Chimerism Studies Research Articles

T cell engineering as therapy for cancer and HIV: our synthetic future.

It is now well established that the immune system can control and eliminate cancer cells. Adoptive T cell transfer has the potential to overcome the significant limitations associated with vaccine-based strategies in patients who are often immune compromised. Application of the emerging discipline of synthetic biology to cancer, which combines elements of genetic engineering and molecular biology to create new biological structures with enhanced functionalities, is the subject of this overview. Various chimeric antigen receptor designs, manufacturing processes and study populations, among other variables, have been tested and reported in recent clinical trials. Many questions remain in the field of engineered T cells, but the encouraging response rates pave a wide road for future investigation into fields as diverse as cancer and chronic infections.

Matched sibling allogenic transplant for an adult case of leukocyte adhesion deficiency (LAD-1)

We report first successful allogenic hematopoetic stem cell transplant of adult male with leukocyte adhesion deficiency. This patient was symptomatic for recurrent pyoderma gangrenosum since 10 years. He had received local treatments, systemic antibiotics, steroids, and cyclosporin but pyoderma was refractory leading to delay in hematopoetic stem cell transplant. The graft source was peripheral blood from 6/6 matched sibling. The conditioning regimen used was Busulphan, Cyclophosphamide, and antithymocyte globulin. Neutrophil engraftment occurred on day +14 and platelet engraftment occurred on day +24. Chimerism study of day +30 with VNTR showed 100% donor chimerism with normalization of CD18 expression.

Human biofluid concentrations of mono(2-ethylhexyl)phthalate extrapolated from pharmacokinetics in chimeric mice with humanized liver administered with di(2-ethylhexyl)phthalate and physiologically based pharmacokinetic modeling

Di(2-ethylhexyl)phthalate (DEHP) is a reproductive toxicant in male rodents. The aim of the current study was to extrapolate the pharmacokinetics and toxicokinetics of mono(2-ethylhexyl)phthalate (MEHP, a primary metabolite of DEHP) in humans by using data from oral administration of DEHP to chimeric mice transplanted with human hepatocytes. MEHP and its glucuronide were detected in plasma from control mice and chimeric mice after single oral doses of 250mg DEHP/kg body weight. Biphasic plasma concentration–time curves of MEHP and its glucuronide were seen only in control mice. MEHP and its glucuronide were extensively excreted in urine within 24h in mice with humanized liver. In contrast, fecal excretion levels of MEHP glucuronide were high in control mice compared with those with humanized liver. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated urine MEHP concentrations in humans were consistent with reported concentrations. This research illustrates how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of pharmacokinetics or toxicokinetics of the primary or secondary metabolites of DEHP.

Both PD-1 ligands protect the kidney from ischemia reperfusion injury.

Acute kidney injury (AKI) is a common problem in hospitalized patients that enhances morbidity and mortality and promotes the development of chronic and end-stage renal disease. Ischemia reperfusion injury (IRI) is one of the major causes of AKI and is characterized by uncontrolled renal inflammation and tubular epithelial cell death. Our recent studies demonstrated that regulatory T cells (Tregs) protect the kidney from ischemia reperfusion-induced inflammation and injury. Blockade of programmed death-1 (PD-1) on the surface of Tregs, prior to adoptive transfer, negates their ability to protect against ischemic kidney injury. The present study was designed to investigate the role of the known PD-1 ligands, PD-L1 and PD-L2, in kidney IRI. Administration of PD-L1 or PD-L2 blocking Abs prior to mild or moderate kidney IRI significantly exacerbated the loss of renal function, renal inflammation, and acute tubular necrosis compared with mice receiving isotype control Abs. Interestingly, blockade of both PD-1 ligands resulted in worse injury, dysfunction, and inflammation than did blocking either ligand alone. Genetic deficiency of either PD-1 ligand also exacerbated kidney dysfunction and acute tubular necrosis after subthreshold ischemia. Bone marrow chimeric studies revealed that PD-L1 expressed on non-bone marrow-derived cells is critical for this resistance to IRI. Finally, blockade of either PD-1 ligand negated the protective ability of adoptively transferred Tregs in IRI. These findings suggest that PD-L1 and PD-L2 are nonredundant aspects of the natural protective response to ischemic injury and may be novel therapeutic targets for AKI.

Autosomal Short Tandem Repeat (STR) Variation Based on 15 Loci in a Population from the Central Region (Riyadh Province) of Saudi Arabia

Introduction: The small size of Short Tandem Repeats (STRs), their ubiquitous genome-wide distribution and polymorphic nature enhances their value in human forensic/population genetics applications. Objectives: This study aims to investigate the short tandem repeat variation based on 15 loci in a population from the central region of Saudi Arabia. Methods: Allele frequency variation for 15 Short Tandem Repeat (STR) loci was examined in 190 unrelated Saudi volunteers. Results: This study summarizes the allele distribution in the Saudi population and compares them to other populations located in Asia, Africa, the Middle East and Europe. The standard forensic parameters of Observed Hetrozygosity (Ho), Expected Heterozygosity (He) and Gene Diversity Index (GD) were determined for the following 15 STR loci: D8S1179, D21S1, D7S820, CSF1PO, D3S1358, TH0, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S5, D5S818 and FGA. The most frequent alleles in the Saudi population were: 8 repeats (0.558) at TPOX, 12 (0.411) at D13S317, 12 (0.385) at CSF1PO, 11 (0.382) at D16D539 and 10 (0.358) at D7S820. The 15 markers utilized in this study are highly informative as evidenced by their high power of discrimination (PD) values with D2S1338, D19S433 and FGA having the highest PD values. The relationship between the Saudi population and other geographically distributed populations, assessed by a Multidimensional Scaling (MDS) plot, showed that the Saudi population clustered with groups from Yemen, Iraq, Qatar, Oman and Bahrain. Conclusion: TPOX, D13S317, CSF1PO, D16D539 and D7S820 markers were found suitable for forensic analysis, paternity testing and can also be used for chimerism study after allogenic bone marrow transplantation for Saudi population. On the other hand, the population admixture with other ethnic origins might explain the variable degree of genetic distances of this population and other Arab-related groups.

Systematic Review of Cases of Transfusion Associated Graft-Versus-Host-Disease (TA-GVHD): Analysis of Patient Characteristics and Outcomes

Introduction:TA-GVHD is a rare, usually fatal complication from blood transfusions wherein donor lymphocytes engraft and mount an immune response against recipient tissues. Immunocompromised hosts who are unable to recognize and destroy foreign lymphocytes are at theoretical risk of TA-GVHD, and a variety of immune incompetencies thus form the indications for cellular blood component irradiation. Much of our understanding on who is at risk of TA-GVHD as well as policies regarding who should receive irradiated components derive from case reports and small case series. We performed a systematic review of cases of TA-GVHD to refine evidence-based guidelines for the use of gamma-irradiated blood components, assess the usefulness of the Centers for Disease Control (CDC) criteria in making the diagnosis of TA-GVHD and to define the minimum details of a case of TA-GVHD that should be reported.Methods:Our systematic review, which was registered with PROSPERO, included all reported cases of TA-GVHD from 1947 until September 2013 with no restrictions on language or date. Titles and abstracts were reviewed by two independent reviewers. References were assessed for additional cases. Data collected included patient demographics, details regarding the transfusion(s), clinicolaboratory features outlined in the CDC criteria (rash, diarrhea, fever, hepatomegaly, liver dysfunction, marrow aplasia and pancytopenia), patient outcomes and results of HLA and chimerism studies.Results:After duplicates were removed, 2130 publications were available for review, with 348 cases of TA-GVHD included in the analysis. The median age was 58 years (IQR 18, 68) with 61.6% males. The most frequent underlying diagnoses were non-cardiac surgery (n=81, 23.3%), cardiac surgery (n=71, 20.4%) and hematologic malignancy (n=66, 19.0%). According to our institutional guidelines for irradiation of blood components (Massachusetts General Hospital, Sunnybrook Health Sciences Centre, The Hospital for Sick Children and University Health Network), 121 cases (34.5%) met the criteria for requiring irradiation of blood components.Among the 245 patients in whom time of symptom onset was reported, the first symptom of TA-GVHD occurred a median of 11 (IQR 8, 14) days from the implicated transfusion. Patients had an average of 3.6 symptoms and/or signs of TA-GVHD using the CDC definition, with rash being the most common finding in 80.2% of cases (Figure 1). A biopsy was performed in 67.5% of cases, with skin as the most common site in 97.0% of cases. Chimerism studies were done in 103 cases (29.7%) to confirm the diagnosis. According to the CDC case definition, 215 (62.0%) had definitive and 73 (21.0%) had probable TA-GVHD. The case imputability based on the CDC definition was definite, probable, possible and doubtful in 28.5, 2.3, 50.0 and 2.0% of cases, respectively. The implicated blood component was red blood cells in 132 cases (37.9%), whole blood in 92 cases (26.4%), and platelets in 20 cases (5.7%). The treatment for TA-GVHD was immunosuppression therapy (IST) in 48.9%, bone marrow transplant (BMT) in 2.3%, and no intervention in 46.7%. Of those treated with IST or BMT, 10.7% survived, whereas 5.6% of the untreated survived, p = 0.063. Overall, 29 patients (8.4%) survived; of these, 3 (10.3%) received BMT, 16 (55.2%) IST and 10 (31.0%) no therapy.Conclusions:To our knowledge this is the largest systematic review of reported cases of TA-GVHD. This review describes the diversity of patient characteristics and presentations of TA-GVHD. Firstly, patients at risk for TA-GVHD encompass all levels of immune integrity, and reliance on the patient’s underlying diagnosis to dictate the need for irradiated blood components limits mitigation, as blood component or donor-recipient factors may be more important considerations. Secondly, the analysis of cases revealed limitations and inconsistencies in reported details, underscoring the need for consensus on reporting criteria, without which TA-GVHD case confirmation is challenging. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Product Attributes and Donor Factors in Reported Cases of Transfusion-Associated Graft Versus Host Disease: A Systematic Review

▪ INTRODUCTIONTransfusion-associated graft-vs-host disease (TA-GVHD) is a rare and often fatal complication of transfusion of cellular blood products. The relative contributions of product and donor factors to the risk of TA-GVHD remain uncertain. METHODSSystematic review of all reported cases of TA-GVHD in the published literature prior to Oct 2013, without language restrictions. Cases attributed to granulocyte transfusions, passenger lymphocyte syndrome, or GVHD following stem-cell transplant (unless traced to blood components rather than the graft) were excluded. Data collected included patient demographics and health information, details of transfusion event(s) and blood component(s), clinicolaboratory features of the TA-GVHD presentation and outcome, and results of human leukocyte antigen (HLA) and chimerism studies. HLA typing was evaluated where reported for both donor (product) and recipient at either class I or class II loci. Donor/recipient pairs were categorized as D=0 when there were no identified donor HLA antigens foreign to the recipient, or D>0 when donor cells contained one or more HLA antigens not found in the recipient. This classification applied separately to HLA class I and class II loci for each case. RESULTSAfter removing duplicates, 2130 citations discovered by the search were examined by two independent reviewers, with 394 identified as publications of interest for complete review. An additional 21 publications were found from the initial review, for a total of 415 publications. Of these, 195 publications described 348 unique cases for inclusion.Component: The component implicated in TA-GVHD was identified in 248 (71%) cases: Red cells (RBC) in 132 (38%); whole blood (WB) in 92 (26%); platelets in 20 (6%); buffy- coat product in 2 (0.6%); and plasma and plasma-reduced blood in one case each. In 100 (29%) cases, the blood component was either not specified or not identified among several potentially responsible components.Storage: Component storage time was reported in 158 (45%) cases. Of these, the implicated product was either described as “fresh” or </=10 days old in 148 (94%). 10 (6%) cases reported a storage time >10 days (maximum 14 days).Related donor: In 63 cases, the donor was either related (n=61) or deliberately HLA-matched (n=2) to the recipient, while in 113 cases the donor was unrelated. The remaining cases either reported a “possible” related donor or did not report the donor-recipient relationship.Leukoreduction/Irradiation: Leukoreduction status was reported in 135 (39%) cases. Of these, the implicated product was leukoreduced in 23 (17%) (10 bedside, 2 pre-storage, 11 not specified). The product was irradiated in 9 cases.HLA: HLA typing of recipient and donor, by serological or molecular techniques, was available for 84 cases (74 cases Class I, 62 Class II). Among patients with HLA data available, 20 (24%) had an underlying diagnosis warranting irradiation by current standards, while 64 (76%) did not. The category of D=0 was found in 47 (64%) of cases with reported class I typing; 44 (71%) of cases with reported class II typing; and 60 (71%) overall (Figure 1). There were 9 cases in which the category of D=0 could be ruled out for both HLA I and II. In the remaining 15 cases, the category of D=0 at either HLA I or II could not be definitively ruled in or out based on reported data. When considering those in whom the presence or absence of D=0 could be definitely determined, while D=0 at either HLA class I or class II was present in 55 of 57 (96%) of recipients without an indication for blood component irradiation, D=0 was present in only 5 of 12 (42%) of recipients with an indication for irradiation, p< 0.0001 (Table 1). CONCLUSIONSThe most common components implicated in TA-GVHD were WB and RBC. Most units were non-leukoreduced and stored for <10 days. Most cases of TA-GVHD occurred in recipients without a standard indication for irradiation. The absence of a foreign donor antigen at either HLA class I or class II occurred in a large majority of cases and was significantly more common in TA-GVHD among recipients without an indication for irradiation compared with those in whom irradiation would be indicated, suggesting that this donor-recipient relationship is the predominant risk factor in the development of TA-GVHD. Policies for irradiating cellular blood components based solely on the diagnosis of the recipient may fail to address all relevant risk factors for TA-GVHD. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Successfull Engraftment and Clearance of Donor Specific Antibodies after Haploidentical Related Stem Cell Transplantation for an Adult Patient with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is one of the most common genetic disorders in the world and despite advances in best supportive care it remains a disease with high risk of morbidity and mortality. Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only curative treatment modality. Reports of adults are limited because of the lack of suitable matched donors and the high treatment related toxicity (TRT) after conventional myeloablative conditioning regimen resulting from the accumulated disease specific end organ damage. Initial reports of non myeloablative conditioning regimen showed disappointing results because of the high risk of graft failure (GF) and disease recurrence. Recently, the development of reduced toxicity conditioning regimen (RTC) with lower TRT and the use of haploidentical family donors has widened the applicability of alloHSCT. Since SCD patients (pt) are highly alloimmunosized, donor HLA-specific antibodies (DSA) are often detectable and might compromise engraftment in HLA mismatched transplants.Material and Methods: We report a twenty two year old male pt with severe sickle cell-ß thalassemia and chronic blood transfusion related alloimmunization who received a family donor bone marrow alloHSCT from his haploidentical father. To reduce the risk of graft rejection, the preparative regimen consisted in a prephase-conditioning sequential immunoablation using two courses of a four day immunosuppressive treatment with fludarabine 40 mg/m2/d and dexamethasone 40 mg/d, six and three weeks before the start of the RIT conditioning regimen consisting of antithymocyte globuline (ATG, rabbit) 1.5 mg/kg from days -12 to -10; thiotepa 5 mg/kg on day -9; fludarabine 40 mg/m2 and intravenous busulfan 100 mg/kg from days -6 to -3. Graft versus host disease (GVHD) prophylaxis was provided by posttransplantation cyclophosphamide 50 mg/kg on day +3 and +4; cyclosporine and mycofenolate mofetil were started at day +5. The transplant work-up program included routine HLA antibody screening and prophylactic desensitization treatment with Rituxan combined with high dose immunoglobulin infusions after each cycle of immunoablation; additionally, plasmapheresis were performed at day -13 and -1, as well as one cycle of red blood cell exchange before the start of the RIT. Disease response was weekly measured by HbS levels using hemoglobin electrophoresis. Chimerism studies were done monthly by peripheral blood isolated CD3+ cells PCR analysis of variable number of nucleotide tandem repeats. DSA were examined using the luminex-based single antigen assay and MFI>=1000 were considered positive.Results: The time to neutrophil recovery over 500 x 103/mm3 and platelet recovery over 50 x 103/mm3 were 29 and 43 days respectively; transfusion independency was reached at day +36. No GF occurred with predominantly donor mixed chimerism (82%) at last follow-up. HbS levels (baseline 68%) continuously decreased after the start of the preparative regimen to < 1% at day +77. DSA were substantially reduced after desensitization treatment and importantly dropped down further after alloHSCT to undetectable levels. Of note, no increase of DSA posttransplantation was observed. With a follow up of 140 days, the pt developed no GVHD or early TRT.Conclusions: We presume that our preparative regimen approach in association with posttransplantation cyclophosphamide provides sufficient T-cell depletion and tolerance induction to enhance durable donor engraftment. The presence of DSA should not be a barrier to HLA haploidentical related alloHSCT. DisclosuresNo relevant conflicts of interest to declare.

Factors Influencing Reconstitution of Mucosal-Associated Invariant T Cells Following Allogeneic Hematopoietic Stem Cell Transplantation

Background: Mucosal-associated invariant T (MAIT) cells are innate-like T cells characterized by high expression of CD161 and a semi-invariant T cell receptor (TCR) comprised of a Vα7.2-Jα33 alpha chain and a limited Vβ repertoire that enables their activation by riboflavin metabolites produced by distinct bacterial and fungal species. MAIT cells are infrequent in cord blood, but undergo TCR-dependent accumulation in neonates in response to gastrointestinal (GI) commensal colonization to comprise approximately 10% of T cells in adult blood. The GI localization of MAIT cells, their capacity to secrete IL-17, and their activation by microbial metabolites suggests a role in mucosal immunity that may be particularly important after allogeneic hematopoietic stem cell transplantation (HCT) when the GI mucosal barrier is compromised and adaptive immunity is impaired. After HCT, the composition of the GI microbiota may be modified by antibiotics, mucositis and immunosuppression, yet its impact on MAIT cell reconstitution, function and post-transplant immunity remain unknown.Aims: To characterize and identify factors influencing MAIT cell reconstitution and function after HCT.Methods: Blood and stool samples were collected from healthy donors and HCT patients prior to and at distinct times after HCT. Absolute counts of MAIT cells, identified as CD3+/CD161hi/Vα7.2+events, were determined in peripheral blood using flow cytometry performed in conjunction with a complete blood count. The bacterial composition of stool was characterized using bacterial 16S rRNA gene PCR with high throughput sequencing and phylogenetic assignment of the amplified fragments. TCR signaling pathway activation in MAIT cells and conventional T cells was evaluated using flow cytometry analysis of phosphoprotein expression after stimulation through the TCR-CD3 complex with anti-CD3/anti-CD28 monoclonal antibodies. TCR Vβ repertoire assessment was performed using high throughput TCRBV gene sequencing.Results: High throughput TCRBV gene sequencing showed that MAIT cells from different donors (n = 3) shared TCRBV sequences, consistent with their capacity to be activated by common GI microbial TCR ligands. Despite GI microbial colonization, MAIT cells from adult donor blood were quiescent and did not proliferate to TCR stimulation. Phosphoprotein flow cytometry established that phosphorylation of proximal TCR signaling pathway molecules (CD3ζ, Lck, and ZAP-70) was diminished and responsible for impaired TCR signaling in adult MAIT cells compared to conventional αβ T cells. MAIT cell proliferation was restored by TCR stimulation in the presence of IL-1β, IL-12, IL-18 and IL-23, raising the possibility that the post-HCT inflammatory environment might be permissive for MAIT cell proliferation driven by GI microbial TCR ligands. We examined the kinetics of MAIT cell reconstitution in HCT patients (n = 163). MAIT cell numbers were lower in patients before conditioning compared to healthy individuals, and were further depleted on the day of stem cell infusion; however, they proliferated in the post-HCT environment in association with induction of Ki67 expression and reached a plateau after day 30 post-HCT (healthy, 56.8/μL; day 30, 6.7/μL). MAIT cell reconstitution after peripheral blood stem cell (PBSC) transplantation was similar comparing myeloablative (MA) and reduced intensity conditioning (RIC) regimens and related compared to unrelated donors, but was highly variable between individuals. Short tandem repeat PCR chimerism studies showed that MAIT cells were of donor origin early after MA and RIC PBSC transplantation. MAIT cell reconstitution was markedly impaired in recipients of cord blood, which contains few MAIT cells, compared to those receiving PBSC, in which MAIT cells are plentiful, suggesting that early MAIT cell reconstitution is primarily derived from mature cells transferred with the HCT graft. Analysis of stool samples from HCT recipients (n = 17) has shown that the relative abundance of distinct gut bacterial species is highly variable between recipients and changed during the course of HCT. Analyses of the relationship between the microbiota and MAIT cell reconstitution will be presented.Conclusions: MAIT cell recovery following HCT varies between different types of transplants and may be influenced by the transferred graft source, the post-HCT environment, and the gut microbiome. DisclosuresNo relevant conflicts of interest to declare.

Infusion of a Non HLA-Matched Off-the-Shelf Ex Vivo Expanded Cord Blood Progenitor Cell Product Following Myeloablative Cord Blood Transplantation Is Safe, Decreases the Time to Hematopoietic Recovery, and Results in Excellent Overall Survival

Background: Myeloablative cord blood transplant (CBT) recipients experience delayed hematopoietic recovery and an increased risk of transplant related mortality (TRM). With the primary goal of generating increased numbers of hematopoietic stem and progenitor cells (HSPC) to supplement a conventional cord blood (CB) graft and thereby enhance engraftment and reduce early TRM, we have developed methods to ex vivo expand CB-derived HSPC in the presence of Notch ligand for 14 days. The product is then harvested and cryopreserved for future clinical use. We have previously reported that infusion of non-HLA matched, off-the-shelf ex vivo expanded CB progenitor cells in patients undergoing myeloablative CBT is safe and associated with faster neutrophil (ANC) recovery. We now present updated outcomes data with a median follow-up of 3.2 years (range 1.9-3.7).Methods: Between September 2010 and August 2012, 15 patients with hematologic malignancies were enrolled in this single center Phase I trial to assess the safety of infusing a non-HLA matched expanded CB HSPC product to augment conventional CBT. Conditioning consisted of Fludarabine (75mg/m2), Cytoxan (120mg/kg) and TBI (13.2 Gy). On the day of transplant, 1 or 2 unmanipulated CB units were infused first followed 4 hours later by infusion of the expanded CB product. Donor(s)/host chimerism studies were performed weekly from day 7 to 28, then at days 80, 180 and 1 year on peripheral blood flow sorted into myeloid and lymphoid fractions. Graft versus host disease (GVHD) prophylaxis consisted of cyclosporine and MMF beginning on day -3. ANC and platelet engraftment were calculated using cumulative incidence rates to accommodate competing risks. Disease-free survival (DFS) was analyzed using Kaplan-Meier estimates.Results: The median age was 21 years (range, 5-45) and weight 59 kg (range, 23-89). Other patient characteristics and median infused cell doses are shown in Table 1. Four patients (26%) received only 1 unmanipulated CB unit along with the expanded graft. All patients engrafted at a median of 19 days (range, 9-31 days), significantly faster than the 25 days (range, 14-45) observed in 41 recipients of two unmanipulated units otherwise treated identically (p=0.01) (Figure 1A).Similar to our initial expansion trial using partially HLA-matched expanded CB cells, early (day 7) myelomonocytic (CD33 and CD14) recovery was almost entirely (98–100%) due to cells arising from the expansion product. Cells derived from the expansion product were no longer detected at day 14 in all but 2 patients. The median time to platelet recovery (>20 x 10^9/L) was 35 days (range 21–45).The cumulative incidence of platelet recovery by day 100 was significantly higher than that observed in our concurrent control group 92% (CI 95%: 59-99) vs. 69% (95%: 51-81), respectively (p=0.005) (Figure 1B). The probability of 3-year DFS was 86% (95% CI, 57-97). No TRM was observed throughout the study period (Figure 1C), although 2 patients relapsed at days 53 and 219 posttransplant and subsequently died after further therapy. All patients presented with grade II acute GVHD at a median time of 32 days (14-86), with no grade III-IV aGVHD observed (Figure 1D). The skin was the most commonly affected organ (n=12). Eight (53%) patients were treated for pre-engraftment syndrome at a median time of 6 days (range 4-9) and five (33%) patients had GVHD after day 100. At 2 years only 2 patients are still on immunosuppressive therapy for mild chronic GVHD.Conclusions: The results of this Phase I safety study demonstrated that infusion of an off-the-shelf non-HLA matched expanded CB HSPC product to augment a CB graft in the myeloablative setting was safe and led to more rapid neutrophil and platelet recovery compared to our concurrent control group. Importantly, no TRM was observed among the 15 patients on this study, with only 2 deaths due to relapsed disease, leading to an excellent OS of 86% at a median follow-up of 3.2 years. Quite interesting is the observation of no grade 3-4 acute GVHD, even with infusion of a non-HLA matched expanded cell product that may induce increased alloreactivity of the unmanipulated CB unit(s). However, in this preliminary pilot study, it appeared that infusion of the non HLA-matched product was associated with less severe acute GVHD. Based on these very promising results, a prospective multicenter randomized trial utilizing this product is underway. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Allogeneic Stem Cell Transplantation with Immunodepleted Grafts for Lymphoproliferative Malignancies

Introduction: The treatment of lymphoproliferative disorders that fail initial therapy remains unsatisfactory. Allogeneic stem cell transplantation can be curative but has been associated with high treatment related mortality, particularly from graft vs. host disease (GvHD). Immunodepletion of the peripheral blood stem cell (PBSC) graft with alemtuzumab effectively prevents GvHD.Methods: Patients with lymphoma (excluding diffuse large B-cell) in second or subsequent response were offered allogeneic stem cell transplantation if they had a human leukocyte antigen (HLA) compatible donor. Haematopoietic stem cells were mobilized with filgrastim (5-10 ug/kg) and PBSC were collected by apheresis. Patients were conditioned with myeloablative doses of chemotherapy or radiotherapy. For GvHD prophylaxis mobilized donor blood stem cells were incubated ex vivo with alemtuzumab (in the bag) for 30 minutes and infused intravenously. Post transplantation patients received therapeutic blood levels of cyclosporine until day 90. The end points of the study were frequency of GvHD, transplant mortality, disease recurrence and overall survival (OS) rates.Results: Sixty one consecutive patients with lymphoma (follicular 17, marginal zone 2, Waldestrom’s macroglobulinaemia 2, transformed 16, peripheral T-cell 16 [all ALK negative] and lymphoblastic 8) underwent allogeneic transplantation. None had received a previous transplant. PBPC were from siblings in 53 patients and 8 from unrelated donors. Twenty patients were female and the median age was 47 years. The median CD34+ cell number infused was 4.71 (1.31 – 17.88) x10^6/kg. The median dose of alemtuzumab added into the bag was 10 mg. All recovered the blood counts at median of 13 days. Seventeen (28%) patients died, 11 (18%) of transplant related causes. Chimerism studies performed in 42 patients showed full donor chimerism in each case. GvHD (> grade 1) was seen in 8 patients (13%). Cytomegalovirus reactivation occurred in 21% but all responded to gancyclovir. Seventeen patients died, 11 (18%) of treatment related complications. Eight patients had disease recurrence, two of three achieved further response after donor leukocyte infusion and six died of the malignancy. At median follow up of 2260 15-4412) days 71% are alive in response. There was no difference in survival among the 4 histological groups or between chemotherapy and radiotherapy based conditioning. In univariate analysis female donor gender (for male patients) and GvHD were significant adverse factors for survival. Cox analysis showed that occurrence of GvHD (p= 0.001) was an independent significantly adverse factor for survival.Conclusions: We conclude that ex vivo immunodepletion of grafts with alemtuzumab leads to reduced rates of GvHD and results in lesser treatment related mortality despite myeloablative conditioning. Recurrence rates appear low suggesting that in the absence of GvHD prescribing dose intense conditioning and allogeneic stem cell transplantation earlier, before the malignancy is refractory, could be an effective strategy for patients with advanced lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Donor Cell Leukemia: A Prospective Study of Its Identification and Treatment

Leukemia relapses occurring in donor cells, so called donor cell leukemias (DCL) after allogeneic hematopoietic stem cell transplantation have been reported in several cases and still are considered as rare diseases. Cytogenetic analysis, flow cytometry and molecular testing have been used to confirm this event in the cases so far reported. The etiology of DCL is unclear, and the reported literature does not suggest a common mechanism. The incidence of this condition is largely unknown, as well as the results of its treatment. We have prospectively searched for DCL in a 12-year period, in a single institution. In a group of 106 consecutive patients allografted because of leukemia; we have identified 7 cases of DCL; six of them were allografted because of relapsed acute lymphoblastic leukemia (ALL) and one because of paroxysmal nocturnal hemoglobinuria / aplastic anemia; these figures suggest that the real incidence of DCL has been underestimated in previous studies. All the patients were allografted from HLA-identical siblings, employing a reduced-intensity conditioning regimen. The cases of DCL appeared one to 40 months (median 10) after the allograft; the number of blast cells when the leukemic activity ensued was above 50% in all cases, whereas the chimerism studies revealed more than 90% cells of donor origin. The origin of the leukemia cells was shown by microsatellites in all cases and in three with a sex mismatch it was confirmed by the enumeration of XX / XY cells in the leukemic cells. The six patients with lymphoblastic DCL were treated prospectively with a pediatric-inspired combined chemotherapy schedule designed for “de novo” ALL patients. A complete response was obtained in 3/6 patients with lymphoblastic DCL these patients being alive in a complete remission at 11, 12 and 98 months after the diagnosis of DCL. The patient with hairy cell DCL has had a benign course, not needing any treatment. In the whole group the median survival (SV) has not been reached, the overall SV being 57% at 98 months. The long-term DCL survivors remain full chimeras and did not need a second transplant. It is concluded that the prevalence of DCL may be higher if it is prospectively looked for, and that acceptable therapeutic results are obtained if patients are treated as “de novo” leukemias employing combined chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Does HLA-C Matching Predict the Engrafting Unit in Double Umbilical Cord Blood Transplants?: A Single Institution Retrospective Analysis

▪Background: Lower total CD34+ cell dose and increased HLA-mismatch are known predictors of engraftment failure and higher transplant related mortality (TRM) in cord blood (CB) recipients. To compensate for cell dose, double unit grafts (DUCBT) are commonly used in adult. However, in the majority of patients (pts), only one of the two CB units engrafts. Identification of the factors that predict which unit will engraft remains elusive, although increased recipient-donor HLA-matching and larger CD3+ cell dose have been associated with the predominating unit in a single center retrospective analysis (Ramirez et al, 2012). Historically, CB units are selected by maximizing matching at the HLA-A and -B antigen and -DRB1 allelic level. Evidence supports that matching at HLA-C appears to decrease TRM, and many clinicians incorporate HLA-C antigen matching into unit selection. It is unclear, however, if HLA-C matching predicts the engrafting unit in DUCBT. This study retrospective study evaluates whether HLA-C matching is associated with the “winning” CB unit.Design: Clinical data was reviewed from all pts with a hematologic malignancy receiving a DUCBT at Moffitt Cancer Center between November 13, 2009 and August 29, 2013. Chimerism studies identified the predominating unit (> 65% single unit) between day 21 and day 28. Subsequent chimerism analyses performed at a median of day 100 confirmed unit predominance. Unit selection required intermediate resolution antigen match at HLA-A, -B, -C, and high resolution allele match at -DRB1. Units were a minimum of 4/8 matched to the patient and each other with a minimum cell dose of 1.5 x 107 total nucleated cell dose (TNC) /kg. Serology for donor specific antibodies against both units was negative.Results: Excluding 6 pts who were missing HLA-C typing on one or both CB units, 54 pts with hematologic malignancies (ALL=6, MDS/AML=29, Other=19) received chemotherapy and total body irradiation as part of a myeloablative conditioning (MAC) or reduced intensity conditioning (RIC) with or without thymoglobulin (ATG) followed by a DUCBT (MAC=14, RIC=23, RIC+ATG=17). Median age was 52 (range 22-69) years. Seven pts demonstrated persistent mixed chimerism in the myeloid and/or lymphoid cell lines beyond day 100. A total of 20 pts with available chimerism data received at least one CB unit matched to the recipient at HLA-C, with one patient excluded due to persistent mixed chimerism. Six pts received both CB units matched at HLA-C, but of the 13 pts receiving one matched and one mismatched unit, the HLA-C matched unit was the engrafting unit 69% (9/13) of the time. Comparing similar HLA mispairings, a matched unit engrafted over a mis-matched unit at HLA-A 50% (5/10) of the time, at HLA-B 38% (5/13) of the time, and at HLA-DRB1 50% (3/6) of the time. TNC dose (larger vs smaller with a required difference of at least 0.03 x 107 TNC/kg), order of infusion (first vs second unit), and overall CB unit HLA matching (4/8-8/8), were assessed as potential predictors for engraftment. Of evaluable patients, the CB unit with the larger TNC engrafted 44% (16/36) of the time, and the first unit infused was the engrafting unit 54% (21/39) of the time. In patients with an unequal overall match grade between the CB units, the better HLA-matched CB unit engrafted 64% (14/22) of the time. Survival analysis of all pts revealed that those who received at least one CB unit antigen matched at HLA-C (n=20) had a 100 day and 1 year overall survival (OS) of 85% (95% Confidence Interval(CI): 67–97%) and 55% (95% CI: 33–75), respectively, whereas pts receiving two HLA-C mismatched CB units (n=34), 100 day OS was 62% (95% CI: 45–77) with 1 year OS 44% (95% CI: 28–61) (Fig 1).Conclusion: HLA-C antigen matching appears to help predict the “winning” unit in settings of HLA-match disparity and DUCBT. Confirmation in a larger population of DUCBT recipients is necessary. To date, the effects of HLA matching and other variables influencing engraftment have been predominately evaluated in recipients of single unit transplants and studies in DUCBT have been limited. Further investigation assessing HLA matching as well as donor-donor interactions is best served through a multicenter data resource. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Separate cellular localizations of human T-lymphotropic virus 1 (HTLV-1) Env and glucose transporter type 1 (GLUT1) are required for HTLV-1 Env-mediated fusion and infection.

Interaction of the envelope glycoprotein (Env) of human T-lymphotropic virus 1 (HTLV-1) with the glucose transporter type 1 (GLUT1) expressed in target cells is essential for viral entry. This study found that the expression level of GLUT1 in virus-producing 293T cells was inversely correlated with HTLV-1 Env-mediated fusion activity and infectivity. Chimeric studies between GLUT1 and GLUT3 indicated that the extracellular loop 6 (ECL6) of GLUT1 is important for the inhibition of cell-cell fusion mediated by Env. When GLUT1 was translocated into the plasma membrane from intracellular storage sites by bafilomycin A1 (BFLA1) treatment in 293T cells, HTLV-1 Env-mediated cell fusion and infection also were inhibited without the overexpression of GLUT1, indicating that the localization of GLUT1 in intracellular compartments rather than in the plasma membrane is crucial for the fusion activity of HTLV-1 Env. Immunoprecipitation and laser scanning confocal microscopic analyses indicated that under normal conditions, HTLV-1 Env and GLUT1 do not colocalize or interact. BFLA1 treatment induced this colocalization and interaction, indicating that GLUT1 normally accumulates in intracellular compartments separate from that of Env. Western blot analyses of FLAG-tagged HTLV-1 Env in virus-producing cells and the incorporation of HTLV-1 Env in virus-like particles (VLPs) indicate that the processing of Env is inhibited by either overexpression of GLUT1 or BFLA1 treatment in virus-producing 293T cells. This inhibition probably is due to the interaction of the Env with GLUT1 in intracellular compartments. Taken together, separate intracellular localizations of GLUT1 and HTLV-1 Env are required for the fusion activity and infectivity of HTLV-1 Env. The deltaretrovirus HTLV-1 is a causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although HTLV-1 is a complex retrovirus that has accessory genes, no HTLV-1 gene product has yet been shown to regulate its receptor GLUT1 in virus-producing cells. In this study, we found that a large amount of GLUT1 or translocation of GLUT1 to the plasma membrane from intracellular compartments in virus-producing cells enhances the colocalization and interaction of GLUT1 with HTLV-1 Env, leading to the inhibition of cell fusion activity and infectivity. The results of our study suggest that GLUT1 normally accumulates in separate intracellular compartments from Env, which is indeed required for the proper processing of Env.

Pharmacokinetics and effects on serum cholinesterase activities of organophosphorus pesticides acephate and chlorpyrifos in chimeric mice transplanted with human hepatocytes

Organophosphorus pesticides acephate and chlorpyrifos in foods have potential to impact human health. The aim of the current study was to investigate the pharmacokinetics of acephate and chlorpyrifos orally administered at lowest-observed-adverse-effect-level doses in chimeric mice transplanted with human hepatocytes. Absorbed acephate and its metabolite methamidophos were detected in serum from wild type mice and chimeric mice orally administered 150mg/kg. Approximately 70% inhibition of cholinesterase was evident in plasma of chimeric mice with humanized liver (which have higher serum cholinesterase activities than wild type mice) 1day after oral administrations of acephate. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated plasma concentrations of acephate and chlorpyrifos in humans were consistent with reported concentrations. Acephate cleared similarly in humans and chimeric mice but accidental/incidental overdose levels of chlorpyrifos cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in mice. The data presented here illustrate how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of toxicological potential of organophosphorus pesticides.

PFM.51 Fatal transfusion-associated graft vs host disease (TA-GvHD) following emergency intrauterine transfusion of maternal blood: case report and survey of UK practice

For intrauterine transfusion (IUT) specific red cells are provided by UK Blood Services, irradiated to prevent TA-GvHD.A fetus (21/40) was referred to a fetal medicine centre (FMC), severely anaemic (parvovirus)...

Interleukin-1β mediated amyloid plaque clearance is independent of CCR2 signaling in the APP/PS1 mouse model of Alzheimer's disease

Neuroinflammation is a key component of Alzheimer's disease (AD) pathogenesis. Particularly, the proinflammatory cytokine interleukin-1 beta (IL-1β) is upregulated in human AD and believed to promote amyloid plaque deposition. However, studies from our laboratory have shown that chronic IL-1β overexpression in the APPswe/PSEN1dE9 (APP/PS1) mouse model of AD ameliorates amyloid pathology, increases plaque-associated microglia, and induces recruitment of peripheral immune cells to the brain parenchyma. To investigate the contribution of CCR2 signaling in IL-1β-mediated amyloid plaque clearance, seven month-old APP/PS1/CCR2−/− mice were intrahippocampally transduced with a recombinant adeno-associated virus serotype 2 containing the cleaved form of human IL-1β (rAAV2-IL-1β). Four weeks after rAAV2-IL-1β transduction, we found significant reductions in 6E10 and Congo red staining of amyloid plaques that was confirmed by decreased levels of insoluble Aβ1–42 and Aβ1–40 in the inflamed hippocampus. Bone marrow chimeric studies confirmed the presence of infiltrating immune cells following IL-1β overexpression and revealed that dramatic reduction of CCR2+ peripheral mononuclear cell recruitment to the inflamed hippocampus did not prevent the ability of IL-1β to induce amyloid plaque clearance. These results suggest that infiltrating CCR2+ monocytes do not contribute to IL-1β-mediated amyloid plaque clearance.

Blocking Metabotropic Glutamate Receptor Subtype 7 (mGlu7) via the Venus Flytrap Domain (VFTD) Inhibits Amygdala Plasticity, Stress, and Anxiety-related Behavior

The metabotropic glutamate receptor subtype 7 (mGlu7) is an important presynaptic regulator of neurotransmission in the mammalian CNS. mGlu7 function has been linked to autism, drug abuse, anxiety, and depression. Despite this, it has been difficult to develop specific blockers of native mGlu7 signaling in relevant brain areas such as amygdala and limbic cortex. Here, we present the mGlu7-selective antagonist 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one (XAP044), which inhibits lateral amygdala long term potentiation (LTP) in brain slices from wild type mice with a half-maximal blockade at 88 nm. There was no effect of XAP044 on LTP of mGlu7-deficient mice, indicating that this pharmacological effect is mGlu7-dependent. Unexpectedly and in contrast to all previous mGlu7-selective drugs, XAP044 does not act via the seven-transmembrane region but rather via a binding pocket localized in mGlu7's extracellular Venus flytrap domain, a region generally known for orthosteric agonist binding. This was shown by chimeric receptor studies in recombinant cell line assays. XAP044 demonstrates good brain exposure and wide spectrum anti-stress and antidepressant- and anxiolytic-like efficacy in rodent behavioral paradigms. XAP044 reduces freezing during acquisition of Pavlovian fear and reduces innate anxiety, which is consistent with the phenotypes of mGlu7-deficient mice, the results of mGlu7 siRNA knockdown studies, and the inhibition of amygdala LTP by XAP044. Thus, we present an mGlu7 antagonist with a novel molecular mode of pharmacological action, providing significant application potential in psychiatry. Modeling the selective interaction between XAP044 and mGlu7's Venus flytrap domain, whose three-dimensional structure is already known, will facilitate future drug development supported by computer-assisted drug design.

Cell therapy for cancer – our synthetic future

It is now well established that the immune system can control and eliminate cancer cells. Adoptive T-cell transfer has the potential to overcome the significant limitations associated with vaccine-based strategies in patients who are often immunocompromised. Application of the emerging discipline of synthetic biology to cancer, which combines elements of genetic engineering and molecular biology to create new biological structures with enhanced functionalities, is the subject of this article. Various chimeric antigen receptor designs, manufacturing processes and study populations, among other variables, have been tested and reported in recent clinical trials. Many questions remain unanswered in the field of engineered T-cells, but the encouraging response rates pave a wide road for future investigation.

Skin Recurrence of Transformed Mycosis Fungoides Postumbilical Cord Blood Transplant despite Complete Donor Chimerism.

Background. Allogeneic stem cell transplant is the treatment of choice for systemic cutaneous T-cell lymphoma (CTCL) which provides graft-versus-lymphoma effect. Herein we discuss a case of recurrence of CTCL skin lesions after cord blood transplant in a patient who continued to have 100% donor chimerism in bone marrow. Case Presentation. A 48-year-old female with history of mycosis fungoides (MF) presented with biopsy proven large cell transformation of MF. PET scan revealed multiple adenopathy in abdomen and chest suspicious for lymphoma and skin biopsy showed large cell transformation. She was treated with multiple cycles of chemotherapy. Posttherapy PET scan showed resolution of lymphadenopathy. Later she underwent ablative preparative regimen followed by single cord blood transplant. Bone marrow chimerism studies at day +60 after transplant showed 100% donor cells without presence of lymphoma. However 5 months after transplant she had recurrence of MF with the same genotype as prior skin lesion. Bone marrow chimerism study continued to show 100% donor cells. Conclusion. A differential graft-versus-lymphoma effect in our case prevented lymphoma recurrence systemically but failed to do so in skin. We hypothesize that this response may be due to presence of other factors in the bone marrow and lymph node microenvironments preventing recurrence in these sites.