Donor Age Correlates With Liver-Resident Natural Killer Cell Activity in Adoptive Immunotherapy Using Donor Liver Natural Killer Cells in Liver Transplantation.

BACKGROUND: Natural killer (NK) cells are involved in innate immunity and have been reported to play an important role in hepatocellular carcinoma recurrence and post-liver transplantation (LT) infection. However, the relationship between donor age and liver-resident NK cell activity remains to be elucidated. METHODS: We successfully performed NK cell immunotherapy in 19 living donor LT recipients to prevent post-LT bloodstream infections. Liver mononuclear cells (LMNCs) were collected from the liver graft perfusate and stimulated with interleukin 2 for 3 days. Liver-resident NK cells were analyzed using flow cytometry and a chromium release assay before and after cell culture. RESULTS: The median donor age was 44 years (range, 24-64 years). The graft weight was 492 g (range, 338-642 g), and the median number of LMNCs was 584 million cells (range, 240-1472 million cells). The proportion of NK cells before and after culture was 22% and 33%, respectively. A significant correlation was found between graft weight and the number of LMNCs. However, no correlation was found between donor age and the number or percentage of NK cells in the liver. Moreover, donor age showed a significant inverse correlation with NKp46 and NKp44 expression before culture and with NKp44, tumor necrosis factor-related apoptosis-inducing ligand, and CD69 expression after culture. CONCLUSION: A significant inverse correlation was observed between donor age and NK cell activity in the liver. This information may be useful for cell therapy during LT.

Impact of a new liver immune status index among patients with hepatocellular carcinoma after initial hepatectomy.

Aim: The anti-tumor effects of natural killer (NK) cells vary among individuals. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expressed on liver NK cells is a marker of anti-tumor cytotoxicity against hepatocellular carcinoma (HCC) in immune cell therapy. This study aimed to develop a liver immune status index (LISI) that predicts low TRAIL expression and validates its ability to predict recurrence after initial hepatectomy for primary HCC. Methods: A functional analysis of liver NK cells co-cultured with interleukin-2 for 3 days was performed of 40 liver transplant donors. The LISI, which predicted low TRAIL expression (25% quartile: <33%) in liver NK cells, was calculated using multiple logistic regression analysis. Next, 586 initial hepatectomy cases were analyzed based on the LISI. Results: Our model was based on the Fibrosis-4 index+0.1 (odds ratio [OR], 1.33), body mass index (OR, 0.61), and albumin levels+0.1 (OR, 0.54). The area under the receiver operating characteristic curve (AUC) of the LISI for low TRAIL expression was 0.89. Stratification of the recurrence rates (RR) revealed that LISI was an independent predictive factor of RR (moderate risk: hazard ratio, 1.44; high risk: hazard ratio, 3.02). The AUC was similar for the LISI, albumin-indocyanine green evaluation grade, albumin-bilirubin score, and geriatric nutritional risk index for predicting RR. Among the vascular invasion cases, the LISI was more useful than the other indexes. Conclusion: Our model facilitates the prediction of RR in high-risk patients by providing LISI to predict the anti-tumor effects of NK cells.

The aryl hydrocarbon receptor maintains antitumor activity of liver resident natural killer cells after partial hepatectomy in C57BL/6J mice.

BACKGROUND: Liver-resident natural killer (lr-NK) cells are distinct from conventional NK cells and exhibit higher cytotoxicity against hepatoma via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). However, the mechanism by which partial hepatectomy (PH) significantly suppresses TRAIL expression in lr-NK cells remains unclear. METHODS: This study aimed to investigate the PH influence on the function and characteristics of liver-resident NK (lr-NK) cells using a PH mouse model. RESULTS: Here, we report that PH alters the differentiation pattern of NK cells in the liver, and an aryl hydrocarbon receptor (AhR) molecule is involved in these changes. Treatment with the AhR agonist 6-formylindolo[3,2-b]carbazole (FICZ) inhibited the maturation of NK cells. FICZ increased the immature subtype proportion of NK cells with high TRAIL activity and decreased the mature subtype of NK cells with low TRAIL activity. Consequently, FICZ increased the expression of TRAIL and cytotoxic activity of NK cells in the liver, and this effect was confirmed even after hepatectomy. The participation of AhR promoted FoxO1 expression in the mTOR signaling pathway involved in the maturation of NK cells, resulting in TRAIL expression. CONCLUSION: Our findings provide direct in-vivo evidence that partial hepatectomy affects lrNK cell activity through NK cell differentiation in the liver. Perioperative therapies using an AhR agonist to improve NK cell function may reduce the recurrence of hepatocellular carcinoma after hepatectomy.

Antidonor T-Cell Responses Are Not Attenuated in Elderly Kidney Transplant Recipients.

OBJECTIVES: Although the number of kidney transplants among elderly patients has been steadily increasing, no specific recommendations have been established for treatment of elderly patients. In general, elderly recipients are considered to be at lower risk of cell rejection and require less intense immunosuppression than younger recipients. However, a recent report from Japan reported that chronic T-cell-mediated rejection was more frequent in elderly living-donor kidney transplant recipients. In this study, we investigated the effects of aging on antidonor T-cell responses in living-donor kidney transplantrecipients. MATERIALS AND METHODS: We retrospectively evaluated 70 adultliving-donor kidney transplantrecipients with negative crossmatches and cyclosporine-based immunosuppressive regimens. To evaluate antidonor T-cell responses, serial mixed lymphocyte reaction assays were performed.We compared results in elderly (≥65 years) versus nonelderly recipients. RESULTS: Regarding donor characteristics, elderly recipients were more likely than nonelderly recipients to receive a transplant from their spouse. The number of mismatches at the HLA-DRB1 loci was significantly higher in the elderly group than in the nonelderly group. As a result, the proportion of patients with antidonor hyporesponsiveness in the elderly group did not increase over the postoperative course. CONCLUSIONS: Antidonor T-cell responses in elderly living-donor kidney transplant recipients were not attenuated over time. Thus, caution is required regarding the imprudent reduction of immunosuppressants in elderly living-donor kidney transplant recipients. A rigorously designed, large-scale, prospective study is required to validate these results.

Evaluation of T-Cell Immune Status of Reduced-Dose Cyclosporine and Everolimus Combination Therapy in Kidney Transplant Patients.

BACKGROUND: Kidney transplantation (KT) outcomes have significantly improved with calcineurin inhibitors (CNIs) administration. In recent years, the dose of CNIs has been reduced, and everolimus (EVR) has been used in combination with CNIs to avoid complications from the long-term use of CNIs. However, T-cell immune responses to these protocols have not been fully evaluated. This study evaluated the anti-donor T-cell responses to our CNI-sparing regimen. METHODS: Fifty-five de novo KT patients were enrolled. Three months after KT, the patients were randomly assigned to either the EVR group, which received low-dose cyclosporine (CsA) (n = 28), or the standard-exposure CsA control group (n = 27), which was treated with both mycophenolate mofetil and methylprednisolone. Graft function, adverse events, and immunologic status were evaluated 3 years after KT. Mixed lymphocyte reaction (MLR) assays were performed to evaluate anti-donor T-cell responses in KT patients. RESULTS: Both groups maintained graft function well, but total cholesterol levels tended to increase annually in the EVR group. The incidence of cytomegalovirus (CMV) infection tended to be lower in the EVR group, regardless of the CMV serologic status. Immunologic evaluation by MLR assay showed that anti-donor T-cell responses were adequately maintained in both groups. CONCLUSIONS: EVR starting 3 months after KT can reduce the trough levels of CsA without affecting graft function or compromising the immunosuppressive effects. The EVR combination protocol is expected to reduce CNI toxicity and improve long-term prognosis after KT.

Arteriosclerosis Decreases Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Expression on Liver Natural Killer Cells in Living Donor Liver Transplantation.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is critical for natural killer (NK) cell-mediated anti-tumor and anti-microbe killing. The TRAIL expression on the donor's liver NK cells from the liver perfusate after interleukin-2 stimulation varies between individuals and is unpredictable. This study aimed to clarify the risk factors for low TRAIL expression by analyzing perioperative donor characteristics. METHODS: This retrospective study of living donor liver transplant (LDLT) donors between 2006 and 2022 was performed to analyze low TRAIL expression risk factors. Seventy-five donors who had undergone hepatectomy for LDLT were divided into 2 groups, low and high TRAIL, according to their TRAIL expression on liver NK cells, using median values. RESULTS: The low TRAIL group (N = 38) was older and had lower nutrition and a higher low-density lipoprotein/high-density lipoprotein (LDL/HDL) cholesterol ratio, related to arteriosclerosis, than the high TRAIL group (N = 37). In multivariate analysis, the geriatric nutritional risk index (GNRI) (odds ratio, 0.86; 95% CI, 0.76-0.94; P < .001) and LDL/HDL cholesterol ratio (odds ratio, 2.32; 95% CI, 1.10-4.86; P = .005) were independent predictive factors for low TRAIL expression on liver NK cells. Furthermore, the TRAIL expression of liver NK cells decreased in donors who already had atherosclerosis and in donors at risk of potentially developing atherosclerosis. CONCLUSIONS: The TRAIL expression on liver NK cells in donors had a strong relationship with atherosclerosis and GNRI. Atherosclerosis can reflect the TRAIL expression on liver NK cells.

Adoptive immunotherapy with natural killer cells from peripheral blood CD34+ stem cells to prevent hepatocellular carcinoma recurrence after curative hepatectomy: a study protocol for an open-label, single-arm phase I study.

INTRODUCTION: Hepatocellular carcinoma (HCC) remains a major clinical problem as more than half of these cases recur after radical resection. Natural killer (NK) cells are at the forefront of the innate immune system and attack microcarcinomas and circulating tumour cells. The objective of this study was to evaluate the feasibility and toxicity of peripheral blood CD34+ stem cell-derived NK cell infusion after radical hepatectomy for HCC. METHODS AND ANALYSIS: This is an open-label, single-arm, single-centre phase I study. Patients who have undergone initial hepatectomy for HCC with three or more risk factors for recurrence (≥10 ng/mL of Alpha fetoprotein (AFP), ≥360 mAU/mL of PIVKA-II, multiple tumours and ≥3 peripheral blood circulating tumour cells) will be enrolled and be treated with three peripheral blood CD34+ stem cell-derived NK cell infusions every 3 months. The primary endpoint will be safety assessment including the type and severity of adverse events, frequency of occurrence and duration of occurrence. The secondary endpoints will include survival, effect of immune response and clinical laboratory test results. ETHICS AND DISSEMINATION: Ethical approval of the trial was obtained from the Certified Committee for Regenerative Medicine Hiroshima University in Japan. The trial results will be shared with the scientific community at international conferences and by publication in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: jRCTb060200020.

Adoptive immunotherapy overcomes genetic susceptibility to bloodstream infections due to fc-gamma receptor polymorphisms after liver transplantation.

Single nucleotide polymorphisms (SNPs) in FCGR3A can predict the susceptibility of liver transplant (LT) recipients to bloodstream infections (BSI) and clinical outcomes following living-donor LT (LDLT). Here, we retrospectively analyzed the relationship of adoptive immunotherapy with activated natural killer (NK) cells from perfusate effluents of liver allografts against BSI following LDLT. Higher BSI incidence and lower survival were observed in LT recipients with FcγRIIIa (158F/F or F/V) (n = 81) who did not receive adoptive immunotherapy (n = 55) than in those who did (n = 26) (BSI frequency, 36.4% vs. 11.5%; p = .033; log-rank p = .047). After matching patient background using propensity score, similar results were obtained (BSI ratio, 41.7% vs. 12.5%; p = .049; log-rank p = .039). The predominant BSI pathogens in patients who did and did not receive adoptive immunotherapy were gram-negative rods (n = 3, 100%) and gram-positive cocci (GPC) (n = 15, 65.2%), respectively. The proportion of NK cells administered to patients with BSI was significantly lower than that administered to patients without BSI (Number: 80.3 (29.9-239.2) × 106 cells vs. 37.1 (35.6-50.4) × 106 ; p = .033, percentage; 14.1 (13.3-17.8)% vs. 34.6 (16.5-47)%, p = .0078). Therefore, adoptive immunotherapy with NK cells was associated with the reduced post-transplant BSI related to GPCs due to FcγRIIIa SNP in LT recipients.

Splenic Artery Embolization in Patients After Orthotopic Liver Transplant.

OBJECTIVES: Hypersplenism (thrombocytopenia, leukopenia, anemia) syndrome and ascites occur after orthotopic liver transplant. These conditions can be treated by open splenectomy. Splenic artery embolization has been practiced as an alternative surgical method. MATERIALS AND METHODS: Between January 2013 and January 2015, twenty-one orthotopic liver transplants were performed at the National Scientific Medical Research Center, Astana, Kazakhstan. Of these patients, 3 subsequently received splenic artery embolization 12, 8, and 6 months after transplant: 2 patients who had been diagnosed with primary biliary cirrhosis and 1 patient with hepatitis B virus -related liver cirrhosis. Two patients received a right-lobe living orthotopic liver transplant, and 1 patient received a deceased donor transplant. Indications for splenic artery embolization (ascites, splenomegaly) were based on clinical and ultrasonographic investigation and laboratory findings (thrombocytopenia, platelet count < 60 × 109/L, leukocytopenia, and white blood cell count < 2 × 109/L). Two recipients had leukothrombocytopenia and refractory ascites, and 1 had only thrombocytopenia. Splenic artery embolization was performed via a percutaneous femoral artery approach under local anesthesia. Transcatheter splenic artery branch occlusion was performed by deploying occlusion material. Preoperative spleen size ranged from 17.5 × 8.0 cm to 22.0 × 12.5 cm; ascites volumes were > 1000 mL. RESULTS: In all patients, ascites and platelet levels decreased after splenic artery embolization. In 1 patient with leukopenia, white blood cell count normalized. After embolization, 1 patient had severe abdominal pain requiring analgesia medication, and 2 patients had fever that lasted 3 days. Patients were discharged 6 to 9 days after embolization. One patient developed a perisplenic abscess without fever 1 month after discharge, and the abscess was drained using an ultrasound-guided percutaneous procedure. CONCLUSIONS: Splenic artery embolization is a safe and effective minimally invasive method for treating hypersplenism and ascites in orthotopic liver transplant recipients and an alternative to open splenectomy.

Evaluation of Kidney Allograft in the Early Posttransplant Period Using Ultrasonography.

OBJECTIVES: The aim of this study was to evaluate the kidney allograft after transplant to assess restoration of blood flow, the time required to functionally recovery after surgery, and the ability to differentiate normal from pathologic grafts using color Doppler ultrasonography in the early posttransplant period. MATERIALS AND METHODS: Sixteen kidney recipients underwent renal color Doppler ultrasonography examinations 1, 2, 3, 7, 15, and 30 days after transplant. We evaluated the clinical and biochemical test results of recipients and the functioning allografts and evaluated the acute pathology. Results of resistive index in color Doppler ultrasonography were compared with blood test results. RESULTS: During the early postoperative period after kidney transplant, the average size of the kidney was 10.7 × 5.1 cm, with parenchyma at 1.7 cm. The structure of the parenchyma was nonhomogeneous in 14 patients and homogeneous in 2 patients. The medullary pyramid layer was prominent in 6 patients, moderately prominent in 7 patients, and not prominent in 3 patients. The pyelocaliceal system was condensed in 1 patient. Hematoma in the perinephrium was found in 5 patients, and free fluid was found in 5 patients. Satisfactory vascularization of allografts occurred in 14 patients, with resistive index sensitivity of 93% and specificity of 83%. CONCLUSIONS: Color Doppler ultrasonography and resistive index results were useful in evaluating kidney allografts during the early postoperative period and in confirming their condition despite excessive blood parameter values.

Evidence for the immunosuppressive potential of calcineurin inhibitor-sparing regimens in liver transplant recipients with impaired renal function.

Patients requiring liver transplantation (LT) frequently experience renal insufficiency (RI), which affects their survival. Although calcineurin inhibitor-sparing immunosuppressive regimens (CSRs) are well known to prevent RI, the immune state in recipients receiving CSR remains to be intensively investigated. Among 60 cases of living-donor LT at our institute, 68% of the patients had none to mild RI (non-RI group) and 32% of the patients had moderate to severe RI (RI group). The RI group received a CSR comprising reduced dose of tacrolimus, methylprednisolone, and mycophenolate mofetil, while the non-RI group received a regimen comprising conventional dose of tacrolimus and methylprednisolone. One year after LT, the mean estimated glomerular filtration rate (eGFR) in the RI group had significantly improved, although it was still lower than that of the non-RI group. Serial mixed lymphocyte reaction assays revealed that antidonor T-cell responses were adequately suppressed in both groups. Thus, we provide evidence that CSR leads to improvement of eGFR after LT in patients with RI, while maintaining an appropriate immunosuppressive state.

Possibility of adoptive immunotherapy with peripheral blood-derived CD3⁻CD56+ and CD3+CD56+ cells for inducing antihepatocellular carcinoma and antihepatitis C virus activity.

We recently showed that interleukin (IL)-2-stimulated CD56+ cells derived from the liver exert vigorous cytotoxicity against hepatocellular carcinoma (HCC) by their binding to the tumor necrosis factor-related apoptosis-inducing ligand expressed on natural killer cells and the corresponding death receptors, and exhibit inhibitory effects on hepatitis C virus (HCV) replication by production of a high level of interferon-γ. These findings prompted us to develop a technique to increase the number of such innate components of cellular immunity from peripheral blood mononuclear cells (PBMCs) so that, they can be easily applied for immunotherapy clinically. We expanded CD3⁻CD56+ and CD3+CD56+ cells ex vivo from PBMCs of human volunteers by using media containing IL-2 and anti-CD3 monoclonal antibody. Among the various culture media used, autoserum supplemented X-VIVO 15 most efficiently supported PBMCs expansion and maintained the viability of the expanded cells (approximately 60-fold expansion after 28-d culture). Cultivation of PBMCs in this medium resulted in the highest proportion of CD3⁻CD56 cells among the propagated lymphocytes (approximately 40% after 28-d culture). An experiment using genomic HCV replicon-containing hepatic cells showed that the CD3⁻CD56+ cell-enriched expansion strongly inhibited HCV replication when compared with freshly isolated PBMCs. The additional anti-CD3 monoclonal antibody pulse stimulation induced anti-HCV activity even in the CD3+CD56+ cells among the propagated PBMCs. Further, cytotoxic assay showed that the expansion of CD3+CD56+ and CD3⁻CD56+ cells resulted in vigorous cytotoxicity against HCC. In conclusion, CD56+ cells obtained from the PBMCs show anti-HCV activity in addition to anti-HCC activity.

Adoptive immunotherapy with liver allograft-derived lymphocytes induces anti-HCV activity after liver transplantation in humans and humanized mice.

After liver transplantation in HCV-infected patients, the virus load inevitably exceeds pre-transplantation levels. This phenomenon reflects suppression of the host-effector immune responses that control HCV replication by the immunosuppressive drugs used to prevent rejection of the transplanted liver. Here, we describe an adoptive immunotherapy approach, using lymphocytes extracted from liver allograft perfusate (termed herein liver allograft-derived lymphocytes), which includes an abundance of NK/NKT cells that mounted an anti-HCV response in HCV-infected liver transplantation recipients, despite the immunosuppressive environment. This therapy involved intravenously injecting patients 3 days after liver transplantation with liver allograft-derived lymphocytes treated with IL-2 and the CD3-specific mAb OKT3. During the first month after liver transplantation, the HCV RNA titers in the sera of recipients who received immunotherapy were markedly lower than those in the sera of recipients who did not receive immunotherapy. We further explored these observations in human hepatocyte-chimeric mice, in which mouse hepatocytes were replaced by human hepatocytes. These mice unfailingly developed HCV infections after inoculation with HCV-infected human serum. However, injection of human liver-derived lymphocytes treated with IL-2/OKT3 completely prevented HCV infection. Furthermore, an in vitro study using genomic HCV replicon-containing hepatic cells revealed that IFN-gamma-secreting cells played a pivotal role in such anti-HCV responses. Thus, our study presents what we believe to be a novel paradigm for the inhibition of HCV replication in HCV-infected liver transplantation recipients.