Transplantation Biology Research Articles

Gut Permeability and Glucose Absorption Are Affected at Early Stages of Graft Rejection in a Small Bowel Transplant Rat Model

Intestinal transplantation (IT) faces many challenges, among them, the necessity to early detect rejection processes. Rodent models of IT are used to provide evidence for intervention strategies as well as improve knowledge of small bowel transplant biology. The aim of our study was to determine the kinetics of small bowel rejection using a Heterotopic IT (HIT) model, with emphasis in the characterization of the process using different approaches, including the use of simple functional assays of absorptive and barrier function evaluation that could be eventually translated to the clinic. Allogeneic HIT was performed following standard procedure and rejection was monitored by clinical scoring and histopathology analysis. In order to evaluate graft absorptive function, a solution containing glucose (2 gr/Kg) and ovalbumin (OVA) (150 ug/dose) diluted in 1.5 ml of normal saline was administered by proximal ostomy 5 and 10 to 12 days after HIT. Blood samples were obtained for glucose and OVA determination at different times post-treatment. In allogeneic HIT recipients rejection kinetics was coincident to previously described, with mild rejection beginning by day 5 post-IT, becoming severe by day 10 post-IT (Figure 1). Interestingly, either at 5 or 10 to 12 post-IT days, glucose levels did not change from the basal value (101 +/− 21 mg/dL) in this group. In most of evaluated cases 5 days after HIT and in all cases evaluated at 10–12 post-IT days in allogeneic groups, OVA was detected in the recipient´s serum (24 +/−11 ug/mL, p<0.001 vs. isogenic control) 90 min post treatment. On the other hand, glucose curves were normal with a rise from basal to 240 +/−32 mg/dL at 60 min post-treatment in non-IT controls as well as in isogeneic HIT recipients (p<0.001 vs allogenic group). Furthermore, OVA was undetectable in any serum sample from control groups.FigureThis result indicate that even in early stages of ACR, there is an impairment of barrier function that is evidenced by passage of non-degraded proteins from intestinal lumen to blood. Furthermore, there is impairment on glucose absorptive capacity, even from the initial stages of rejection diagnosed by clinical and histological evaluations. The evaluation of these graft functional features could provide additional tools for rejection diagnosis in the clinics. Normal microscopic (A) and macroscopic (B) gaft appearance 10 days after syngeneic HIT. Microsocpic (C and E) and macroscopic appearance of the transplanted intestine compatible with ACR in allogeneic group.

Major Histocompatibility Complex Class I Chain-Related A (MICA) Molecules: Relevance in Solid Organ Transplantation.

An ever growing number of reports on graft rejection and/or failure even with good HLA matches have highlighted an important role of non-HLA antigens in influencing allograft immunity. The list of non-HLA antigens that have been implicated in graft rejection in different types of organ transplantation has already grown long. Of these, the Major Histocompatibility Complex class I chain-related molecule A (MICA) is one of the most polymorphic and extensively studied non-HLA antigenic targets especially in the kidney transplantation. Humoral response to MICA antigens has repeatedly been associated with lower graft survival and an increased risk of acute and chronic rejection following kidney and liver transplantation with few studies showing conflicting results. Although there are clear indications of MICA antibodies being associated with adverse graft outcome, a definitive consensus on this relationship has not been arrived yet. Furthermore, only a few studies have dealt with the impact of MICA donor-specific antibodies as compared to those that are not donor specific on graft outcome. In addition to the membrane bound form, a soluble isoform of MICA (sMICA), which has the potential to engage the natural killer cell-activating receptor NKG2D resulting in endocytosis and degradation of receptor–ligand interaction complex leading to suppression of NKG2D-mediated host innate immunity, has been a subject of intense discussion. Most studies on sMICA have been directed toward understanding their influence on tumor growth, with limited literature focusing its role in transplant biology. Furthermore, a unique dimorphism (methionine to valine) at position 129 in the α2 domain categorizes MICA alleles into strong (MICA-129 met) and weak (MICA-129 val) binders of NKG2D receptor depending on whether they have methionine or valine at this position. Although the implications of MICA 129 dimorphism have been highlighted in hematopoietic stem cell transplantation, its role in solid organ transplantation is yet to be explored. This review summarizes the currently available information on MICA antibodies, soluble MICA, and MICA-129 dimorphism in a setting of solid organ transplantation.

Mesenchymal Stem Cells Induced In Vitro Generated Regulatory-T Cells: Potential Soldiers of Transplantation Biology

Introduction: CD4+ regulatory T-cells that constitutively express CD25 have a potent mechanism of action of immunomodulation and hence hold a great therapeutic potential for the treatment of autoimmune diseases and tolerance induction in the context of solid organ transplantation. Methods: For in vitro generation of Tregs volunteer donor’s adipose tissue was resected and mesenchymal stem cells (MSC) were generated. MSC were co-cultured with post-renal transplantation recipient’s peripheral blood derived peripheral blood mononuclear cells (PBMC) and with irradiated PBMC from same recipient with the addition of exogenous IL-2. Controls were carried out without use of MSC. In vitro generated Tregs were subjected for quantitative and qualitative analysis of Tregs specific markers. Results: Quantitative analysis revealed increased cell counts of Tregs in comparison to PBMC. Tregs specific markers evaluation using flocytometer showed rise in all Treg specific cell population: CD4+25+127Neg with mean 7.6 ± 6.1%, CD4+FoxP3+ mean of 21.7 ± 15.3% and CD4+CTLA-4+ mean of 16.9 ± 13.7%. Conclusion: We conclude that MSC induce in vitro generation of Tregs. In vitro generated Tregs hold a great promise as “magic bullet” for various disease prevention and/or treatment. Keywords: Regulatory-T cells, In vitro generation, mesenchymal stem cells, co-culture, IL-2.

Does MHC Class I Chain-Related Gene A Matter?

In this issue of Biology of Blood and Marrow Transplantation, Askar et al. evaluate the association of MHC class I chain–related gene A (MICA) polymorphisms with the development of graft-versus host-disease (GVHD) after unrelated donor hematopoietic stem cell transplantation (HCT) [1]. Prior reports have provided conflicting results, leaving the effect of MICA donor-recipient mismatch in HCT unanswered.

MODELING OF A SUBCUTANEOUS XENOGRAFT OF HUMAN SKIN MELANOMA MEL CHER WITH V600E BRAF MUTATION IN IMMUNODEFICIENT MICE FOR PRECLINICAL STUDY THE TARGETING ANTICANCER DRUGS

Introduction. Development of new models of a human disseminated skin melanoma of the with molecular-genetic targets for specific therapy increases productivity of the preclinical researches new the anti-melanoma drugs or their combinations in vitro and in vivo. Such opportunity is realized by adaptation in vivo of the original human pigmented skin melanoma cell line mel Cher and receiving subcutaneous (s. c.) xenograft under monitoring of transplant, morphological, molecular-genetic (V600E BRAF mutation) and chemotherapeutic (sensitivity for the inhibitor of BRAF kinases to a vemurafenib) characteristics. Objective: receiving from the cell line mel Cher s. c. xenogratft of the human pigmented skin melanoma with V600E BRAF mutation and sensitive to specific target therapy. Materials and methods. Human pigmented skin melanoma cell line mel Cher from the Collection of Russian Cancer Research Center and immunodeficient Balb/c nude female mice cultivated in Russian Cancer Research Center was used. Required characteristics are defined by multiple s. c. transplanting in vivo by methods of transplant biology, a light microscopy, molecular-genetics and the experimental chemotherapy. Sensitivity to a BRAF kinase inhibitor to a vemurafenib was estimated under monitoring of the tumor growth rate (Vt/V0) on indexes, adequate for patients: existence of the complete remission and possibility of recurrence. Results. When s. c. transplantation of 107 cell of mel Cher line cytological identical intertwined s. c. xenografts with a stable growth kinetics on 4-9 passages (a latent phase 8 days, exponential - to 14 days, stationary - to 24 days) and existence of a mutation of V600E BRAF have been recieved. Vemurafenib in a single dose of 75 mg/kg caused the complete remission during a 15-day course and within 7 days after its cancellation - with the subsequent recurrence. Conclusion: receiving from the cell line mel Cher s. c. xenogratft of a human pigmented melanoma of skin with a mutation of V600E BRAF and sensitive to specific target therapy is suitable for preclinical studying of the new anti-melanoma drugs specific for this target.

Late treatment effects following bone marrow transplant: Efficacy of implementing international guidelines.

An increasing cohort of haematopoietic cell transplantation (HCT) survivors has raised awareness of long-term and late effects. Updated recommendations for HCT late effects screening were published in 2012 [Majhail etal. Biology of Blood and Marrow Transplantation, 18 (2012):348]. We aimed to assess the clinical efficacy of a dedicated screening clinic to identify problems in HCT survivors using the international guidelines. Clinic letters and test results of the first 59 consecutive patients attending the screening clinic were evaluated. 30 females and 29 males (mean age of 49years, range 22-74) were included. The mean time since transplant was 6years (0.5-18). 49/65 transplants were allogeneic. Primary indications for HCT were myeloid (56%), lymphoid (37%), solid tumour (5%) and auto-immune diseases (2%). 134 complications were reported (mean 2, range 0-8), with 114 documented further actions/referrals. The most commonly reported concerns were pain 18/59 (31%), fatigue 14/59 (24%), sexual function 14/59 (24%) and sleep disturbance 11/59 (19%). Second primary malignancies were recorded in five cases. Implementation and audit of the international late effect screening guidelines confirm the need for systematic long-term physical and psychological screening and care, thus ensuring timely and efficient identification of problems and the opportunity to minimise morbidity effects and optimise health.

Implications of Resident Memory T Cells for Transplantation.

Recent studies have established resident memory T cells (TRM ) as the dominant memory lymphocyte population surveying most nonlymphoid tissues. Unlike other memory T cell lineages, TRM do not recirculate through blood and are permanently confined to their tissue of residence. TRM orchestrate local immune responses and have been shown to accelerate local pathogen control in many experimental infection models. Here we briefly summarize recent advances in TRM differentiation, maintenance, and their protective function. While little is known, we have speculated on the potential implications of TRM for transplantation biology. Areas of emphasis include the role of passenger TRM in controlling latent viral recrudescence in donor organs, donor TRM as a source of graft-versus-host disease, the ability of TRM to potently induce inflammation through sensing and alarm functions, and differentiation of host T cells into TRM in response to local cues inside the allograft. Further investigation of TRM in the context of transplantation might identify therapeutic targets to prolong graft survival.

Single HLA Mismatch Unrelated Donor Allogeneic Stem Cell Transplantation in Caucasian Recipients: Outcomes in HLA-A, -B, -C, -DRB1 and -DQB1 Mismatch Hematopoietic Stem Cell Transplantation: A Study on Behalf of the Francophone Society of Stem Cell Transplantation and Cellular Therapy (SFGM-TC) and the Francophone Society for Histocompatibility and Immunogenetics (SFHI)

▪INTRODUCTION: Matching all alleles of the HLA-A, -B, -C, and -DRB1 loci (8/8 match) is associated with the highest overall survival (OS) rates after unrelated donor (URD) hematopoietic stem cell transplantation (HSCT). In Europe, patients (pts) are also matched at the HLA-DQB1 loci (10/10 match), although there is no evidence of better OS. Data on Caucasian pts receiving a single HLA mismatch URD HSCT are still controversial. We therefore conducted a multicenter retrospective study to assess the impact of a single HLA mismatch (9/10 match) on outcomes after URD HSCT in a large cohort of French pts. METHODSWe collected data from 1092 pts who underwent HSCT between January 2000 and December 2012 at 32 French transplantation centers. Informed consent was obtained in accordance with the Declaration of Helsinki. High-resolution typing was performed for HLA-A, -B, -C, -DRB1, -DQB1 loci for all donor/recipient pairs. Clinical data were obtained through ProMISe (Project Manager Internet Server), an internet-based system shared by all French transplantation centers. Endpoints of interest were classical HSCT outcomes: engraftment, Graft versus host disease (GvHD), treatment related mortality (TRM) and relapse. GvHD free relapse free survival was also studied (defined as alive with no previous grade III-IV aGvHD, no moderate or severe chronic GvHD (cGvHD) and no relapse). Myeloablative conditioning was defined as a combination of agents expected to produce profound pancytopenia and myeloablation within 1-3 weeks after administration; pancytopenia is long lasting, usually irreversible and in most instances fatal, unless hematopoiesis is restored by hematopoietic stem cell infusion (Bacigalupo, et al.. Biology of Blood and Marrow Transplantation, 2009). Models were adjusted for disease risk, recipient age, CMV and sex matching, stem cell source, GVHD prophylaxis and conditioning regimen. For adjusted analysis DQB1 was used as the reference category. Disease risk was classified as standard or high risk using the American Society for Blood and Marrow Transplantation Request for Information 2006 risk scoring schema. RESULTSPopulation characteristics are shown in Table 1. Gender balance was the only difference between groups (p<0.013). Median follow-up was 84 months. Engraftment rates were not different between groups (overall, 91.8% (95% Confidence interval (CI) (90.1 to 93.4)). Overall Cumulative Incidence of aGVHD grade II-IV and grade III-IV were 39.9% (95% CI, (38.0 to 41.8)) and 22.2% (95% CI, (20.2 to 24.2)), respectively. Myeloablative Conditioning (MAC) using Total Body Irradiation (TBI)>4Gy [Hazard Ratio (HR) HR:1.95 (95% CI, 1.45 to 2.63)] and high disease risk [HR :1.34 (95% CI, 1.09 to 1.66)] were significantly associated with an increased cumulative incidence of grade II-IV aGVHD, whereas only MAC with TBI>4Gy was a risk factor for grade III-IV aGVHD [HR:1.91 (95% CI, 1.28 to 2.87)].Overall Cumulative Incidence of cGVHD was 38.1% (95% CI, 35.9 to 40.1) at 84 months. Overall relapse rate was 33.8% (95% CI, 30.5 to 37.1) with no differences according to HLA mismatch subtype. CI of TRM was 36.0% (95% IC, 32.9 to 39.1). Single HLA-A mismatch [HR:1.55 (95% CI , 1.11 to 2.17)], high disease risk [HR:1.40 (1.12 to 1.74)], a female donor for a male recipient [HR:1.41 (1.10 to 1.79)], and the use of a MAC regimen [HR:1.50 (1.08 to 2.08)] were significantly associated with a higher TRM after adjusted analysis. Overall GRFS was 14.5% (12.1 to 17.3). Single HLA-A mismatch [HR:1.25 (95% IC, 1.01 to 1.54)] and high disease risk [HR:1.45 (95% IC, 1.26 to 1.67)] were significantly associated with a lower GRFS (Figure1). CONCLUSIONIn this large cohort of Caucasian pts who received a single HLA mismatch URD HSCT, high disease risk was the major prognostic factor related to higher rates of both acute GvHD and treatment related mortality, and eventually a lower GRFS. HSCT outcomes were similar according to HLA single mismatches except for HLA-A mismatch, which was associated with a significant increase in TRM, and a worse GFRS. This study also highlights the high rates of both TRM and relapse after single mismatch URD HSCT (9/10) which justifies the use of reduced toxicity conditioning regimens, as well as the addition of targeted therapies pre- and post- HSCT in this setting. [Display omitted] [Display omitted] DisclosuresMichallet:Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Astellas Pharma: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria. Peffault De Latour:Amgen: Research Funding; Alexion: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Sustained Natural Killer Cell Recovery Post-Autologous Peripheral Blood Hematopoietic Stem Cell Transplantation Predicts Survival in Non-Hodgkin's Lymphoma

AbstractOur group recently reported a Phase III clinical trial (Porrata et al. Biology of Blood and Marrow Transplantation 2016; 22: 1017-1023) demonstrating that the infusion of the autograft-absolute lymphocyte count (A-ALC) and autograft natural killer cells (A-NKC) are survival prognostic factors in non-Hodgkin lymphoma (NHL) patients treated with autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT). A limitation of our Phase III clinical trial was the short-term follow-up of just two years. Therefore, we evaluated longer follow-up and assessed the role of the infused A-ALC, infused A-NKC, and peripheral blood absolute natural killer cell count (ANKC) recovery on survival post-APBHSCT. Of the 122 patients that participated in the Phase III trial, 111 patients were able to complete APBHSCT and included in the current study. With a median follow-up of 57.2 months (range: 2.1-84.6 months), superior overall survival (OS) and progression-free survival (PFS) was observed in patients infused with an A-ALC ≥ 0.5 x 109 lymphocytes/kg [OS: HR = 0.429, 95%CI, 0.128-0.828, p < 0.01; and PFS: HR = 0.456, 95% CI, 0.177-0.863, p < 0.01] and an A-NKC ≥ 0.09 x 109 cells/kg [OS: HR = 1.58e-3, 95% CI, 1.046e-6-0.179, p < 0.003; and PFS: HR= 1.58e-3, 95% CI, 7.81e-7-0.064, p < 0.003]. Furthermore, patients that maintained an ANKC ≥ 250 cells/µl at 3 months, 6 months, 9 months and 12 months post-APBHSCT also experienced superior OS (Figure 1A) and PFS (Figure 1B)[OS and PFS times in months were evaluated from infusion date of stem cells]. In the multivariate analysis a sustained ANKC ≥ 250 cells/µl was an independent predictor for OS (HR = 0.013, 95% CI, 0.001-0.063, p < 0.0001) and PFS (HR = 0.014, 95% CI, 0.001-0.067, p < 0.0001). This study is an extension of our Phase III clinical trial showing that sustained innate immunity by measuring the ANKC correlated with superior clinical outcomes; thus providing a platform to develop innate immunity immunotherapeutic strategists directing to improve clinical outcomes post-APBHSCT in NHL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reduced-Intensity and Non-Myeloablative Allogeneic Stem Cell Transplantation from Alternative HLA-Mismatched Donors for Hodgkin's Lymphoma: A Study By the SFGM-TC (Francophone Society of Bone Marrow Transplantation and Cellular Therapy)

Allogeneic hematopoietic stem cell transplantation (allo-SCT) following a non-myeloablative (NMA) or reduced-intensity conditioning (RIC) is considered a valid approach to treat patients with refractory/relapsed Hodgkin's lymphoma (HL), in particular those having failed a previous autologous SCT (ASCT). When an HLA-matched donor is lacking, a graft from a haploidentical donor (HAPLO), a mismatched unrelated donor (MMUD) or cord blood (CB) might be considered. In this retrospective, registry-based study, we compared the outcome of patients with HL undergoing a RIC or NMA allo-SCT from a HAPLO, MMUD or CB graft.After detailed review of our database, we found 98 consecutive patients with HL who underwent a NMA/RIC allo-SCT from an alternative HLA-mismatched donor at 24 French and Belgian centers between January 2009 and December 2014. Probabilities of overall survival (OS), event-free survival (EFS) and graft-versus-host disease (GVHD)-free Relapse-free Survival (GRFS) were determined using the Kaplan-Meier method. The cumulative incidences of relapse (CIR), non-relapse mortality (NRM) and cGVHD were studied using a competing risk methodology. We defined GRFS as the probability of being alive without evidence of relapse, grade 3-4 acute GVHD or chronic GVHD (all grades included). Regarding conditioning regimens and intensity, patients in the HAPLO group (n = 34) received a T-cell replete allo-SCT after a NMA (as described by the Baltimore group in Luznik et al, Biology of Blood and Bone Marrow Transplantation, 2008, n = 31, 91%) or a RIC (n = 3, 9%) followed by post-transplant cyclophosphamide (PT CY). Patients in the MMUD group (n = 27) received a variety of NMA (n = 7, 26%) and RIC (n = 20, 74%). All patients in the CB group received a RIC (n = 37, 37%). All but one patient (n = 33) in the HAPLO group received the following GVHD prophylaxis: Cy 50 mg/kg on day +3 and day +4, tacrolimus or CsA and mycophenolate mofetil started on day +5. One patient received only one day of PT CY 50mg/kg at day +3 and also received ATG on day -2 and day -1 for a total dose of 5mg/kg. GVHD prophylaxis for MMUD consisted of CsA or tacrolimus, started on day -3 or day -1 in all patients according to local practice with either mycophenolate mofetil (n = 25, 89%) or methotrexate 15 mg/m2 at day +1, 10 mg/m2 at day+3 and day +6 (n = 3, 11%). GVHD prophylaxis consisted of CsA and mycophenolate mofetil starting on day -3 for all CB patients.Median age at allo-SCT was 28 (range: 16-68). The median number of treatments before allo-SCT was 4 (range: 3-6). An ASCT had been performed prior to allo-SCT in 77% (n = 26), 100% (n = 28%) and 100% (n = 37) of cases in the HAPLO, MMUD and CB group, respectively. Disease status at allo-SCT per Cheson 1999 criteria was complete remission in 51% (n = 51), partial response in 32% (n = 31), stable or progressive disease in 11% (n = 11), while no data was available in 5% (n = 5). Positron emission tomography was positive at allo-SCT in 45% (n = 44), negative in 43% (n = 43) while no data was available in 12% (n = 12). Median follow-up was 31 months (range: 3 - 79). In univariate analysis we observed a significantly higher probability of GRFS in patients who received a HAPLO allo-SCT (52% versus 22% and 31% at three years in the HAPLO, CB and MMUD groups, respectively, p = 0.02, Logrank test). Higher GRFS was also observed in patients receiving a NMA conditioning compared to a RIC (50% versus 27% at three years, p = 0.009, Logrank test). We did not observe significant differences in OS, EFS or NRM, according to donor type. Disease status at allo-SCT significantly impacted OS (p<0.001, Logrank test), CIR (p = 0.02, Gray’s test), EFS (p < 0.001, Logrank test) and GRFS (p = 0.002, Logrank test). After adjustment for significant covariates, MMUD and CB remained associated with significantly lower GRFS (HR = 2.02, p = 0.03 and HR = 2.43, p = 0.009, respectively, Cox model) compared to HAPLO donors.In conclusion, significantly higher GRFS was observed in HL patients receiving a haploidentical NMA/RIC T-cell replete allo-SCT with PT CY. How this separation of the GVH from the graft-versus-lymphoma effect occurs requires further investigations. Haploidentical NMA/RIC T-cell replete allo-SCT with PT CY for advanced HL should now be compared with NMA/RIC allo-SCT from related and unrelated HLA-compatible donors, ideally within prospective trials. DisclosuresMichallet:Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Astellas Pharma: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria. Peffault De La Tour:ALEXION: Consultancy, Honoraria, Research Funding; PFIZER: Consultancy, Honoraria, Research Funding; NOVARTIS: Consultancy, Honoraria, Research Funding.

From HLA typing to anti-HLA antibody detection and beyond: The road ahead.

The complex polymorphism of the HLA genes and the need of a proper identification of anti-HLA antibodies have led to continuously develop novel practical and feasible technologies in the field of organ and tissue transplantation. Technologies to identify HLA molecules have evolved from the serological to the molecular methods and a true innovation in the DNA sequencing has taken place with the development of next generation sequencing. An interesting field to explore is how the information resulting from the HLA-DNA sequencing can be applied in the clinical setting by including the alloimmunization assessment. Indeed, a good characterization of anti-HLA antibody at epitope level can reduce the risk of immunization. Many anti-HLA antibodies are specific for epitopes rather than for HLA antigens and the knowledge of unacceptable epitopes allows to reduce the number of mismatched antigens. Furthermore, high resolution HLA allele typing could help to understand the epitopes against which antibodies are developed. However, the improvements should not only concern the diagnostic tools in the pre-transplantation phase, but also the monitoring of post transplantation outcome. There is a growing interest in developing new non-invasive biomarkers to monitor the rejection. Currently, increasing evidence has focused on miRNAs, epigenetic markers emerged as regulators of molecular events that are differently expressed in biopsies and blood as well as in urinary samples of transplanted recipients. The implementation of next generation sequencing and genome-wide expression analysis together with functional assays may provide useful tools to evaluate the epigenetic modulation in transplantation biology but many efforts are requested for translating in the clinical arena the results obtained in experimental models.

74. The Impact of Different Hinge and Transmembrane Components on the Function of a Novel Fully-Human Anti-CD19 Chimeric Antigen Receptor

T cells expressing anti-CD19 chimeric antigen receptors (CARs) have significant activity against B-cell malignancies in humans, but increased efficacy and decreased toxicity of anti-CD19 CAR T cells are needed. Anti-CD19 CARs that have been clinically tested have single-chain variable fragments (scFvs) derived from murine antibodies or in 1 case a humanized murine antibody. Evidence of human anti-mouse immune responses against CARs with murine scFvs has been reported (Jensen et al. Biology of Blood and Marrow Transplantation 2010; Lee et al. Lancet, 2015), providing a rationale for development of less immunogenic CARs. A CAR with a scfv derived from a fully-human antibody would eliminate these human anti-mouse immune responses and perhaps increase the persistence of anti-CD19 CAR T cells. A non-immunogenic CAR might be particularly important if multiple, temporally-separated doses of CAR T cells are administered to the same patient. We have designed and constructed fully-human anti-CD19 CARs encoded by a lentiviral vector. These CARs specifically recognize CD19, and T cells expressing the CARs exhibit a full range of functions including degranulation, cytotoxicity, proliferation, and release of a variety of cytokines. The extracellular scFv component of a CAR is connected to intracellular domains by an extracellular hinge region and a transmembrane (TM) region. In comparing anti-CD19 CARs, we noticed a difference in cytokine production by CARs with hinge and TM regions from the human CD8α molecule compared to CARs with hinge and TM regions from the human CD28 molecule. We designated a fully-human CAR with CD8α hinge and TM domains hu19-CD828Z, and we designated a CAR that was identical to hu19-CD828Z except that it had CD28 hinge and TM domains hu19-28Z. Compared to T cells expressing hu19-CD828Z, T cells expressing hu19-28Z produced much higher levels of interferon gamma (IFNγ), tumor necrosis factor (TNF), and interleukin-2. For example, after an overnight culture with CD19+ NALM6 cells, T cells expressing hu19-CD828Z yielded a mean of 5688 pg/mL IFNγ in the culture supernatant while T cells expressing hu19-28Z yielded 19396 pg/mL IFNγ (P=0.03). Similarly, after an overnight culture with CD19+ NALM6 cells, T cells expressing hu19-CD828Z yielded a mean of 2033 pg/mL TNF in the culture supernatant while T cells expressing hu19-28Z yielded 5008 pg/mL TNF (P=0.007). Cytokine levels were normalized for the percentage of T cells expressing each CAR in each experiment. This same pattern of hinge and TM domains affecting cytokine production was observed for CARs incorporating a different anti-CD19 scFv. In contrast to cytokine production, significant differences in other T-cell functions including degranulation, cytotoxicity, and proliferation were not found when hu19-CD828Z and hu19-28Z were compared. When T cells expressing hu19-CD828Z and hu19-28Z were assessed in a murine lymphoma model, there was not a statistically-significant difference in tumor eradication or survival. We are currently assessing other measures of T-cell activation to obtain a more mechanistic understanding of the differences in T-cell activation with different hinge and TM domains. Most of the clinical toxicity of anti-CD19 CAR T cells is caused by cytokine release. Compared to anti-CD19 CARs with CD28 hinge and TM domains, anti-CD19 CARs with hinge and TM regions from CD8α cause lower levels of cytokine release from T cells in vitro. This decreased cytokine release might reduce cytokine-mediated toxicity in patients. We have initiated a clinical trial of T cells expressing hu19-CD828Z. We will assess cytokine release, immunogenicity, peak blood levels, and long-term persistence of hu19-CD828Z-expressing T cells in humans.

Cell Manipulation in Pediatric Haploidentical Stem Cell Transplantation: State of the Art

Haploidentical transplantation in children can extend the opportunity for transplantation to almost every patient lacking a human leukocyte antigen (HLA)–matched donor and offer this treatment to every child with an otherwise incurable disease. Although initial attempts were associated with a high transplant-related mortality, recent insights into the biology of haploidentical transplantation, the availability of effective ex vivo large-scale graft-manipulation technology, and improved supportive care strategies have led to significantly better outcomes. Concurrently, the indication for haploidentical transplantation has been extended, including different malignant and nonmalignant conditions. Worldwide donor registries include mainly donors of Caucasian origin. Patients of non-Caucasian origin have a lower chance of finding a suitable unrelated donor. Haploidentical transplantation allows the treatment of children independently of their ethnic background in a timely fashion. One of the major advantages of using a related donor is the possibility of collecting or generating additional cellular products from the same donor to open the possibility of enhancing both the antitumor effects of the graft and the immunologic reconstitution after transplantation.

New frontiers for zebrafish management.

The zebrafish (Danio rerio) is a preeminent model organism with a wide and expanding utility for numerous scientific disciplines. The same features that once endeared this small freshwater minnow to developmental biologists combined with its relatively high genetic similarity to mammals and the advent of new, more efficient methods for genome editingare now helping to spur expanded growth in its usage in various fields, including toxicology, drug discovery, transplant biology, disease modeling, and even aquaculture. Continued maturation and adoption of the zebrafish model system in these and other fields of science will require that methods and approaches for husbandry and management of these fish in controlled settings be refined and improved to the extent that, ultimately, zebrafish research becomes more reproducible, defined, cost-effective, and accessible to the masses. Knowledge and technology transfer from laboratory animal science and commercial aquaculture will be a necessary part of this development.

An Insight Into the Immunologic Events and Risk Assessment in Renal Transplantation.

Organ transplantation has always been considered to be the optimal therapeutic intervention in patients with end-stage organ failure. In the US, approximately 615,000 patients are diagnosed with end-stage renal disease and less than 30% have received a kidney transplant. One of the crucial drawbacks in successful renal transplantation is allograft rejection. Survival rates among transplant recipients have greatly improved due to better understanding of transplant biology and more effective immunosuppressive agents. Post-transplant immune monitoring and optimization of the immunosuppressive therapy using non-invasive biomarkers can effectively predict impending graft rejection and may spare the need for renal biopsy. This article provides an insight into the immunomodulations of renal transplant recipients. It depicts the immune system including several types of kidney rejection and reviews the biomarkers that may serve in near future, as surveillance tools for graft monitoring. Finally, a summary on the main immunosuppressive drugs used in kidney transplant both in the induction and maintenance phases is also covered.

MiRNAs and piRNAs from bone marrow mesenchymal stem cell extracellular vesicles induce cell survival and inhibit cell differentiation of cord blood hematopoietic stem cells: a new insight in transplantation.

Hematopoietic stem cells (HSC), including umbilical cord blood CD34+ stem cells (UCB-CD34+), are used for the treatment of several diseases. Although different studies suggest that bone marrow mesenchymal stem cells (BM-MSC) support hematopoiesis, the exact mechanism remains unclear. Recently, extracellular vesicles (EVs) have been described as a novel avenue of cell communication, which may mediate BM-MSC effect on HSC. In this work, we studied the interaction between UCB-CD34+ cells and BM-MSC derived EVs. First, by sequencing EV derived miRNAs and piRNAs we found that EVs contain RNAs able to influence UCB-CD34+ cell fate. Accordingly, a gene expression profile of UCB-CD34+ cells treated with EVs, identified about 100 down-regulated genes among those targeted by EV-derived miRNAs and piRNAs (e.g. miR-27b/MPL, miR-21/ANXA1, miR-181/EGR2), indicating that EV content was able to modify gene expression profile of receiving cells. Moreover, we demonstrated that UCB-CD34+ cells, exposed to EVs, significantly changed different biological functions, becoming more viable and less differentiated. UCB-CD34+ gene expression profile also identified 103 up-regulated genes, most of them codifying for chemokines, cytokines and their receptors, involved in chemotaxis of different BM cells, an essential function of hematopoietic reconstitution. Finally, the exposure of UCB-CD34+ cells to EVs caused an increased expression CXCR4, paralleled by an in vivo augmented migration from peripheral blood to BM niche in NSG mice. This study demonstrates the existence of a powerful cross talk between BM-MSC and UCB-CD34+ cells, mediated by EVs, providing new insight in the biology of cord blood transplantation.

Implementation of NIH Criteria for Standardization of Chronic Graft-Versus-Host Disease in Croatia: Two-Year Experience

Background: Chronic graft-versus-host disease (cGVHD) is a disorder that affects many organ systems in highly variable fashion occurring in approximately 50% of patients following allogeneic hematopoietic stem cell transplantation (alloHSCT). It is the major cause of non-relapse morbidity and mortality after alloHSCT in individuals otherwise cured of their hematologic diseases, inducing poor quality of life, impaired functional status, inability to work, and need for ongoing chronic care, which has also important impact to health-related costs. cGVHD Consensus Conference held in 2005 at the National Institutes of Health (NIH), USA, produced recommendations regarding cGVHD diagnosis, staging, histopathology, response criteria, biomarkers, ancillary and supportive care, and design of clinical trials. In 2014, second cGVHD NIH Consensus Conference updated these recommendations, published during 2015 as 6 papers in Biology of Blood and Marrow Transplantation (BBMT) journal. Although practitioners are generally familiar with the NIH recommendations, many barriers prevent their greater uptake in clinical practice. In order to overcome these challenges, in 2013 multidisciplinary clinic infrastructure was organized at the University Hospital Center (UHC) Zagreb, Croatia, in collaboration with the NIH leading scientists, using established cGVHD-related grading scales and measurements.Methods: Division of Hematology, UHC Zagreb, Croatia, has experience with alloHSCT since 1983, and 827 patients received alloHSCT until the end of 2014. Since the establishment of multidisciplinary cGVHD team in 2013, patients were enrolled into the Unity through Knowledge Fund (UKF) study protocol (funded by World Bank and Croatian Ministry of Science, Education and Sports) and examined by multiple subspecialists, firstly seen by hematologist, with detailed history and physical exam. Standard cGVHD scoring forms are filled according to NIH Consensus recommendations, and extensive laboratory analyses were done. Patients are seen and evaluated by other sub-specialists (Dental, Dermatology, Rehabilitation, Neurology, Ophthalmology, Gynecology, and other) with further workup as needed. Quality of life questionnaires are filled during the visit. All data are collected in a specially developed database and weekly team meetings were established. Blood and small biopsy tissue samples (skin, mouth) are stored for further research.Results: Using multidisciplinary approach since 2013, 46 (6 pediatric) cGVHD patients were assesed, median age was 41 years; range [9-71], 24 were male and 22 were female. The median time from transplant to enrollment was 20 months [2-258], from cGVHD diagnosis to enrollment 7 months [0.03-234] and from transplant to cGVHD diagnosis 10 months [2-128]. Additional 17 post-alloHSCT patients were eveluated, but without confirmation of cGVHD diagnosis. Among cGVHD patients, 31 (67%) of them received transplants from matched related donors, 27 (59%) of them had myeloablative conditioning, and 26 (57%) received peripheral blood stem cells as graft source. Thirty-five (76%) patients had previous acute GVHD, 11 (24%) had de novo cGVHD, 21 (46%) quiescent and 14 (30%) progressive onset; 41 (89%) were classified as classic and 5 (11%) as overlap; 23 (50%) patients had severe, 19 (41%) moderate, and 4 (9%) mild global cGVHD score. The most involved organs were skin (54%), eyes (50%), lungs (48%) and mouth (39%). Due to internationally peer reviewed UKF grant awarded in 2013 doctoral and postdoctoral researcher were hired, and visits of young clinicians to NIH and other cGVHD centers were realized. Several new research subprojects emerged since formation of our cGVHD team and applications to the new project calls were submitted. Also, 2 international cGVHD symposiums were organized in Zagreb, Croatia, in last 2 years stimulating education and networking.Conclusion: Implementation of NIH criteria for standardizationof cGVHD in Croatia showed remarkable results, not just improving quality of medical documentation and management of these long-terms survivors with complex and long-lasting health issues, but also facilitating further international clinical research and collaboration with cGVHD community, with potential positive impact to health-related costs and benefit to society. DisclosuresNemet:Pliva: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria.

Proceedings of the 13th international transplantation symposia: mTOR-inhibition: what have we learned and how so we best apply the learning.

In Transplantation Research we have recently published a series of articles on the role of mammalian Target of Rapamycin (mTOR) inhibitors [1–3]. This is a new departure for the Journal, on which we hope to build in the future, of publishing online conference supplements. The articles are based on the most recent of a long-running annual conference, supported initially by Wyeth and subsequently by Pfizer, following the introduction of sirolimus (Rapamune). For me, this meeting gave delegates a unique experience, and I doubt if any participant failed to learn something to change their clinical practice. Using a combination of didactic lectures, workshops and interactive sessions for around 300 clinicians and 20–30 faculty members, the design of the meeting promoted dialogue and exchange of information. Perhaps the key aspect of the meeting was to mix faculty, including some of the leading figures in transplantation, with transplant clinicians from a range of backgrounds in an informal atmosphere. Initially, the faculty members were those involved in the development of sirolimus (SRL) and early clinical trials, but later involved those with interests in infection, cardiovascular disease and cancer where there were specific issues or opportunities relating to the use of mTOR-inhibitors; and subsequently, to the developing role of mTOR on cellular function and cancer, intravascular stents, and other areas. The focus of the meeting also evolved with time: initially devoted to SRL, in later meetings this agent became the fulcrum for a meeting that covered other emerging immunosuppressants – including mTOR-inhibitors, such as everolimus, novel agents such as belatacept, and much broader aspects of transplant biology and management.

The emerging role of complement inhibitors in transplantation

The role of complement in the biology of kidney transplantation is becoming more and more significant, especially but not only because we now have access to drugs inhibiting complement. After describing the main characteristics of complement biology, both activation of the complement cascade and the many regulatory factors, we will review the precise role of complement in kidney transplant biology. Complement activation has been involved in ischemia-reperfusion injury, in the recurrence of several diseases such as atypical hemolytic uremic syndrome, C3 glomerulopathies, and antiphospholipid syndrome, as well as the process of antibody-mediated rejection, either acute or chronic. There are many potentially interesting drugs interfering with complement inhibition that have been or may be studied in kidney transplantation. Currently, the bulk of data concerns eculizumab, a monoclonal antibody blocking the complement cascade at the C5. Its efficacy has been demonstrated in the treatment and prevention of recurrence of atypical hemolytic uremic syndrome with an overall good safety profile. Although it has been reported to be efficacious to prevent antibody-mediated rejection, properly designed trials are currently being performed to state this efficacy. In addition, randomized trials are, in the process, regarding the prevention of ischemia-reperfusion injury after kidney transplantation.

Editorial introductions

Current Opinion in Hematology was launched in 1994. It is part of a successful series of review journals whose unique format is designed to provide a systematic and critical assessment of the literature as presented in the many primary journals. The field of hematology is divided into nine sections that are reviewed once a year. Each section is assigned a Section Editor, a leading authority in the area, who identifies the most important topics at that time. Here we are pleased to introduce the Section Editors for this issue. SECTION EDITORS Andrea BacigalupoAndrea BacigalupoDr Bacigalupo is the Head of the Division of Hematology and Stem Cell Transplants at Ospedale San Martino, Genova, Italy. He graduated in 1973 from the Genova Medical School, Italy, and started an allogeneic transplant program in 1976. He has conducted several clinical trials in patients with aplastic anemia and in patients undergoing allogeneic transplants. His main interests are bone marrow failure, Graft-versus-Host-Disease (GvHD) and related complications, and graft versus leukemia reactions. He has also been interested in cytomegalovirus and Epstein–Barr virus infections, looking at early diagnosis and prospective studies on prophylaxis and pre-emptive therapy. Dr Bacigalupo has served as a board member of the European Group for Bone Marrow Transplantation (EBMT) for several years, as secretary, then Chairperson of the Aplastic Anemia Working Party and then as President from 1998 to 2002. He served as President of the Italian Group of Bone Marrow Transplantation (GITMO) from 2002 to 2005. He has been a member of the Italian National Health Council for two terms (2000–2006). He has served on the Regimen Related Toxicity Working Group of the Center for International Blood and Marrow Transplant Research (CIBMTR). Dr Bacigalupo has published over 500 articles in peer-reviewed journals and he is a member of several national and international hematology societies. He is Associate Editor of the journal Bone Marrow Transplantation and Biology of Blood and Marrow Transplantation. He serves on the Board of Haematologica and of the Journal of Clinical Oncology. Jed B. GorlinJed B. GorlinDr Jed B. Gorlin is board certified in pediatrics and blood banking and transfusion medicine. Dr Gorlin is the Vice President of Medical and Quality Affairs for Innovative Blood Resources, USA. He received his undergraduate degree from Stanford University, USA, a MD degree from Yale University, USA, and a MBA from the University of Minnesota's Carlson School of Business, USA. Dr Gorlin trained in pediatrics and pediatric hematology/oncology at Boston Children's hospital, USA, and completed research fellowships at Dana-Farber Cancer Institute, USA, Massachusetts General Hospital, USA, Brigham & Women's Hospital, USA, and the Puget Sound Blood Center, USA. Dr Gorlin served on the boards of AABB and the National Blood Foundation. He is currently the Co-Director of Transfusion Medicine at Hennepin County Medical Center, a level one trauma hospital in Minneapolis, USA, and the Children's Hospitals and Clinics in Minneapolis and St. Paul, USA. He is a consultant to AABB and CDC for projects in Rwanda, Tanzania and Central Asia including Kazakhstan and Kyrgyzstan.