV viral load reduction in lung grafts. Conclusions: Early I/R injury following lung transplant resulted significant alterations of transcriptome landscape unique to different cell types. Myd88/AP-1 plays a critical role in CMV reactivation; thus targeting Myd88 has a great therapeutic potential to prevent I/R injury-mediated CMV reactivation. NIH NIAID R21AI163876. NIH/NIAID P01AI112522.

Management of Peripheral T-cell Lymphomas and the Role of Transplant.

Here, we review the management of peripheral T-cell lymphoma, particularly focusing on the role of autologous and allogeneic stem cell transplant. Peripheral T-cell lymphomas are a rare subset of non-Hodgkin's lymphomas that are treated with curative intent. While therapy has been based on other aggressive lymphoid malignancies, outcomes are generally poorer than B-cell lymphomas with 5-year overall and progression-free survival of 30-40% and 20-30%, respectively. In effort to improve outcomes, transplant has been used in both the frontline and salvage settings. Although not studied in randomized studies, consolidation with autologous stem cell transplant in first remission has been associated with an approximate 5-year overall survival of 50-60% and 5-year progression-free survival of 40-45%. Unfortunately, most patients relapse, and, in this setting, allogeneic transplant remains the only curative option for those who are transplant-eligible. Multiple series have now shown that 3-year overall survival with allogeneic transplant is approximately 60%. However, outcomes with transplant are associated with disease control at the time of transplant. In contrast to B-cell malignancies, treatment decisions for peripheral T-cell lymphomas are supported mostly by phase II studies, retrospective series, and expert opinion. For patients with peripheral T-cell lymphoma able to achieve sufficient disease control, autologous stem cell transplantation in first remission and allogeneic stem cell transplantation in relapsed disease offer modest benefit over chemotherapy alone.

Regulatory and transitional B cells: potential biomarkers and therapeutic targets in organ transplantation.

Regulatory B cells (Bregs) play a prominent role in various disease settings. While progress has been hindered by the lack of a specific Breg marker, new findings highlight their role modulating the alloimmune response and promoting allograft survival. Herein, we focus on the recent advances in Breg biology and their role in transplantation. We review studies showing that T-cell immunoglobulin and mucin domain 1 (TIM-1) is an inclusive and functional Breg marker in mice that may have human relevance. We highlight the utility of the B cell interleukin-10/tumor necrosis factor-alpha (IL-10/TNFα) ratio in identifying underlying immunological reactivity and predicting clinical outcomes in kidney transplantation. This may identify patients requiring more immunosuppression and provide insight into potential therapeutic approaches that can modulate the Breg: B effector cell (Beff) balance. Emerging data support Bregs as potent modulators of immune responses in humans. Their ability to promote allograft survival must await development of approaches to expand Bregs in vitro/in vivo . The low IL-10/TNFα ratio reflecting decreased Breg/Beff balance, predicts acute rejection (AR) and poorer outcomes in renal transplantation. It remains to be determined whether this paradigm can be extended to other allografts and whether therapy aiming to correct the relative deficiency of Bregs will improve outcomes.

Innovative strategies to improve hematopoietic stem cell transplant outcomes in myelofibrosis.

Myelofibrosis (MF) is a clonal myeloproliferative neoplasm characterized by inflammation, marrow fibrosis, and an inherent risk of blastic transformation. Hematopoietic allogeneic stem cell transplant is the only potentially curative therapy for this disease, however, survival gains observed for other transplant indications over the past two decades have not been realized for MF. The role of transplantation may also evolve with the use of novel targeted agents. The chronic inflammatory state associated with MF necessitates pretransplantation assessment of end-organ function. Applying the transplant methodology employed for other myeloid disorders to patients with MF fails to acknowledge differences in the underlying disease pathophysiology. Limited understanding of the causes of poor transplant outcomes in this cohort has prevented refinement of transplant eligibility criteria in MF. There is increasing evidence of heterogeneity in molecular disease grade, beyond the clinical manifestations which have traditionally guided transplant timing. Exploring the physiological consequences of disease chronicity unique to MF, acknowledging the heterogeneity in disease grade, and using advanced prognostic models, molecular diagnostics and other organ function diagnostic tools, we present an innovative review of strategies with the potential to improve transplant outcomes in this disease. Larger, prospective studies which consider the impact of molecular-based disease grade are needed for MF transplantation.