CMV-positive Donors Research Articles

Evaluation of Deceased Donor Kidney Transplantation in the Eurotransplant Senior Program in Comparison to Standard Allocation.

BackgroundThe organ shortage and long waiting times have dramatically increased the age of potential kidney transplant recipients. The Eurotransplant Senior Program (ESP) was initiated to allocate kidneys from deceased donors aged ≥65 years to recipients with a comparable age independent of pre-transplant human leucocyte antigen (HLA) matching; however, parameters affecting the long-term benefits of this strategy remain poorly defined.Material/MethodsWe retrospectively evaluated outcome and risk factors for mortality in kidney recipients aged ≥65 years that were transplanted according to the ESP protocol relative to patients aged >50 years transplanted according to the Eurotransplant kidney allocation system (ETKAS) criteria at the University Freiburg Medical Center, Germany, between 2008 and 2018.ResultsGraft survival, graft function, the maintenance immunosuppressive therapy, and the incidence of rejections and infections did not differ between groups. Infectious diseases were the main cause of death in both groups; however, infection-associated mortality was more than double in the ESP group, and 5-year patient survival was 61.4% in the ESP group compared to 83.2% in the ETKAS group. Multivariate analysis identified age, the number of HLA mismatches, and the CMV serostatus with a seropositive donor and negative recipient as the main risk factors for mortality.ConclusionsA comparable immunosuppressive regimen used in ESP and ETKAS patients was associated with similar rejection rates and infectious disease complications, and infections were the most common cause of death in both groups. CMV-negative patients receiving an organ from a CMV-positive donor and patients with a high number of HLA mismatches require close follow-up to reduce mortality.

Abstract 3562: LOAd732 - a novel oncolytic adenovirus with enhanced immunostimulatory properties and resistance to immunosuppression

Abstract Immunotherapy aims at activating or re-activating the immune system to create an anti-tumor response that could lead to eradication of the tumor cells. Central is the activation of dendritic cells (DCs) capable of presenting tumor antigens to T cells that can expand and kill tumor cells. However, the tumor is a harsh environment for the adaptive immune system and many of the conditions needed for the establishment of an immune response are lacking. Oncolytic viral therapy presents an opportunity to overcome the immunosuppression in tumors by destroying tumor cells, releasing antigens and immunostimulatory factors. The effects of the oncolytic virus can be further amplified by the introduction of transgenes expressed by the virus. LOAd oncolytic adenoviruses belong to a platform of Ad5/35 adenoviruses that have replication restricted to tumor cells. The viruses express human immunostimulatory transgenes under a CMV promotor that enables expression in all infected cells. LOAd703, encoding trimerized (TMZ)-CD40L and 4-1BBL, is under clinical investigation in solid tumors. Herein we present the data from the investigation of our novel oncolytic adenovirus LOAd732 that encodes for three human transgenes; TMZ-CD40L, 4-1BBL and IL-2. Infection of the human cell lines Mel526 and Mel624 with LOAd732 led to cell killing as was evaluated by MTS viability assay and to expression of the transgenes, TMZ-CD40L, 4-1BBL and IL-2 evaluated by flow cytometry and ELISA. Moreover the immmunostimulatory function of LOAd732 was investigated by infecting immature dendritic cells, differentiated from CD14+ monocytes using GM-CSF and IL-4. Flow cytometry assay revealed that DCs infected with LOAd732 showed a mature profile with expression of activation markers and co-stimulatory molecules such as CD80, CD83, CD86, CD70, ICAM-1, and MHC molecules. Analysis of supernatants from the cultures with multiplex immunoassay (MDS technology) showed that LOAd732 DCs produce IL-12p70, IL-15, IL-21 and CXCL10. To assess the functionality of the DCs infected by LOAd732, the capacity of the cells to expand antigen-specific T cells were investigated in a CMV model system where DCs from CMV positive donors are pulsed with a CMV peptide and co-cultured with autologous T cells. LOAd732 infected DCs could expand CMV-specific CD8+ T cells. Since the tumor microenvironment often is immunosuppressive, TGF-β and IL-10 was added to the co-cultures to model these conditions. The addition of the immunosuppressing cytokines did not affect LOAd732 capacity to expand the antigen-specific T cells. In conclusion, LOAd732 infected DCs express activation markers and co-stimulatory molecules and produce cytokines necessary for the initiation of an immune response. Moreover the DCs are functional and can expand antigen-specific T cells and protect them from TGF-β and IL-10 suppression. Citation Format: Jessica Wenthe, Emma Eriksson, Tanja Lövgren, Rafael Moreno, Ramon Alemany, Angelica Loskog. LOAd732 - a novel oncolytic adenovirus with enhanced immunostimulatory properties and resistance to immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3562.

Disseminated Pulmonary Kaposi Sarcoma After Lung Transplantation: The Increased Risk Donor

<h3>Introduction</h3> Kaposi sarcoma (KS) is a rare complication after lung transplantation (LTx). It is caused by human herpes virus 8 (HHV-8). We report a case of primary disseminated pulmonary KS after LTx from an increased risk donor. <h3>Case Report</h3> A 66-year-old male with end-stage COPD underwent a bilateral LTx from an increased risk donor with positive HBsAb, HBcAb and Lues Ab, in November 2020. Apart from Valganciclovir (vGCV) and co-trimoxazole, entecavir and benzylpenicillin were given as prophylaxis. Routine immunosuppression included basiliximab, tacrolimus, mycophenolate mofetil (MMF) and prednisolone. vGCV prophylaxis was discontinued at 3 months (CMV-positive donor and recipient). The patient was treated effectively for acute rejection with pulse methylprednisolone in February 2021. Unfortunately, he was readmitted with dyspnea and lung function decline in April 2021. CT-scan showed bilateral consolidations and pleural thickening and the PET-scan showed an intense uptake in both lungs and pleura. Transthoracic needle biopsy showed HHV-8 positive spindle cell lesions. Subsequently, our patient developed skin lesions on his chest and in his mouth compatible with KS (biopsy-proven). In retrospect, the recipient was HHV-8 seronegative before LTx. Therefore, the diagnosis primary HHV-8 infection leading to disseminated pulmonary Kaposi sarcoma was made. Which appeared after cessation of the vGCV prophylaxis. Valganciclovir was initiated and immunosuppression was adjusted: MMF discontinued and tacrolimus switched to sirolimus (target through levels 15 µg/l). Nevertheless, KS was progressive and liposomal doxorubicin 20mg/m2 was started. Our patient deteriorated and was transferred to the ICU for intubation. Bronchoscopy revealed brown endobronchial lesions compatible with KS. The effect of the treatment could not be assessed, because the patient decided to refuse further treatment. He died soon after his decision was made. <h3>Summary</h3> Scarcity of donor organs led to the acceptance of an increased risk donor. Prophylaxis against hepatitis B and Lues was given, but after discontinuation of vGCV our patient developed a primary HHV-8 infection leading to disseminated KS and ultimately the death of our patient. HHV-8 testing of donor serum and lifelong prophylaxis might have prevented this clinical course and should be considered in recipients of increased risk donors.

Cytomegalovirus infection after renal transplantation.

Renal transplant patients show a high prevalence of cytomegalovirus (CMV) infection after the procedure. This study was conducted to assess the prevalence and factors associated with the incidence of CMV infection among renal transplant patients. A total of 100 patients were recruited in this study. The CMV load in the blood of each patient was assessed using the technique of polymerase chain reaction (PCR). The serostatus of all recipients and donors was examined preoperatively and those of the recipients again postoperatively. The association of CMV load was assessed with the following factors: age, gender, alanine aminotransferase (ALT) and serum creatinine levels, types of immunosuppressive and induction regimens, preoperative diabetes status, and serological virologic response (SVR) at 12 weeks postoperatively. Our findings showed that CMV incidence was significantly higher in middle-aged patients (62 of 66 patients, 93.9%; p=0.0001). Furthermore, about 88.2% of patients induced by anti-thymocyte globulin (ATG) showed a high viral load, significantly higher than the proportion of CMV-positive patients induced by basiliximab (p=0.001). In addition, a higher proportion of CMV-negative recipients who received the graft from CMV-positive donors and vice-versa were CMV-positive postoperatively. Administration of Valcyte 450 showed 100% efficiency in decreasing the CMV load in the patients. Among all the assessed factors, only the age of the recipients, type of induction therapy used, and the preoperative serostatus of both donors and recipients were significantly associated with the postoperative CMV incidence among the patients.

Post-Transplant Cyclophosphamide (PT-Cy) for Graft Versus Host Disease (GVHD) Prophylaxis in Allogeneic Transplantation from HLA Identical Sibling and Matched Unrelated Donor for Patients with Acute Leukemia, on Behalf of ALWP-EBMT

Background: Post-transplant Cyclophosphamide (PT-Cy) is being used successfully in the setting of unmanipulated haploidentical transplant as graft versus host disease (GVHD) prophylaxis. Experience with the application of PT-Cy for prevention of GVHD post allogeneic stem cell transplantation (HSCT) from matched sibling donors (MSD) or unrelated donor (UD) is rather limited and it involves mainly single center experience and small numbers of patients.Methods: Study aim was to evaluate the yield of PT-Cy as GVHD prophylaxis post HSCT from MSD and UD transplants for acute leukemia. We analyzed 423 patients (pts) with acute leukemia who received PT-CY alone or in combination to other drugs as GVHD prophylaxis and reported to the ALWP of the EBMT. 78 pts received PT-Cy alone (group 1); 204 received PT-CY in combination with one immunosuppressive (IS) drug -cyclosporine A (CSA) or and methotrexate (MTX) or mycophenolate mofetil (MMF)(group 2), while 141 pts received PT-Cy in combination with 2 IS drugs- CSA+MTX or CSA+MMF (group 3). Transplants were performed from 2007-2015 and median follow up is 16 months.Results: There were some differences among groups: pts in group 1 were younger (median age 37 years, p<0.001) were transplanted in more recent year (2014, p<0.001), with a MSD (80%, p<0.001), and CMV positive donor (73%, p=0.008), received more frequently a reduced intensity conditioning (RIC) (56%, p<0.001) and bone marrow (BM) as source of stem cells (74%, p<0.001), with no ATG (100%, p<0.001). Probability of OS at 2 year was 50%, 52.2% and 62.4%, for the 3 groups, respectively, p=0.06.Overall the cumulative incidence (CI) of grade II-IV acute GVHD was 27.9%, and the CI of chronic GVHD was 33.4%, and 16.5% the CI of extensive cGVHD. The CI of relapse at 2 years was 33.5% and non-relapse mortality (NRM) 18.5%, disease recurrence and GVHD were the most common causes of death in the whole cohort.At 2 years the probability of leukemia free survival (LFS) and overall survival (OS) was 48% and 55%.In multivariate analysis, the addition of IS to the PT-CY did not impact the risk of acute GVHD. Diagnosis of ALL (HR 0.57, p<0.001), unrelated donor (HR 1.65, p=0.02), RIC regimen (HR 1.72, p=0.01) and donor CMV positive (HR 1.77, p=0.01) were associated with the risk of grade II-IV acute GVHD. The use of PBSC (HR 2.41, p=0.01) and the absence of ATG (HR 1.69, p=0.03) were independently associated with an higher risk of chronic GVHD (p=0.005). In comparison to PT-Cy alone the addition of 1 or 2 IS drugs as GVHD prophylaxis were associated with reduced risk of extensive chronic GVHD (PT Cy+ 1 drug, HR 0.25, p=0.02, PT Cy+ 2 drugs, HR 0.77, p=0.01) as well the use of BM (HR 0.21, p<0.001) and the use of ATG (HR 0.22, p<0.001). Notably, the addition of 1 or 2 IS drugs to PT-CY did not impact the risk of relapse. Advance disease status (HR 2.42, p<0.001), was the only factor associated with increased risk of relapse. As for NRM addition of CSA+MTX or CSA+MMF to the PT-Cy (HR 0.35, p=0.04), diagnosis of AML (HR 0.35, p=0.001), and disease status at transplant (HR 2.94, p<0.001), and patients CMV serology (HR 2.04, p=0.04), influenced the NRM.Factors associated with increased OS were the use of PT-Cy in combination with CSA+MTX or CSA+MMF (HR 0.49, p=0.02), diagnosis of AML (HR 0.59, p=0.001), and disease status at HSCT ((CR1 vs CR2 HR 0.58, p=0.02), (CR1 vs advanced disease (HR 0.40, p<0.001)).Conclusions: PT-CY is effective GVHD prophylaxis in MSD and MUD HSCT for acute leukemia. The addition of IS drugs CSA+MTX or CSA+MMF to the PT-Cy shapes its effect and further decrease the risk of severe chronic GVHD, reducing transplant related mortality and improving overall survival. DisclosuresMohty:Sanofi: Honoraria, Speakers Bureau.

Abstract 16993: Impact of Donor and Recipient Cytomegalovirus Immune Status on Long-Term Graft Survival in Pediatric Heart Transplantation

Introduction: While CMV infection has been associated with graft loss in adult heart transplants, its role in children is less understood. We aim to better understand the effect of donor and recipient CMV immune status at transplant on survival in pediatric recipients. Methods: We analyzed 6,951 pediatric heart-only transplants in the Scientific Registry of Transplant Recipients (1987-2019). Patients were stratified by recipient and donor CMV status at transplant. The primary endpoint was freedom from graft loss. Results: Of the various donor and recipient CMV statuses, only CMV-positive recipients had decreased graft survival compared to CMV-negative ones (at 15-yrs: 47% vs. 53%, HR 1.14). However, when fully adjusted by Cox regression, recipient status was no longer a risk factor ( p = 0.12). In this model, anti-CMV treatment with ganciclovir or valganciclovir was associated with a lower risk of graft loss (HR 0.80, CI 0.71 - 0.90, p &lt; 0.005). When stratified by the four donor-recipient CMV status combinations, antiviral use was associated with improved survival in both donor-positive groups: most strongly in donor-positive/recipient-positive transplants (at 15-yrs: 51% vs. 39%, p &lt; 0.005) and to a lesser extent in donor-positive/recipient-negative transplants (at 15-yrs: 53% vs. 51%, p &lt; 0.005). The association is maintained when examined by era, such as with more frequent use of anti-CMV therapy after 1995. No association with antiviral use was observed in either donor-negative transplant group. Conclusion: CMV-positive recipients had greater long-term graft loss, although this may be confounded by other transplant characteristics. Anti-CMV therapy is associated with improved long-term graft survival in transplants with CMV-positive donors. This finding may explain the lack of association between donor CMV status and graft loss in that an association may be masked by antiviral use. Further investigation of anti-CMV therapy may mitigate risk of graft loss.

Early Infection after Allogeneic Transplant Increases NRM without Influencing Relapse Rate

Intro: Relapse post allogeneic transplant is the leading cause of early mortality in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Known risk factors of relapse include persistent disease at the time of transplant, use of anti-thymocyte globulin, reduced intensity conditioning, and absence of chronic graft versus host disease (GVHD). Early infection has been reported to increase non-relapse mortality (NRM) while protecting against hematologic disease relapse (Auletta et al., Blood 2016). We sought to determine the influence of patient features and post-transplant early infection (bacterial, fungal, and viral) on acute GVHD, relapse, and mortality. Methods: Adult patients (N=146) with myeloid disorders (AML, MDS, MPN/CMML) who received an initial allogeneic transplantation from 2013-2018 at Oregon Health &amp; Science University were retrospectively identified and followed until 6/30/20 for acquired infections, hematologic relapse, and survival outcomes. Time-to-event endpoints, starting from transplant date, were modeled with Cox regression for single events (overall survival [OS], cause-specific hazards for competing events) and Fine-Gray subdistribution regression for events with a competing risk (early infection vs. death, acute GVHD vs. death, relapse vs. NRM). Infection status (one or more confirmed infections) in the first 100 days post-transplant was considered as a time-dependent covariate. Results: Among 146 adult patients, 77% had AML, 54% were male, and the median age was 59 years (range 18 - 72). The Karnofsky score was 90-100 in 44% and 60-80 in 51% of patients. Using 2017 ELN risk guidelines for AML and IPSS-R for MDS and MDS/MPN, 11% of patients were classified as low risk, 33% as intermediate, and 50% as adverse. The median time from diagnosis to transplant was 5 months (range 2 - 203). At the time of transplant, 63% of patients had attained some degree of response (i.e., complete remission [CR] or hematologic improvement). However, 66% had minimal residual disease (defined as persistent cytogenetic abnormalities or leukemia-associated mutations detected by NGS). Fifty-one percent of patients had myeloablative conditioning and 90% received peripheral blood stem cell grafts with methotrexate and calcineurin-based GVHD prophylaxis. Donors were HLA-matched in 86% of patients, with haploidentical and umbilical cord blood grafts representing 10% and 3%, respectively. Forty-six percent of donors were positive for cytomegalovirus (CMV). Seventy-five percent (n=109) of patients had at least one infection in the first 100 days after transplant. Cumulative incidence rates of any infection are displayed in Table 1. The median time from transplant to first infection was 22 days, ranging from 0 to 73 days. Forty-three patients (29%) relapsed during study follow-up. The median time from transplant to relapse was 6.1 months (IQR 3.1 - 12.0). Over a median follow-up time of 18.7 months, 78 patients (53%) died, with median time to death of 4.1 months after transplant. Forty-six (59%) of the deaths were attributed to causes other than hematologic disease (i.e., infection, GVHD, other cancer). Low prognostic risk and total body irradiation during conditioning were associated with early infection (Table 2). In the multivariable setting, a higher rate of death was associated with male gender (HR=1.67; p=0.040), having at least a 6-month lag between diagnosis and transplant (HR=2.79; p&amp;lt;0.001), and contracting an infection of any type (HR=2.54; p=0.003; Table 3). There was no association between early infection and acute GVHD, which occurred in 85 patients (58.2%) with a median time to onset of 34 days. A greater incidence of acute GVHD was related to older recipient age (HR=1.88 for ≥50 years vs. &amp;lt;50; p=0.014), being male (HR=2.62; p&amp;lt;0.001), not having achieved a CR (HR=1.66; p=0.020), and undergoing transplant ≥ 6 months after diagnosis (HR=1.72; p=0.014). Infection did not correlate with the rate of relapse (HR=1.02, p=0.942) but significantly elevated the rate of NRM (HR=5.72, p&amp;lt;0.001). Time-independent variables associated with a higher probability of relapse were failure to attain a pre-transplant CR (HR=1.87; p=0.038) and having a CMV-positive donor (HR=2.11; p=0.016). Conclusion: Our retrospective study found that post-transplant early infection in AML and MDS/MPN patients did not impact relapse rate but drastically increased the rate of NRM. Disclosures Saultz: Kyn Theraputics: Research Funding. Maziarz:Novartis, Incyte, CRISPR Therapeutics, Artiva Biotherapeutics, and AlloVir: Consultancy; Novartis and Athersys: Other: DSMB participant; Novartis and Juno: Research Funding; Incyte, Kite, BMS/Celgene, PACT Pharma, Orca BioSystems, and Omeros: Honoraria; Athersys: Patents &amp; Royalties.

Abstract 1630: FceR1g negative NK-cells (g-NK) enhance antibody-dependent cellular cytotoxicity and in vivo efficacy of therapeutic monoclonal antibodies against hematologic malignanices

Abstract Monoclonal antibodies (mAbs) are a central component of hematologic malignancy therapy; however, not all patients initially respond to these agents and resistance is a major clinical issue. A major mechanism of action for these mAbs in vivo is NK cell antibody-dependent cellular cytotoxicity (ADCC). Recent studies led to the discovery of a novel subset of human NK cells that lack expression of FceR1g (“g-NK cells”) and have a multi-fold increase in ADCC activity after CD16 crosslinking. Here, we validate the potency of g-NKs as an off-the shelf cellular immunotherapy to enhance the efficacy of therapeutic mAbs. g-NK cells were enriched and expanded using a proprietary method from CMV-positive blood donors selected for high levels of circulating g-NKs. For ADCC cytotoxicity assays, expanded NK cells were sorted by flow cytometry into populations of “conventional” NK cells (cNK), adaptive (NKG2C+) NK cells, and g-NK cells. After sorting, NK-cells were co-cultured 4 hr at 37° C with Raji or MM.1S target cells (1e4 cells) at varying NK-cell:target cell ratios. For in vivo persistence studies a single dose of 1e7 g-NK or cNK cells was injected into NOD scid gamma (NSG) mice. For in vivo efficacy studies with rituximab (RIT), NSG mice were inoculated with 5e5 luciferase-labeled Raji lymphoma cells and dosed weekly with 15e6 g-NK cells +/-200 ug RIT or vehicle. For in vivo efficacy studies with daratumumab (DARA) and elotuzumab (ELO), NSG mice were inoculated with 1e6 luciferase-labeled MM.1S cells and dosed every 6d with expanded g-NKs (20e6), resting cNKs (3e5, equivalent to physiological levels of NK cells/kg in humans, and +/- 10 ug DARA or 10 ug ELO. In all studies, mice received IL-15 intraperitoneally every 3 days. Our expansion method was able to expand g-NKs from selected CMV-seropositive donors ~800-fold (n=8). By flow cytometry we found an increase in the percentage of g-NK from 28% of NK cells at input to 82% post-expansion. In vitro ADCC assays demonstrated that g-NKs enhanced cytotoxicity of RIT to Raji cells (70% at 1:1 E:T) when compared to both NKG2C+ NKs (29%) and cNKs (27%) (p&amp;lt;0.001). Notably, we saw similar ADCC of g-NK cells whether used fresh or thawed after viable freezing (70% vs. 63%, p=0.71). In addition, g-NK cells enhanced cytotoxicity of DARA and ELO to MM.1S cells (58% and 54% at 1:1 E:T) when compared to cNK (14% and 12% at 1:1 E:T) (p&amp;lt;0.001). We confirmed in NSG mice that g-NK persistence was markedly improved (&amp;gt;90%) over cNKs in blood at multiple time points (p&amp;lt;0.001), and spleen at Day 22 (p&amp;lt;0.001). Efficacy studies using Raji cells showed the combination of g-NK and RIT led to a &amp;gt;90% reduction in tumor burden versus RIT alone (p&amp;lt;0.001). Evaluation of expanded g-NKs in a disseminated orthotopic xenograft model of multiple myeloma found a dramatic reduction (&amp;gt;70%) in tumor burden with DARA+g-NK vs. DARA alone or DARA+cNK (p&amp;lt;0.001). Similar results were found with ELO. Here we demonstrate that adoptive transfer of expanded g-NK cells markedly enhances anti-tumor ADCC of therapeutic mAbs in several preclinical models. Importantly, because adoptive transfer of NK-cells in humans does not result in severe graft-versus-host disease, we propose that this long-lived, highly potent NK-cell therapy could be administered in an “off-the-shelf” manner to supercharge mAb efficacy. Citation Format: Austin Basil Bigley, Nadia H. Agha, Shanae Spade, Gaetano Dipierro, Ronald Martell, Byron C. Hann, Nina Shah, Arun P. Wiita. FceR1g negative NK-cells (g-NK) enhance antibody-dependent cellular cytotoxicity and in vivo efficacy of therapeutic monoclonal antibodies against hematologic malignanices [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1630.

The Value of a Nulliparous Uterus.

The Value of a Nulliparous Uterus.

Skewed Repertoire Reconstitution and Gene Expression of HLA-A2402 CMV-CTLs after Allogeneic HCT

<h3>Background</h3> Cellular immunity is important for the control of Cytomegalovirus (CMV) reactivation after allogeneic hematopoietic cell transplantation (HCT). However, the actual <i>in vivo</i> dynamics of CMV-specific cytotoxic T-cell (CMV-CTL) clones are still unclear. Thus, we investigated the T-cell receptor (TCR)-beta repertoire and gene expressions of HLA-A*2402-restricted CMV-CTLs in allo-HCT, using next-generation sequence methods (NGS). <h3>Patients</h3> The clone repertoire and dynamics of HLA-A*2402-restricted CMV-CTL were analyzed in 20 cases (30-1000 cells in each). First, we focused on the clone diversity in the group who had never experienced CMV reactivation after HCT from CMV-positive donors (the transferred-immunity group without CMV reactivation, n=7 cases). Next, we focused on the group who received HCT from CMV-negative donors (the naively-introduced immunity group after CMV reactivation, n=13 cases). Patients who received HCT from CMV-positive donors but experienced CMV reactivation were excluded, since there might simultaneously exist the transferred and introduced immunities. <h3>Results</h3> First, we focused on the clone diversity in the transferred immunity group (n = 7) and identified 78 clones. The top 2 clones in each case at one month after HCT exclusively accounted for >75% (range: 78-100%) of CMV-CTLs in all cases (Fig.1). In addition, these top 2 clones were also dominant in their corresponding donors (n = 3), suggesting that the transferred CMV immunity from donors would play a critical role for the prevention of CMV reactivation. Next, looking at the naively-introduced immunity group (n = 13) where 139 clones were identified, the similar skewed CMV-CTL repertoire reconstitution was also observed, and the top 2 clones accounted for >75% in 10 of the 13 cases (range: 61-100%) at 3 months after HCT (Fig.1). Focusing on the diversity of CMV-CTLs in the late phase after HCT (range: 6 -24 months, n=16), some novel clones appeared and became one of dominant clones in several cases, but the top 1 dominant clones at the early phase in each case stayed at top in 13 of the 16 cases. TRBV7 and BJ1-2 were frequently used in their CDR3 of CMV-CTLs (Fig. 2). In terms of specific amino acid sequences of CDR3 of TCR-ƒÀ, no common motif was found through all cases. However, [TSG, Q(G)GG, DPG, and NQG] motifs were frequently observed 3 months after HCT in the naively-introduced group, while [G(Q)GG or DPG] was observed even among the different cases of the transferred-immunity group. Single-cell RNA sequences demonstrated relatively uniform population in terms of gene expression even if different CMV-CTL clones existed in a patient (Fig.3). <h3>Conclusion</h3> <i>In vivo</i> clone-monitoring and gene expression of CMV-CTLs using NGS could provide an insight into the mechanism of immunological reconstitution following HCT.

A case of severe nephrotic syndrome caused by isolated CMV glomerulopathy with ganciclovir resistant UL 97 mutation

Introduction: While many cases of cytomegalovirus (CMV) nephropathy have been reported in the literature, isolated CMV glomerulopathy is considered a rare finding. Case Presentation: We report the case of a CMV-negative recipient of kidney allograft from a CMVpositive donor (D+/R-), the recipient subsequently developed severe nephrotic syndrome secondary to biopsy-proven isolated CMV glomerulopathy. Conclusion: The patient developed CMV viremia with ganciclovir resistant UL97 mutation. His treatment course was resistant to recommended dose of intravenous ganciclovir, so therapy was changed to foscarnet with resolution of his viremia and reduction in proteinuria.

GCV/VCVG prophylaxis against CMV DNAemia in pediatric renal transplant patients: A systematic review and meta-analysis.

CMV disease continues to stand as a significant threat to the longevity of renal transplants in children. More pediatric recipients are CMV-negative with CMV-positive donor serologies resulting in a HR mismatch. The length of prophylaxis with GCV or VGCV required to optimally prevent recurrence of CMVDNAemia remains unknown. This study is a meta-analysis comparing GCV/VGCV prophylaxis regimens provided for <6months, from 6 to <12months, and ≥12months after transplant in order to prevent CMVDNAemia. The search conducted involved PubMed, EMBASE, ISI Web of Science, and Cochrane Central Register from inception through December 2017. Search terms Kidney Transplantation, CMV, GCV, and VGCV provided 204 studies for abstract review. Studies excluded were those which did not itemize pediatric data separately, single case reports, and duplicate studies. Pooled analysis of five retrospective studies and one prospective study identified that there is no statistically significant difference in the incidence of CMV DNAemia when comparing <6months of prophylaxis and >12months of prophylaxis (23% and 15%, respectively, P=0.23). Regardless of the length of prophylaxis, there was no statistical difference in the incidence of CMV DNAemia in the HR patients (6 to <12months vs <6months, P=0.62; 6 to <12months vs ≥12months, P=0.78; ≥12months vs <6months, P=0.83). This study identifies no optimal length of prophylaxis for HR mismatch pediatric renal transplant patients as many develop CMV DNAemia.

Reduced-Intensity Conditioning Followed by Related and Unrelated Allografts for Hematologic Malignancies: Expanded Analysis and Long-Term Follow-Up

Reduced-intensity conditioning (RIC) extends the curative potential of allogeneic hematopoietic cell transplantation (HCT) to patients with hematologic malignancies unable to withstand myeloablative conditioning. We prospectively analyzed the outcomes of 292 consecutive patients, median age 58 years (range, 19 to 75) with hematologic malignancies treated with a uniform RIC regimen of cyclophosphamide, fludarabine, and total body irradiation (200 cGy) with or without antithymocyte globulin and cyclosporine and mycophenolate mofetil graft-versus-host disease (GVHD) prophylaxis followed by allogeneic HCT at the University of Minnesota from 2002 to 6. Probability of 5-year overall survival was 78% for patients with indolent non-Hodgkin lymphoma, 53% for chronic myelogenous leukemia, 55% for Hodgkin lymphoma, 40% for acute myelogenous leukemia, 37% for myelodysplastic syndrome, 29% for myeloma, and 14% for myeloproliferative neoplasms. Corresponding outcomes for relapse were 0%, 13%, 53%, 37%, 39%, 75%, and 29%, respectively. Disease risk index (DRI) predicted both survival and relapse with superior survival (64%) and lowest relapse (16%) in those with low risk score compared with 24% survival and 57% relapse in those with high/very-high risk scores. Recipient cytomegalovirus (CMV)-positive serostatus was protective from relapse with the lowest rates in those also receiving a CMV-positive donor graft (29%). The cumulative incidence of 2-year nonrelapse mortality was 26% and was lowest in those receiving a matched sibling graft at 21%, with low (21%) or intermediate (18%) HCT-specific comorbidity index, and was similar across age groups. The incidence of grades II to IV acute GVHD was 43% and grades III to IV 27%; the highest rates were found in those receiving an unrelated donor (URD) peripheral blood stem cell (PBSC) graft, at 50%. Chronic GVHD at 1 year was 36%. Future approaches incorporating alternative GVHD prophylaxis, particularly for URD PBSC grafts, and targeted post-transplant antineoplastic therapies for those with high DRI are indicated to improve these outcomes.

Significance of Cytomegalovirus Specific T Lymphocytes in Kidney Transplant Recipients

Introduction CMV viremia is routinely used to confirmed CMV infection after kidney transplantation. Also, immune assays can be applied to predict viremia development or guiding prophylaxis and preemptive therapies. Most of the immunological assays rely on functional methods that detect INFγ after stimulation of lymphocytes with viral peptides. There is also MHC multimers assay that identifies antigen-specific lymphocytes crucial for controlling virus latency. It was proved for allogeneic stem cell transplant recipients that levels 2 to 10 cells/μl can successfully protect against CMV infection. Unfortunately, it is not for kidney transplant recipients. We hypothesize that CD28 expression may serve as an additional functional marker for viral-specific lymphocytes, as CD28 occurs with CMV infection and it is linked with early, intermediate and late antigen-specific T lymphocytes differentiation status. Thus, the aim of these study was to verify whether the CMV specific T lymphocytes or extended phenotype of CMV specific T lymphocytes may be used as a marker of CMV viremia in kidney transplant recipients. Materials and Methods CMV viremia was monitored in 52 kidney transplant recipients every three months, in two-years lasting follow-up. CMV specific lymphocytes were measured as CD8+ lymphocytes using MHC multimer assay. Further, CMV specific lymphocytes were assessed for CD28 expression (Figure 1).Values were reported both as percentages as well as absolute cell count. Results and Discussion CMV specific CD8+ T cells, expressing CD28 were superior for identification of patients at risk of viremia development after kidney transplantation. A cut-off value below 3 cells/μl successfully predict CMV viremia with acceptable specificity and sensitivity confirmed by ROC analysis, AUC values 0,774 – 0,951 depends on time posttransplant (Figure 2).The overall risk of CMV viremia was strongly associated with donor/recipient (D/R) serological status before transplantation, analyzed with Kaplan-Meier survival analysis (Figure 3).The highest risk of viremia development was associated with CMV primary infection (D+/R-). Interestingly, CMV positive recipients transplanted from CMV positive donor (D+/R+) had a higher risk of viremia when compared to uninfected donors (D-/R+), despite increased levels of CMV specific T lymphocytes (Figure 4).Conclusion Collectively, CMV specific T lymphocytes testing enables personalized assessment of recipient’s immune system. This data may be used for CMV viremia risk stratification, that could be applied for tailored pharmacotherapy after kidney transplantation. National Centre for Research and Development, Poland (No. STRATEGMED1/233368/1/NCBR/2014). National Science Centre, Poland (No. NN402420738 and NN402562440).

Factors associated with immune hemolytic anemia after pediatric liver transplantation.

Immune-mediated hemolytic anemia following SOT is a rare disorder, the risk factors for which are unknown. Our purpose was to analyze a seemingly increased incidence in our center with the aim to identify predisposing factors. This recipients single-center retrospective study reviewed the medical records of 96 pediatric LT between 2000 and 2013. IHA was defined as acute anemia with a positive direct antiglobulin test. Seven cases of immune-mediated hemolytic anemia were identified (incidence 8.5%). Three cases presented during the first 3months following LT (early IHA), and 4 presented later (late IHA). All patients with late IHA required rituximab. Using univariate analysis, the following factors were associated with IHA onset: BA (P=.04), younger age (P=.04), and the use of IGL-1 preservation solution (P=.05). Late IHA was associated with viral infections occurring beyond 3months following LT, younger age, and BA (P=.01). Overall, CMV infection was associated with the development of both early and late IHA: CMV-negative recipients who received an organ from a CMV-positive donor were more likely to develop IHA (P=.035), and de novo CMV infection during the first year post-LT was associated with late IHA (P=.03). IHA is a rare complication following pediatric LT, occurring more frequently in younger patients and patients with an initial diagnosis of BA. CMV-negative recipients and patients who experience a de novo CMV infection in the first year following LT seem particularly vulnerable. IGL-1 preservation solution may be associated with an increased likelihood of developing IHA, a novel finding which warrants further investigation.

Allogeneic haematopoietic stem cell transplant in patients with lower risk myelodysplastic syndrome: a retrospective analysis on behalf of the Chronic Malignancy Working Party of the EBMT.

We report a retrospective analysis of 246 myelodysplastic syndrome (MDS) patients in the EBMT (The European Society for Blood and Marrow Transplantation) database who were transplanted for International Prognostic Scoring System (IPSS) low or intermediate-1 disease. The majority of these patients (76%) were reclassified as intermediate or higher risk according to R-IPSS. The 3-year overall survival (OS) and PFS were 58% and 54%, respectively. In a multivariate analysis, adverse risk factors for PFS were marrow blast percentage (hazard ratio (HR): 1.77, P=0.037), donor/recipient CMV serostatus (donor-/recipient+: HR: 2.02, P=0.011) and source of stem cells (marrow and non-CR: HR: 5.72, P<0.0001, marrow and CR: HR: 3.17, P=0.027). Independent risk factors for OS were disease status at time of transplant and the use of in vivo T-cell depletion (TCD). Patients who did not receive TCD and were transplanted from an unrelated donor had worse OS (HR: 4.08, P<0.0001). In conclusion, 'lower' risk MDS patients have better outcome than those with 'higher risk' after haematopoietic stem cell transplant (HSCT). Selecting the right source of stem cells, a CMV-positive donor for CMV-positive patients and using in vivo TCD results in the best outcome in these patients. More studies are needed to evaluate the role of HSCT in these patients as compared with conventional treatment.

Polymorphism in programmed cell death 1 gene is strongly associated with lung and kidney allograft survival in recipients from CMV-positive donors

Cytomegalovirus (CMV) has a role in chronic rejection and graft loss in kidney transplant (KTx) and lung transplant (LTx) recipients. In addition, donor CMV seropositivity is an independent risk factor for renal graft loss. The anti-CMV response might modulate this risk. Expression of programmed cell death 1 (PD-1), a receptor involved in viral-specific T-cell exhaustion, is influenced by a single nucleotide polymorphism called PD-1.3 (wild-type allele G, variant allele A). We performed a retrospective study to assess the impact of PD-1.3 on graft outcome in donor CMV seropositive (D+) and donor CMV seronegative (D-) KTx and LTx. We also performed a case-control study to evaluate the anti-CMVpp65 response according to genotype. PD-1.3 was determined in 1,119 KTx and 181 LTx. In 481 D+ KTx, A allele carriers (24%) experienced significantly less graft failure compared with GG carriers (p = 0.001). Multivariate analysis showed that this association was independent of donor and recipient age, acute rejection episodes, and number of human leukocyte antigen mismatches (hazard ratio, 0.381; 95% confidence interval, 0.209-0.696; p = 0.002). Analysis in 85 D+ LTx showed similar results: A allele carriers had better survival (hazard ratio, 0.302; 95% confidence interval, 0.128-0.716; p = 0.006) and greater 6-month forced expiratory volume (71% ± 17% vs 54% ± 16%, p = 0.001). In D- recipients, PD-1.3 did not affect KTx or LTx outcome. Finally, AA recipients had a stronger anti-CMVpp65 T-cell response than matched GG recipients (p = 0.003). The A variant allele in PD-1.3 single nucleotide polymorphism improved graft survival in kidney and lung transplant recipients receiving grafts from CMV-positive donors.

Cytomegalovirus-negative kidney transplant recipients are at an increased risk for malignancy after kidney transplantation.

The effect of cytomegalovirus (CMV) serology status on malignancy risk in kidney transplanted patients is not clear yet. In a nested case-control study, CMV serology status was compared between patients with a malignancy and 2:1 matched control patients without a malignancy. In a cohort study, the hazard of malignancy was compared between patients that were CMV-negative but had a CMV-positive donor and other patients, using Cox analysis. Fifty-two of 599 patients transplanted in our center between 2001 and 2014 developed a malignancy. Nine (17.3%) of the 52 patients that developed cancer were CMV-negative but had a-CMV-positive donor compared with 6 (5.8%) of the 104 matched control patients (odd ratio 3.42, 95% confidence interval [CI] 1.15-10.2, P=.021). By univariate Cox model, there was a trend toward increased cancer risk in CMV-negative patients with a positive donor (hazard ratio [HR] 1.95, 95% CI 0.95-4.0, P=.07), but after adjusting for multiple covariates, CMV-negative status was significantly associated with increased risk of cancer (HR 2.55, 95% CI 1.23-5.26; P=.012). CMV-negative patients that had a CMV-positive donor were found to have a higher risk of malignancy after kidney transplantation.

Ganciclovir-Resistant Cytomegalovirus Infection in a Kidney Transplant Recipient Successfully Treated with Foscarnet and Everolimus.

Cytomegalovirus (CMV) infection remains a major cause of morbidity, graft failure, and death in kidney transplant recipients. We describe a case of a 53-year-old CMV-seronegative man who underwent renal transplant from a CMV-positive donor and who developed ganciclovir- (GCV-) resistant CMV infection. Foscarnet was started while immunosuppressive therapy was modified with the introduction of everolimus minimizing tacrolimus dosage. Only two weeks after the start of this treatment regimen was the patient's viral load negative. At two-year follow-up the patient has no clinical or laboratory signs of CMV infection and a good and stable renal function or graft survival. In our case, administration of an mTOR inhibitor combined with foscarnet led to rapid and persistent viral clearance without compromising short- and medium-term graft function. This combination therapy supports the need for the kidney transplant community to individualize a target therapy for each type of GCV-resistant CMV infection.

Response to antiviral therapy in haematopoietic stem cell transplant recipients with cytomegalovirus (CMV) reactivation according to the donor CMV serological status

Pre-emptive antiviral treatment efficiently prevents occurrence of cytomegalovirus (CMV) disease in allogeneic stem cell transplant recipients. However, successive treatment courses can be necessary. The current study was aimed at determining factors that could influence the response to antiviral treatment, in particular the donor CMV serostatus. A total of 147 consecutive CMV-seropositive recipients (R+) were included and prospectively monitored for 6 months after transplantation. Reactivation of CMV occurred in 111 patients, 61 of 78 with a CMV-positive donor (D+) and in 50 of 69 with a CMV-negative donor (D–) (p 0.45). Baseline viral loads and initial viral doubling times did not differ between D+/R+ and D–/R+. Fifteen D+/R+ and four D–/R+ had self-resolving CMV infections. A total of 92 patients received antiviral treatment and 81 (88%) had a significant decrease in CMV load under therapy. Repeated CMV episodes were observed in 67% of those and were significantly more frequent in D–/R+ than in D+/R+ (p <0001). Half-life of CMV under treatment was significantly longer in D–/R+ than in D+/R+. Treatment failure observed in eight recipients was associated with low leucocyte count at reactivation onset, and was significantly more frequent in D–/R+ (six patients) than in D+/R+ (two patients) (p <0.0001). CMV strains resistant to antivirals were found in two D–/R+. Donor CMV serostatus influenced neither CMV reactivation occurrence nor the kinetics of CMV DNA load in the early phase of CMV replication but had a significant impact on response to antiviral therapy. Virological drug-resistance remained rare.