Lymphocyte Engraftment Research Articles

Humanization with CD34-positive hematopoietic stem cells in NOG-EXL mice results in improved long-term survival and less severe myeloid cell hyperactivation phenotype relative to NSG-SGM3 mice.

NSG-SGM3 and NOG-EXL mice combine severe immunodeficiency with transgenic expression of human myeloid stimulatory cytokines, resulting in marked expansion of myeloid populations upon humanization with CD34+ hematopoietic stem cells (HSCs). Humanized NSG-SGM3 mice typically develop a lethal macrophage activation syndrome and mast cell hyperplasia that limit their use in long-term studies (e.g., humanization followed by tumor xenotransplantation). It is currently unclear to what extent humanized NOG-EXL mice suffer from the same condition observed in humanized NSG-SGM3 mice. We compared the effects of human CD34+ HSC engraftment in these two strains in an orthotopic patient-derived glioblastoma model. NSG-SGM3 mice humanized in-house were compared to NOG-EXL mice humanized in-house and commercially available humanized NOG-EXL mice. Mice were euthanized at humane or study endpoints, and complete pathological assessments were performed. A semiquantitative multiparametric clinicopathological scoring system was developed to characterize chimeric myeloid cell hyperactivation (MCH) syndrome. NSG-SGM3 mice were euthanized at 16 weeks after humanization because of severe deterioration of clinical conditions. Humanized NOG-EXL mice survived to the study endpoint at 22 weeks after humanization and showed less-severe MCH phenotypes than NSG-SGM3 mice. Major differences included the lack of mast cell expansion and limited tissue/organ involvement in NOG-EXL mice compared to NSG-SGM3 mice. Engraftment of human lymphocytes, assessed by immunohistochemistry, was similar in the two strains. The longer survival and decreased MCH phenotype severity in NOG-EXL mice enabled their use in a tumor xenotransplantation study. The NOG-EXL model is better suited than the NSG-SGM3 model for immuno-oncology studies requiring long-term survival after humanization.

FRI374 Immunomodulatory Effects Of Progesterone And Glucocorticoids In Pregnancy Immunity

Abstract Disclosure: A. Yasuda: None. T. Seki: None. H. Kashiwagi: None. N. Kitajima: None. Y. Kametani: None. Immune tolerance to a semi-allogeneic fetus is crucial for a successful pregnancy. However, the mechanisms of tolerance to semi-allografts in the co-existence with normal defenses against cancer and pathogens is not clear. During pregnancy, levels of pregnancy-related proteins and hormones such as progesterone (P4), estrogen, testosterone, and glucocorticoids (GC) increase. We hypothesized that the most abundant pregnancy related hormone in the placenta, P4, might play an important role in the local tolerance in the placenta. Thus, we aimed to compare the effects of P4 and cortisol (COR) on human lymphocytes in vivo, to clarify their role in the systemic regulation of immunity during pregnancy. Healthy donor peripheral blood mononuclear cells (PBMCs) were treated with P4 or COR for 6 h. These PBMCs were washed and transplanted intravenously into 8- to 9-week-old NOG-hIL-4-Tg (formal name, NOD.Cg-PrkdcscidIl2rgtm1Sug/Tg(CMV-IL4)/Jic) mice. Human epidermal growth factor receptor-2 (HER2) peptide was emulsified in complete Freund's adjuvant and administered intraperitoneally to these PBMC-NOG-hIL-4-Tg mice at the same day. As a negative control, an equal volume of PBS was added to the culture and transplanted into NOG-hIL-4-Tg mice. Boosters were administered using incomplete Freund's adjuvant 2 weeks after the primary immunization. Peripheral blood was collected 4 weeks after transplantation and 2 weeks after booster administration. T and B cell profiles were analyzed using FCM 28 days later. The results revealed that significantly higher numbers of human CD45+ cells engrafted in the spleens of P4-treated mice compared to COR-treated mice 4 weeks post-transplantation. Transplanted human CD45+ cells included T and B cells, and CD8+ T cells were significantly higher in P4-treated mice compared to both control and COR-treated mice. Moreover, antigen-specific IgG production was maintained in the P4-treated mice, suggesting that the T cell/B cell function was maintained in the mice. Compared to control and P4-treated mice, COR-treated mice tended to engraft fewer human CD45+ cells. Moreover, B cell engraftment tended to be lower in COR-treated mice than in control mice. Antigen-specific IgG production was not maintained. There results suggest that lymphocytes which experienced a high P4 environment maintain engraftment and function of human lymphocytes in the NOG-hIL-4-Tg mice, while high COR environment rather dampens the engraftment and function of them. The high-P4 environment limited in the placenta may support pregnant immunity to protect the semi-allogeneic fetus from pathogen-induced infectious disease or cancer. Presentation: Friday, June 16, 2023

The impact of Treosulfan-based conditioning for inborn errors of immunity: Is dose monitoring crucial?

In children with inborn errors of immunity (IEI) who will receive a hematopoietic stem cell transplant (HSCT) treosulfan-based conditioning is currently preferred. The aim of this study was to investigate early and late outcomes in pediatric IEI patients receiving pre-HSCT treosulfan and to examine the effect of treosulfan dose monitoring on outcomes. Seventy-three pediatric patients receiving this management between 2015 and 2022 were included. Overall survival rate was 80%, and event-free survival was 67.8%. A larger treosulfan dose AUC after first application increased the rate of early toxicity (p=.034) and slowed lymphocyte engraftment (r=.290; p=.030). Underlying disease, treosulfan AUC, donor type, stem cell type, number of immunosuppressive agents, the dose of anti-thymocyte globulin, and post-transplantation cyclophosphamide did not to increase risk of acute graft-versus-host disease. The risk of mixed chimerism (MC) in patients with autoimmune lymphoproliferative syndrome and leukocyte adhesion deficiency were higher than those with severe combined immunodeficiency (p=.021 and p=.014, respectively). The risk of MC was lower in those receiving peripheral blood stem cells (SC) compared with bone marrow derived SC (OR=.204, p=.022). The AUC of the treosulfan dose was not associated with poorer late outcomes. Treosulfan is an agent that can be used safely in the IEI patient group, level measurement appears essential to identify early toxicities. Prospective studies with more extended follow-up periods are needed.

Iron Overload and Iron-Induced Microbiome Changes May Affect Lymphoid Levels Post Bone Marrow Transplant and Graft-Versus-Host Disease Survival in Mice

Introduction: Bone Marrow Transplant (BMT) represents the only cure for many malignant and non-malignant hematopoietic disorders. This treatment is often accompanied by multiple red blood cell transfusions, which can lead to iron overload. Iron overload is associated with poorer outcomes following BMT, such as increased risk of infections or Graft-versus-Host Disease (GVHD); however, it is not yet clear whether this correlation is caused by iron itself or is simply confounded by patients with more severe disease requiring more transfusions. Previous studies using mouse models, have shown that transfusional iron overload reduces the population of beneficial bacteria in the gut microbiome or decreases the production of microbiome-produced metabolites (i.e. indoles) that have been associated with BMT outcomes and GVHD risk. Here, we developed a reductionist mouse model of iron overload and BMT to investigate the effect of iron and iron-induced changes to gut microbiota and their metabolites on BMT outcomes and GVHD survival. Methods: Female Balb/C mice were infused twice a week, for two weeks, with a total of 50 mg of iron dextran or saline followed by lethal irradiation (9 Gy). Cohorts of mice (n=10 per group) received 1x10^10 T cell-depleted bone marrow cells with or without 5x10^6 T cells from UBC-GFP C57Bl6 mice to establish allogenic BMT and GHVD models, respectively. Mice were then monitored daily for survival with a formal severity score evaluated twice a week. Analysis of donor red blood cells, platelets, and white blood cells (neutrophils, B and T cells) from peripheral blood was also performed once a week to check for engraftment. To examine the impact of iron-induced microbiota changes, fecal microbiome transplant (FMT) was performed in mice receiving BMT. Briefly, mice were treated with an antibiotic cocktail (ampicillin 1 g/L, kanamycin 1 g/L, metronidazole 1g/L, vancomycin 0.5g/L) by gavage 2 days before the start of FMT. Feces from control and iron overloaded mice were collected freshly and weighed. Stool pellets were then resuspended in PBS (100 mg feces/ml PBS), vortexed and then spun down at 800g for 3 minutes. Mice received 100ul of supernatant by gavage. Additional cohorts of mice were administrated with indole-3 proprionic acid (IPA) in the drinking water at a concentration of 0.1 mg/ml prior to irradiation throughout the experiment and monitored for survival after receipt of allogeneic T cells to induce GVHD. All mice received enrofloxacin (10mg/kg) starting on the day of BMT and for the following 3 days by intraperitoneal injection. Results: There was a significant reduction in overall lymphoid (CD45+ CD172a- CD11b-/low GFP+), B (CD45+ CD172a- CD11b-/low CD19+ GFP+), and T (CD45+ CD172a- CD11b-/low CD3+ GFP+) cells in iron overloaded mice compared to control mice (p<0.0001 at day +28) by flow cytometry analysis in peripheral blood after BMT. Although FMT from control feces into iron overloaded mice did not affect lymphoid numbers, FMT from iron overloaded feces into control mice reduced the number of donor lymphoid and B cells (p<0.05 at day +28; Figure 1A). Iron overload reduced survival compared to control mice in the GHVD mouse model (p<0.05 Mantel-Cox test). Administration of IPA in iron overloaded mice did not significantly affect overall survival (Figure 1B). Conclusions: Our results show that iron overload negatively impacts lymphocyte engraftment in a mouse model of BMT. Furthermore, iron overload also reduces survival in mice with GVHD. Iron overload was previously shown to reduce indole levels in mice. Although we did not observe a significant impact of exogenous indole administration on GVHD survival, FMT from iron overloaded into control mice reduced levels of lymphoid and B cells after BMT implicating gut microbiota in this outcome. Taken together, these results suggest that iron overload is an important factor that negatively affects BMT outcomes. The extent to which iron-induced gut microbiota changes are involved in the mechanisms responsible for this effect requires further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Gene Edited T Cell Therapies for Inborn Errors of Immunity

Inborn errors of immunity (IEIs) are a heterogeneous group of inherited disorders of the immune system. Many IEIs have a severe clinical phenotype that results in progressive morbidity and premature mortality. Over 450 IEIs have been described and the incidence of all IEIs is 1/1,000–10,000 people. Current treatment options are unsatisfactory for many IEIs. Allogeneic haematopoietic stem cell transplantation (alloHSCT) is curative but requires the availability of a suitable donor and carries a risk of graft failure, graft rejection and graft-versus-host disease (GvHD). Autologous gene therapy (GT) offers a cure whilst abrogating the immunological complications of alloHSCT. Gene editing (GE) technologies allow the precise modification of an organisms’ DNA at a base-pair level. In the context of genetic disease, this enables correction of genetic defects whilst preserving the endogenous gene control machinery. Gene editing technologies have the potential to transform the treatment landscape of IEIs. In contrast to gene addition techniques, gene editing using the CRISPR system repairs or replaces the mutation in the DNA. Many IEIs are limited to the lymphoid compartment and may be amenable to T cell correction alone (rather than haematopoietic stem cells). T cell Gene editing has the advantages of higher editing efficiencies, reduced risk of deleterious off-target edits in terminally differentiated cells and less toxic conditioning required for engraftment of lymphocytes. Although most T cells lack the self-renewing property of HSCs, a population of T cells, the T stem cell memory compartment has long-term multipotent and self-renewal capacity. Gene edited T cell therapies for IEIs are currently in development and may offer a less-toxic curative therapy to patients affected by certain IEIs. In this review, we discuss the history of T cell gene therapy, developments in T cell gene editing cellular therapies before detailing exciting pre-clinical studies that demonstrate gene editing T cell therapies as a proof-of-concept for several IEIs.

Selected Articles from This Issue

Prostate cancer is the most frequently diagnosed cancer and second leading cause of cancer death in US males. Androgen receptor (AR) is the best-known therapeutic target for prostate cancer. Here, Cole and colleagues have identified a novel small molecule, (+)-JJ-74-138, that directly binds to the AR and non-competitively inhibits AR activity. Furthermore, this small molecule shows efficacy in vitro and in vivo in models resistant to clinically available AR antagonists that competitively inhibit the binding of androgens to AR. Further development of non-competitive AR antagonists like (+)-JJ-74-138 shows promise treating tumors resistant to current AR targeting therapies.Epigenetic modulation has recently emerged as an immune evasion mechanism in cancer. The co-dependence of G9A and EZH2 histone methyltransferases in regulating gene transcription led to the development of the dual G9A/EZH2 inhibitor, HKMTI-1-005. In this highlighted manuscript, Spiliopoulou and colleagues demonstrate that treatment with HKMTI-1-005 derepresses transcription of immunostimulatory gene networks and achieves robust engraftment of tumor-infiltrating lymphocytes in the Trp53‒/‒ ID8 mouse model of ovarian cancer. Immune infiltration was accompanied by tumor growth inhibition and an improvement in survival. Collectively these results support further development of epigenetic modifiers as an anti-cancer strategy.The high mortality of PDAC attributes to early metastasis at diagnosis and chemoresistance partly driven by mutant p53. PDAC cells are predisposed to ferroptosis. New therapeutics capable of inducing ferroptosis and inhibiting PDAC metastasis are highly desirable. This study by Li, et al reports a small molecule compound MMRi62 bearing these two capabilities. This study shows MMRi62 inhibits PDAC proliferation in vitro and in vivo by induction of ferroptosis via induced lysosomal degradation of ferritin heavy chain and inhibits metastasis in vivo which involves proteasomal degradation of mutant p53.Zammarchi and colleagues describe the development of ADCT-601, a novel pyrrolobenzodiazepine dimer-based antibody-drug conjugate targeting AXL, a well-known tyrosine kinase receptor implicated in numerous aspects of tumor development, progression, and resistance to cancer therapies. Zammarchi and colleagues present original data outlining the mode of action of ADCT-601 when used as monotherapy, both in vitro and in vivo, in tumor models of different origins, including a monomethyl auristatin E-resistant lung cancer model, and in combination with olaparib in an ovarian cancer model; preclinical evaluation of its safety and tolerability is also reported. Altogether, the data presented warrant further evaluation of ADCT-601 in the clinical setting.

Engrafting Horse Immune Cells into Mouse Hosts for the Study of the Acute Equine Immune Responses.

Simple SummaryFor decades, studies using research mice as models for disease have been critical to our current understanding of disease processes and associated immune responses, highlighting the ways in which mouse physiology is different from human and other species. Recent work has been directed at creating mice that can host human immune cells, allowing the study and manipulation of the human immune response without harm to patients. The purpose of this study was to explore to use of mouse hosts for horse immune cells. Horses are difficult to study immunologically as they are expensive to keep, and keeping their environment free of immune triggers is very difficult. Using mice allows us to increase our study numbers and control the environment which improves study reproducibility. In this study, we transferred both horse blood lymphocytes as well as horse bone marrow into specially modified mouse hosts. We found that mice are able to host horse immune cells and that these transferred cells are active. Future work can now build on this study to understand the horse immune response to infectious agents using mice, helping to identify new therapeutic tools to help equine patients.Immunological studies in the horse are frequently hampered by lack of environmental control, complicated study design, and ethical concerns when performing high risk studies. The purpose of the current study was to investigate the utility of a xenograft model for studying acute equine immune responses. Immunocompromised non obese diabetic (NOD). sudden combined immunodeficiency (scid).gamma-/- (NSG) mice were engrafted with either equine peripheral blood lymphocytes (PBLs) or equine bone marrow to determine an optimal protocol for equine lymphocyte engraftment. We found that both PBL and bone marrow grafts populated the host mice successfully. Bone marrow transplants were technically more challenging and required further processing to retard graft versus host disease. Graft vs host disease was apparent at 28 days post-PBL transfer and 56 days post-bone marrow transfer. The results of these studies support the use of mouse hosts to study acute equine immune responses and that different engraftment techniques can be used depending on the experimental design.

Native Spleen Preservation During Visceral Transplantation Inhibits Graft-Versus-Host-Disease Development: Clinical and Experimental Study.

We aimed to assess whether native spleen preservation during visceral transplantation (VT) affects graft-versus-host-disease (GVHD) incidence. GVHD is one of the most severe and frequently lethal hematological complications after VT procedures. Because there is no specific treatment for GVHD, it is imperative to develop a strategy to reduce donor lymphocyte engraftment and proliferation. Our study included both clinical and experimental data. A total of 108 patients were divided into 3 groups: a native spleen preservation group, a native spleen removal with no donor spleen group, and a donor spleen included (allogeneic spleen) group. We also used an allogeneic VT rat model, in which recipients were divided into 2 groups: a native spleen preservation (+SP) group and a native spleen removal (-S) group. Skin rash appearance, histopathological changes, chimerism, and spleen effects on circulating allogeneic T-cells were assessed. The patients with native spleen preservation showed a lower rate of GVHD ( P <.001) and better survival ( P <.05) than those in the other groups. Skin and histological signs of GVHD were lower in the rats in the +SP group ( P <.05). The donor T-cell frequency in the bloodstream and skin was also significantly reduced when the native spleen was preserved ( P <.01 and P <.0001, respectively). The clinical and experimental data indicate that recipient spleen preservation protects against GVHD after VT, and donor cell clearance from the bloodstream by spleen macrophages could be the underlying mechanism. Therefore, spleen preservation should be considered in VT procedures, whenever possible.

Primary immunodeficiencies: HSCT experiences of a single center in Turkey.

Primary immunodeficiency diseases (PID) are characterized by the occurrence of frequent infections and are caused by many genetic defects. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment option for the majority of PID. As a Pediatric Hematology-Oncology-Immunology Transplantation Unit, we wanted to present our HSCT experience regarding treatment of primary immunodeficiency diseases. 58 patients were included in the study between January 2014 and June 2019. We searched 9/10 or 10/10 matched-related donor (MRD) firstly, in the absence of fully matched-related donor. We screened matched unrelated donor (MUD) from donor banks. MRD was used in 24 (41.3%) patients, MUD in 20 (34.4%) patients, and haploidentical donors in 14 (24.1%) patients. Demographic data, HSCT characteristics, and outcome were evaluated. While 16 patients had severe combined immunodeficiency (SCID), the remaining was non-SCID. Of the 58 patients, 38 were male and 20 were female. Median age at transplantation was 12months (range: 2.5-172months). Combined immunodeficiencies consisted 67.2% of patients. Mean follow-up time was 27months (6months-5years). Median neutrophil, lymphocyte, and thrombocyte engraftment days were similar in comparison of both donor type and stem cell source. The most common complication was acute GvHD in 15 (25.8%) patients. In total, five patients (31%) belonging to the SCID group and 10 patients (23.8%) belonging to the non-SCID group died. Our total mortality rate was 15 (25.8%) in all patients. We would like to present our HSCT experiences as a pediatric immunology transplantation center. Existing severe infections before transplantation period, BCGitis, and CMV are important issues of transplantation in Turkey. However, the follow-up time is shorter than some studies, our results regarding complications and survival are similar to previous reports.

Repopulation of Lymphocytes and Natural Killer Cells on Day 15 Following First Autologous Stem Cell Transplantation in Myeloma Patients Correlates with the Number of Reinfused Lymphocytes and Natural Killer T Cells in the Autologous Graft

IntroductionAutologous hematopoietic stem cell transplantation (aHSCT) is still considered standard of care for eligible myeloma patients following induction treatment. Beside the effect of a high dose melphalan, several studies proposed a beneficial effect of immune graft versus myeloma effect following aHSCT. The higher number of lymphocytes and natural killer (NK) cells on day 15 after aHSCT correlated with a better longterm outcome. We have previously published the data on the influence of different mobilization regimens on the number of collected lymphocytes and NK cells and their number on day 15 post aHSCT. In this abstract we present the data on the correlation between the dose of reinfused lymphocytes, NK cells and CD34+ cells and the number of lymphocytes and NK cells on day 15 after aHSCT.MethodsWe prospectively enrolled 48 newly diagnosed myeloma patients following induction treatment with bortezomib and dexamethasone. They were mobilized with either filgrastim (10 mcg/kg body weight daily), pegfilgrastim (12 mg one-time injection) or cyclophosphamide (4 g/m2) followed by filgrastim (10 mcg/kg body weight daily).The lymphocyte and NK cell (CD16+/56+) counts in the stem cell concentrate and on day 15 post aHSCT in peripheral blood were determined with the flow cytometer. Pearson r correlation was used to associate the dose of infused cells and their number on day 15 after aHSCT in peripheral blood. Scatter plots and linear regression were used for the data presentation. SPSS Statistics v21 (IBM, USA) was used for the analysis. The study complied with the Declaration of Helsinki and was approved by the local ethical committee. All patients signed a written informed consent.ResultsWe established a correlation between the dose of infused lymphocytes in the autologous graft and their number in the peripheral blood on day 15 after aHSCT (Figure 1; Pearson r = 0.27, p = 0.04). There was also a correlation between the dose of infused NK cells and their number in the peripheral blood on day 15 post aHSCT (Figure 2; Pearson r = 0.6, p < 0.01). However, there was no correlation between the number of reinfused CD34+ cells and the number of lymphocytes on day 15 after aHSCT (Figure 3).ConclusionThe early immune repopulation is important for better overall survival after the aHSCT in multiple myeloma and lymphoma patients. The data from our study showed that the higher numbers of reinfused lymphocytes and NK cells, but not the total number of CD 34+ cells, significantly impact earlier lymphocyte and NK cell engraftment. We concluded that with the aim to achieve the best possible outcomes of aHSCT for our patients, we must make sure to reinfuse not only sufficient number of CD 34+ cells, but also of lymphocytes and NK cells. [Display omitted] [Display omitted] [Display omitted] DisclosuresSkopec:amgen: Consultancy, Honoraria; novartis: Honoraria.

Reactivation of Human Herpes Virus-6 After Pediatric Stem Cell Transplantation: Risk Factors, Onset, Clinical Symptoms and Association With Severity of Acute Graft-Versus-Host Disease.

To study clinical symptoms, timing and consequences of human herpesvirus-6 (HHV-6) reactivation after pediatric allogeneic stem cell transplantation (SCT), HHV-6 was investigated by plasma polymerase chain reaction in a cohort of 106 pediatric SCT recipients. HHV-6 viremia was detected post-SCT in 48% of the patients with a median time of onset at 20 days after SCT. In week 3 and 4 post-SCT, HHV-6 is the most common infectious agent detected. In up to 30% of the patients with fever of unknown origin, HHV-6 was the only detected infectious agent to explain fever. Patients transplanted with an unrelated donor or receiving serotherapy were at increased risk of HHV-6 reactivation. The onset of HHV-6 reactivation coincided with the appearance of lymphocytes and monocytes in peripheral blood. Treatment with alemtuzumab (MabCampath) delayed both lymphocyte and monocyte engraftment and, concomitantly, onset of HHV-6 reactivation was delayed in those cases. HHV-6 reactivation was not associated with an increased incidence of acute graft-versus-host disease (GvHD). However, progression to grade II-IV GvHD was in 9 of 10 patients associated with HHV-6 reactivation before GvHD (P = 0.006) and HHV-6 was the only infection with such an association. HHV-6 frequently reactivates after pediatric SCT around the time of mononuclear cell engraftment and is associated with an increased severity of GvHD. HHV-6 may explain fever of unknown origin in 30% of the patients early after SCT. Assessment of HHV-6 reactivation in patients early after SCT can be instrumental for clinical decision making.

Higher Stem Cell Dose Infusion after Intensive Chemotherapy Does Not Improve Symptom Burden in Older Patients with Multiple Myeloma and Amyloidosis.

Autologous hematopoietic stem cell transplantation (ASCT) for multiple myeloma (MM) is associated with high symptom burden, particularly for older patients and those with amyloid light-chain (AL) amyloidosis. Symptom burden peaks during leukopenia. We hypothesized that higher doses of CD34(+) stem cells would be associated with an improved symptom outcome. Patients undergoing ASCT for MM who were ≥60 years old or had AL amyloidosis were randomized to receive either a standard (4 to 6× 10(6) cells/kg) or high dose (10 to 15× 10(6) cells/kg) of CD34(+) cells after melphalan 200 mg/m(2). Symptom burden was assessed via the MD Anderson Symptom Inventory MM module. Eighty patients were enrolled. Median CD34(+) cell doses were 5.1× 10(6) cells/kg (standard dose) and 10.5× 10(6) cells/kg (high dose). The most severe symptoms during the first week were fatigue, lack of appetite, drowsiness, disturbed sleep, and pain. The area under the curve for the mean composite severity score of these symptoms was similar between treatment arms (P= .819). Median times to neutrophil, lymphocyte, and platelet engraftment were also similar between groups. IL-6 increased similarly for both groups throughout the ASCT course. Infusion of higher autologous stem cell dose after high-dose chemotherapy does not yield a difference in symptom burden or engraftment time in the first few weeks after ASCT.

Impact of Different Mobilization Methods on Graft Composition and Outcome after Autologous Stem Cell Transplantation: Implications for Upfront Use of Plerixafor

Early lymphocyte recovery (ELC) is associated with improved outcomes of hematologic malignancies after autologous hematopoietic stem cell transplantation (auto-SCT). ELC, its composition and impact on outcome depends on many variables; however there is limited data on ELC after different mobilization strategies (G-CSF [G] vs. G + high dose cyclophosphamide [GC] vs. G + plerixafor [GP]). Results from a recent study showed that GP based mobilization can affect the number and subsets of immune competent cells contained in the graft. We studied whether these differences are associated with immune reconstitution (ELC), engraftment, or long-term outcomes.We retrospectively identified patients undergoing auto-SCT at the Nashville VA Transplant Center between January 2000 and December 2010 in our CIBMTR database. Disease response was determined by standard CIBMTR response criteria. At our center, GP mobilization is reserved for patients who failed prior mobilization, to rescue G or GC mobilization, or as upfront usage in heavily pre-treated patients. Our patient cohort primarily included patients with multiple myeloma (MM) and lymphoma (LY).We had evaluable data on 333 patients (MM=196; LY=127; others=10). Comparative analysis of different mobilization methods are summarized in Table 1. Median number of regimens pre-SCT for MM was 2 (range 1-5) and for lymphoma 2 (range1-7). Among LY patients, 60 (47.3%) patients were in complete remission (CR), 58 (45.7%) in partial remission (PR) and 9 (7%) had stable disease (SD). Among MM patients, 69 (35.2%) were in CR or very good partial remission (VGPR) pre-transplant, 105 (53.5%) were in PR, and 14 (7.1%) had SD. There was no significant difference between disease response status among different mobilization methods for either the MM or LY patients. A higher absolute WBC count was seen in grafts after GP mobilization compared to G or GC (p=0.01), despite a majority of patients having received GP mobilization after failed G or GC mobilization, or as a rescue regimen (n=20 [89%]). Similarly, absolute lymphocyte counts were higher in grafts mobilized after GP compared to G or GC (p=0.01). All patients engrafted and there was no difference in time to WBC or platelet engraftment between mobilization methods. Although the GP cohort was more heavily treated than the other cohorts (>2 regimens for GP 82%, vs. G 72% vs. GC 58% [p=0.02]), progression-free survival (PFS) and overall survival (OS) of G vs. GC vs. GP at 2-years was not significantly different between MM and LY cohorts (Table 1).In summary, grafts mobilized with GP exhibited major differences in graft composition in conjunction with favorable post- transplant outcomes compared with grafts mobilized with G or GC. GP mobilization accelerated lymphocyte engraftment in this heavily treated group compared to G or GC. For patients proceeding to transplant heavily pre-treated, GP is a better mobilization method to ensure a robust graft is collected while avoiding the need for multiple stem cell collections and providing similar outcomes as patients less heavily treated and mobilized by G or GC. A prospective randomized controlled trial would elucidate whether progression free survival and overall survival might be improved by utilizing GP mobilization as a first-line therapy rather than as a rescue method.Table 1Graft composition and outcomes of different stem cell mobilization methodsVariableG (n=97)GC (n=213)GP (n=23)P valueNumbers of regimens pre-SCT, median2.2 (95% CI, 2.0-2.4)1.9 (1.8-2.0)2.3 (1.9-2.7)0.02WBC in graft, median (range)184.8 (12-777.7)138.6 (11-542)286.1 (186-400.3)0.01Absolute lymphocyte in graft (x103), median (range)128.1 (13-321.1)73.9 (3.4-433.6)161.2 (47.4-302.0)0.01ANC >500 (days), median (range)16 (11-25)15 (7-86)18 (13-24)0.16Platelets >20 (days), median (range)13 (9-22)12 (7-18)12 (10-21)0.07OS (2 year)Lymphoma74.577.972.70.054Multiple myeloma89.674.4720.76PFS (2 year)Lymphoma58.257.646.10.1Multiple myeloma66.349600.21 DisclosuresNo relevant conflicts of interest to declare.

A Radio-Resistant Perforin-Expressing Lymphoid Population Controls Allogeneic T Cell Engraftment, Activation and Onset of Gvhd

IntroductionImmunosuppressive pre-transplantation conditioning is essential for donor cell engraftment in allogeneic bone marrow transplant (BMT). The role of residual post-conditioning recipient immunity in determining engraftment is poorly understood. Although recipient perforin has previously been shown to modulate myeloid engraftment from mouse bone marrow, and adoptive transfer of natural killer (NK) cells can ameliorate the onset of graft-versus host disease (GVHD), the role of perforin-expressing endogenous recipient NK and NKT cells in modulating donor T cell engraftment has not been described. Using an MHC-mismatched mouse model, we examined the role of perforin-expressing NK cells in regulating both the rate and activation status of donor lymphocyte engraftment after allogeneic transplantation.MethodsAn MHC-disparate model of BMT was established transplanting BALB/c donor BM (H-2Kd), into female 6-14 week old C57BL/6 WT and C57BL/6.perforin KO (H-2Kb) recipients. On day 0, recipient mice were administered a split dose of lethal radiation using a caesium source (2 x 6 gray), and injected i.v. with 5e6 T cell depleted BM (TCD-BM) cells from MHC-mismatched or syngeneic donors. On day 2, recipient mice were injected i.v. with 1e6 BALB/c purified splenic T cells at a 2:1 CD4+:CD8+ T cell ratio. Mice were monitored daily, and killed at selected time points post-transplant (typically day 5-7 for short term engraftment, or day 20 for long term engraftment) to examine donor lymphoid cell engraftment.ResultsAn HLA-mismatched BMT mouse model demonstrated that both the rate and proportion of donor lymphoid cell engraftment, and expansion of effector memory donor T cells in both spleen and BM were significantly increased within 5-7 days post BMT in perforin-deficient (pfn-/-) recipients, compared with wild-type (WT). Critically, we found that the absence of perforin resulted in the increased production of pro-inflammatory cytokines, in particular IL-6, from engrafting donor T cells. IL-6 has recently been identified as a primary driver of both mouse model and clinical GVHD. Correlating with pro-inflammatory cytokine secretion, effector memory donor T lymphocytes expanded more rapidly in pfn-/- recipients than in WT mice, possibly arising from more rapid proliferation or selective survival of these donor cells. In WT recipients, donor lymphoid cell engraftment was enhanced to that seen in pfn-/- recipients, by depleting NK cells prior to BMT, demonstrating that a perforin-dependent, NK-mediated host-versus graft effect limits the rate of donor cell engraftment and T cell activation. We found that radiation-resistant NKT cells survived in the BM of lethally irradiated mice and may drive NK cell activation, resulting in the host-versus graft effect. Furthermore, reduced pre-transplant irradiation doses in pfn-/- recipients permitted long-term donor lymphoid cell engraftment and improved survival (Figure 1).Figure 1: Reduced total body irradiation in perforin-deficient mice permits rapid donor lymphoid cell engraftment. On day 0, WT or pfn-/- (KO) mice were irradiated with variable doses (12-6 gray), and injected i.v. with 5e6 TCD-BM cells from BALB/c donors. On day 2, recipient mice were injected i.v. with 1e6 splenic BALB/c T cells. On day 7 (A) and day 20 (B) after BMT, the BM cells of WT or KO mice were stained for H-2Kd (donor cells) and engraftment determined as a percentage of donor lymphocytes. (C) Survival of KO mice administered 9 gray (closed circles), 7.5 gray (triangles) and 6 gray (lines) after BMT described above. [Display omitted] [Display omitted] [Display omitted] ConclusionsThe challenge in clinical HSCT is to promote reliable donor engraftment and full immunological reconstitution whilst minimizing pre-transplant conditioning and associated toxicity. Our findings suggest that suppression of perforin activity or selective depletion of recipient NK cells prior to BMT may allow the combined advantage of reduced transplant toxicity whilst maintaining donor engraftment and promoting the graft versus tumour effect. DisclosuresNo relevant conflicts of interest to declare.

Day 45 Lymphocyte Recovery Is Independently Associated with Transplant-Related Mortality after Adult Myeloablative Double-Unit Cord Blood Transplantation (dCBT)

▪Background: Our center has reported that the infused viable CD34+ cell dose of the dominant unit determines the speed & success of neutrophil recovery after dCBT (Purtill et al, Blood 2014). However, how unit characteristics affect lymphocyte recovery has not been determined, & the effect of both delayed neutrophil & lymphocyte recoveries on transplant-related mortality (TRM) requires investigation.Methods: The association between 1) dominant unit characteristics (TNC, CD34+, CD3+ cell doses & 8-allele HLA-match) & lymphocyte recovery, & 2) the effect of both neutrophil & lymphocyte recoveries on TRM were analyzed in 98 adult myeloablative dCBT recipients (median age 42 years, range 16-69) transplanted for hematologic malignancies. The analysis was restricted to adults given the significant differences in patients (pts), grafts, & neutrophil recovery in children.Results: The cumulative incidence of day 45 neutrophil engraftment ≥ 0.5 x 109/L was 96% (95%CI:89-99) with a median recovery of 25 days (range 12-43). The day 45 cumulative incidence of lymphocyte engraftment ≥ 0.2 x 109/L was also 96% (95%CI:89-99, median 25 days, range 12-59). Dominant unit infused viable CD34+ cell dose determined both neutrophil (as previously reported) & also lymphocyte recovery. To analyze if the speed & success of neutrophil & lymphocyte recovery are important in determining subsequent TRM in engrafting pts, we performed a day 45 landmark analysis of the 93 patient subset who achieved neutrophil engraftment & were alive 45 days post-dCBT (5 pts with graft failure or early death were excluded). In univariate analysis, a trend toward increased day 45-180 TRM was observed among the 51 pts with delayed neutrophil engraftment > 25 days [HR 2.71 (95%CI 0.88-8.42), p = 0.084], but there was no association between the absolute neutrophil count on day 45 & subsequent TRM. Conversely, while the time to lymphocyte recovery ≥ 0.2 x 109/L was not significant, the day 45 lymphocyte count correlated significantly with day 45-180 TRM. A lymphocyte cut-point of 0.3 x 109/L was identified below which TRM risk was markedly increased: 17 dCBT recipients with a day 45 lymphocyte count < 0.2 had a TRM HR 12.04 (95%CI: 4.33-33.52), p < 0.001 (Figure). Age > 40 years & recipient CMV seropositivity were also associated with a trend toward increased day 45-180 TRM in univariate analysis whereas regimen intensity, disease status, & dominant unit HLA-match were not. Patient demographics (age, weight, disease stage, CMV serostatus, & conditioning intensity) & dominant unit characteristics (infused TNC, viable CD34+, CD3+ cell doses) were similar in pts with day 45 lymphocyte counts > or < 0.2 x 109/L. Low lymphocyte count < 0.2 x 109/L was not explained by differences in corticosteroid therapy. The causes of day 45-180 TRM in pts with low day 45 lymphocytes were organ failure (n = 5) & acute GVHD (n = 4) with one patient dying of infection. On multivariate analysis, day 45 lymphocyte count < 0.2 x 109/L was independently associated with a greater day 45-180 TRM risk (p < 0.001, Table) while a trend was observed for delayed neutrophil engraftment (p = 0.069). Recipient age > 40 years & CMV serostatus were not significant (p = 0.196 & p = 0.418, respectively). The relationship between lymphocyte recovery & TRM was maintained when the 20 pts on therapy for acute GVHD by day 45 were excluded.Conclusion: Both early neutrophil & lymphocyte recovery are critically important factors in the success of adult myeloablative dCBT. While it is well appreciated that early failure of neutrophil recovery is associated with a very high mortality rate, this analysis of pts engrafting with neutrophils suggests a low lymphocyte count (<0.2) at day 45 was associated with a high risk of early TRM and was an independent and more powerful TRM risk factor than the speed of prior neutrophil recovery. The exact mechanisms that underlie this observation require further investigation but these findings emphasize the importance of considering both neutrophil and lymphocyte recovery in the assessment of the quality of early CB engraftment.TableVariablenHR (95%CI)pSpeed of ANC recovery< 25 days42Reference0.069≥ 25 days512.94 (0.92-9.39)Lymphocyte count at day 45> 0.2 x 109/L76Reference< 0.001< 0.2 x 109/L1712.10 (4.21-34.81)* Recipient age and CMV serostatus were included in the initial model but removed using a backward conditional method. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Respiratory syncytial virus in hematopoietic cell transplant recipients: factors determining progression to lower respiratory tract disease.

Background. Respiratory syncytial virus (RSV) lower respiratory tract disease (LRD) is a life-threatening complication in hematopoietic cell transplant (HCT) recipients. Lymphopenia has been associated with an increased risk of progression from upper respiratory tract infection (URI) to LRD. Methods. This study retrospectively analyzed the significance of lymphocyte engraftment dynamics, lung function, smoking history, corticosteroids, antiviral treatment, viral subtypes, and RSV-specific neutralizing antibodies for the progression to LRD in 181 HCT recipients with RSV URI. Results. In multivariable models, smoking history, conditioning with high-dose total body irradiation, and an absolute lymphocyte count (ALC) ≤100/mm3 at the time of URI onset were significantly associated with disease progression. No progression occurred in patients with ALCs of >1000/mm3 at URI onset. Lymphocyte engraftment dynamics were similar in progressors and nonprogressors. Pre- and posttransplant donor and posttransplant recipient RSV subtype-specific neutralizing antibody levels, RSV viral subtypes, and corticosteroids also were not significantly associated with LRD progression. Conclusions. Host and transplant related factors appear to determine the risk of progression to LRD more than viral factors. Dysfunctional cell-mediated immunity appears to be important in the pathogenesis of progressive RSV disease after HCT. A characterization of RSV-specific T-cell immunity is warranted.

Engraftment of canine peripheral blood lymphocytes into nonobese diabetic-severe combined immune deficient IL-2R common gamma chain null mice

To study the canine immune system we generated a mouse model engrafted with canine lymphocytes using NOD SCID IL2R common gamma chain −/− (NSG) mice as recipients (Ca-PBL-SCID). Engraftment of canine peripheral blood lymphocytes (PBLs) was determined post-injection with 107 peripheral blood mononuclear cells (PBMCs) into irradiated NSG mice using flow cytometry and fluorescently labeled antibodies specific to canine helper T cells (CD45+ CD4+), cytotoxic lymphocytes (CD45+ CD8+), regulatory T cells (CD45+ CD4+ Foxp3+), and B cells (CD45+ Ig+ CD21lo). Canine CD45+ lymphocytes were detectable as early as day 1 in the peritoneal cavity, and beginning at 9 days in the blood, bone marrow, and spleen. CD4+ T cells, of which Foxp-3+ CD25hi cells constituted a minor percentage, were the predominant lymphocyte population at 9 days post engraftment contrasting with increasing proportions of CD8+ CTL's and Ig+ B cells beginning at 16 days. Canine immunoglobulin was initially detected in the serum of Ca-PBL-SCID mice at 9 days post-engraftment and peaked in concentration at day 36. From day 28 to 52 post-engraftment 30% of the Ca-PBL-SCID mice became markedly anemic and thrombocytopenic, yet gross and histopathologic examination of bone marrow, kidneys, spleen, liver, and intestine revealed no obvious lesions. Blood smear evaluation revealed agglutination of mature red blood cells, reticulocytes and a regenerative anemia. These findings demonstrate that NSG mice are capable of engraftment of canine PBLs yet develop graft versus host disease similar to Hu-PBL-SCID mice.

Haploidentical Stem Cell Transplantation After Negative Depletion Of T Cells Expressing The αβ Chain Of The T-Cell Receptor (TCR) For Adults With Hematological Malignancies

Introduction For many years, T cell depletion (TCD) of hematopoietic stem cells (HSCs) has been based on either positive or negative selection of mobilised peripheral blood cells (PBPCs). After CD34+ cell selection, the T cell repertoire is very narrow since the number of T lymphocytes in the graft has to be particularly low to prevent GvHD and ATG in the conditioning exerts an additional in vivo T cell depletion. Thus the immune recovery is slow and patients tend to remain susceptible to opportunistic infections for several months after HSCT. To hasten and improve post-transplant immune reconstitution broad repertoire various strategies of adoptive donor T cell immunotherapy (e.g. engineering with a suicide gene; depleting alloreactivity by means of photodynamic purging or through the use of freshly purified regulatory T cells) have been investigated over the past years. More recently, selective elimination of αβ+ T cells has been performed to achieve a 4,5–5 log TCD and to retain in the graft NK, dendritic cells, monocytes and γδT lymphocytes. Under this approach, a rapid immunological reconstitution and very promising outcome have been reported in pediatric patients. With the aims of confirming these results even in adults, we have recently launched this programme and here we report our preliminar clinical data. Methods Thirteen patients, median age 40 years (range 19-65), with AML (n=9), ALL (n=2), HL (n=1) or Rhabdomyosarcoma (n=1) entered the study. All but two patients, who were in first remission, were in advanced-stage disease at transplant with five patients in chemoresistant relapse. Conditioning consisted of ATG 1,5 mg/kg from day -13 to day -10, Treosulfan 12gr/sqm from -9 to –7, Fludarabine 30mg/sqm from -6 to -2 and Thiotepa 5mg/Kg on days -5 and -4. Ten μg/kg G-CSF was used to mobilize PBPCs from one-haplotype mismatched donors (4 mothers, 4 brothers, 2 sisters, 1 son, 1 daughter and 1 cousin). Mobilized mononuclear cells were incubated with a biotinylated anti-TcRαβ antibody and subsequently with an antibiotin antibody conjugated to magnetic microbeads (Miltenyi Biotec, Germany). Under a strong magnetic field, TcRαβ T lymphocytes were retained, whereas all nonmagnetized cells were recovered. Short sirolimus (1mg/day x3 weeks) was used as additional GVHD prophylaxis in 3 cases whose grafts contained more than 2x105/kg αβ+Tcells. Results Grafts contained a median of 12,3x106/kg CD34+ cells(range7-19), 6 x106 CD3+Tcells/kg (range 2,3-13)with 10,4x104/kg αβ+T cells (range 1,38-62) and 5,8x106 γδ+Tcells/kg (range2,1-12,6), 6x104B cells/kg (range 0,2–32) and 34x108 CD56+NKcells/kg (range10-91). All but one patient, who required a second graft from the same donor to boost hematopoietic reconstitution, achieved a full donor sustained engraftment. Median time to reach 500 neutrophils and 50,000 platelets was 13 (range 9-18) and 11 days (range 9-13), respectively. Four patients had skin grade I/II aGVHD. No patients has so far developed chronic GvHD. Median CD4+ cell counts at 30, 60, 90 and 120 days since the transplant were 33, 122, 190 and 251 n/mL, respectively. CMV reactivation occurred in only 2 cases (in one, CMV serology was unfavourable: CMV-negative donor/CMV-positive recipient). Overall, 3 patients have so far died (2 non-hematologic causes and 1 early relapse). Ten survive disease-free at a median follow-up of 104 days (range 30-178). Conclusions The infusion of αβ/CD19-depleted grafts was safe and effective also in adult setting, resulting into rapid donor hematopoietic engraftment and early expansion of donor-derived γδT lymphocytes, without life-threatening infectious complications. Disclosures: No relevant conflicts of interest to declare.

Disruption Of CD9 Expression Affects Adhesion, Migration and Engraftment Of Pre-B Lymphocytes

IntroductionCD9 is a membrane protein, member of the tetraspanin family. Recent publications have reported the role of CD9 on engraftment of hematopoietic stem cells, and on cancer stem cell potential. The expression of CD9 has been correlated to the risk of metastases and to a poor clinical outcome in various types of cancer. Surprisingly, CD9 protein is downregulated in ETV6/RUNX1 pre-B acute lymphoblastic leukemia. The purpose of our study is to investigate the effect of CD9 expression on migration and engraftment abilities of pre-B lymphocytes. Materials and MethodsThe CD9-positive Nalm6 and REH (ETV6/RUNX1) pre-B cells were used. By lentiviral transduction of shRNA targeting mRNA, we generated Nalm6 and REH cell lines depleted in CD9 protein. Engraftment tests were performed in vivo using Nod Scid Gamma immunodeficient mice. REH and Nalm6 cells were detected in bone marrow by CD10 and respectively CD19 or HLA-DR labelling. Ability of the different cell lines to adhere on fibronectin and to migrate through double chambers system in response to CXCL12 were measured in vitro. We also investigated the presence of membrane villosities on REH and REH shCD9 cell surface by scanning electron microscopy. Finally, F-actin polymerization after CXCL12 stimulation was measured by rhodamin-phalloidin labelling. ResultsIn vivo engrafments tests showed that the human cells detected in bone marrow is strongly enriched in CD9 positive cells compared to the initially injected population. This result suggests that CD9 facilitates pre-B lymphobasts homing. An in vitro analysis of adhesion on fibronectin demonstrated that cellular adhesion is dependent on membrane expression of CD9. As well, the more CD9 is expressed, the higher the migration rate in response to CXCL12 chemokine is. The analysis of membrane villosities on REH cell surface revealed that cells over expressing CD9 had longer villosity than shCD9 transducted cell lines. Moreover, F-actin labelling after CXCL12 stimulation showed an increased F-actin polymerization in CD9-positive cells and the formation of actin extensions. ConclusionWe provide novel evidence that CD9 is a key player of pre-B lymphoblasts engraftment, adhesion and CXCL12 dependant migration. CD9 expression is related to actin remodelling. We are now investigating a potential link between CD9 and RAC1 activation in response to CXC12. Therefore, the expression level of CD9 could impact leukemic blasts abilities to spread and be responsible of relapses.This work is supported by CNRS, University of Rennes 1, University Hospital of Rennes, la Ligue Régionale contre le Cancer (committee 22, 35 and 56) (MPA, VG, MBT), SFR Biosit UMS 3480 (VG, MBT), Association Laurette Fugain (VG) and Europe Career Integration Grant (MBT). Disclosures:No relevant conflicts of interest to declare.

Prevention of acute graft‐versus‐host disease by humanized anti‐CD26 monoclonal antibody

CD26 (DPP4) is a T cell costimulatory molecule as well as T cell activation marker, and CD26(+) T cells are accumulated in inflamed tissues, such as rheumatoid synovitis and autoimmune thyroiditis. In the present study, we found accumulation of CD26(+) T cells in graft-versus-host disease (GVHD) target organs. To expand our in vitro findings to an in vivo system, we examined CD26-dependent organ injury in a xenogeneic GVHD (x-GVHD) murine model. Following intraperitoneal injection of human peripheral blood mononuclear cells into non-obese diabetic severe combined immunodeficiency/γ(c) (-/-) mice (hu-PBL-NOG mice), the mice exhibited the onset of GVHD symptoms associated with the presence of CD26(high) human lymphocytes in the peripheral blood and GVHD target tissues. Administration of humanized anti-human CD26 monoclonal antibody (mAb) decreased x-GVHD severity and prolonged survival in hu-PBL-NOG mice without loss of engraftment of human T cells, while increasing doses of CTLA4- immunoglobulin fusion protein diminished engraftment of human lymphocytes. Importantly, anti-CD26 mAb treatment preserved the graft-versus-leukaemia effects in studies using cotransplantation of P815 murine leukaemic cells. In addition, CD26(+) lymphocytes infiltrated the GVHD patients' target tissues. Altogether, our data indicate a role for CD26 in the regulation of GVHD and point to CD26 as a novel target for therapeutic intervention in this disease.