Recipient Peripheral Blood Mononuclear Cells Research Articles

Cellular dynamics in pig-to-human kidney xenotransplantation

Xenotransplantation of genetically engineered porcine organs has the potential to address the challenge of organ donor shortage. Two cases of porcine-to-human kidney xenotransplantation were performed, yet the physiological effects on the xenografts and the recipients' immune responses remain largely uncharacterized. We performed single-cell RNA sequencing (scRNA-seq) and longitudinal RNA-seq analyses of the porcine kidneys to dissect xenotransplantation-associated cellular dynamics and xenograft-recipient interactions. We additionally performed longitudinal scRNA-seq of the peripheral blood mononuclear cells (PBMCs) to detect recipient immune responses across time. Although no hyperacute rejection signals were detected, scRNA-seq analyses of the xenografts found evidence of endothelial cell and immune response activation, indicating early signs of antibody-mediated rejection. Tracing the cells' species origin, we found human immune cell infiltration in both xenografts. Human transcripts in the longitudinal bulk RNA-seq revealed that human immune cell infiltration and the activation of interferon-gamma-induced chemokine expression occurred by 12 and 48h post-xenotransplantation, respectively. Concordantly, longitudinal scRNA-seq of PBMCs also revealed two phases of the recipients' immune responses at 12 and 48-53 h. Lastly, we observed global expression signatures of xenotransplantation-associated kidney tissue damage in the xenografts. Surprisingly, we detected a rapid increase of proliferative cells in both xenografts, indicating the activation of the porcine tissue repair program. Longitudinal and single-cell transcriptomic analyses of porcine kidneys and the recipient's PBMCs revealed time-resolved cellular dynamics of xenograft-recipient interactions during xenotransplantation. These cues can be leveraged for designing gene edits and immunosuppression regimens to optimize xenotransplantation outcomes. This work was supported by NIH RM1HG009491 and DP5OD033430.

Exosomes released by environmental pollutant-stimulated Keratinocytes/PBMCs can trigger psoriatic inflammation in recipient cells via the AhR signaling pathway.

Introduction: Exosomes, pivotal in intercellular communication during skin disease pathogenesis, have garnered substantial attention. However, the impact of environmental pollutants, such as benzo[a]pyrene (BaP) and 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), on exosome release amid inflammatory skin diseases remains unexplored. This study addresses this gap by examining the influence of BaP and TCDD on exosome function, specifically focusing on immune-related pathway alterations in normal recipient keratinocytes and peripheral blood mononuclear cells (PBMCs). Methods: HaCaT cells were treated with exosomes from BaP- or TCDD-treated keratinocytes. Proinflammatory cytokines and chemokines, including TNF-α, IL-1β, IL-6, IL-8, CXCL1, and CXCL5, were assessed. The involvement of the p65NF-κB/p38MAPK/ERK signaling pathway in recipient keratinocytes was investigated. Aryl hydrocarbon receptor (AhR) silencing was employed to elucidate its role in mediating the proinflammatory response induced by exosomes from BaP- or TCDD-treated keratinocytes. Results and discussion: Treatment with exosomes from BaP- or TCDD-treated keratinocytes induced a significant increase in proinflammatory cytokines and chemokines in HaCaT cells. The upregulation implicated the p65NF-κB/p38MAPK/ERK signaling pathway. AhR silencing attenuated this response, suggesting a role for AhR in mediating this response. In PBMCs from healthy controls, exosomes from BaP-stimulated PBMCs of psoriatic patients led to increased expression of proinflammatory cytokines and modulation of Th1/Th17 cell distribution via AhR activation. These findings unveil a novel dimension in the interplay between environmental xenobiotic agents (BaP and TCDD) and exosomal functions. The study establishes their influence on psoriatic inflammatory responses, shedding light on the underlying mechanisms mediated through the AhR signaling pathway in recipient keratinocytes and PBMCs.

Characterization of "Off-Target" Immune Modulation Induced by Live Attenuated Yellow Fever Vaccine.

Live attenuated vaccines alter immune functions and are associated with beneficial outcomes. We previously demonstrated that live attenuated yellow fever virus (YFV) vaccine (LA-YF-Vax) dampens T-cell receptor (TCR) signaling in vitro via an RNA-based mechanism. We examined study participants before and after LA-YF-Vax to assess TCR-mediated functions in vivo. Serum samples and peripheral blood mononuclear cells (PBMCs) were obtained before and after LA-YF-Vax (with or without additional vaccines) or quadrivalent influenza vaccine. TCR-mediated activation was determined by interleukin 2 release or phosphorylation of the lymphocyte-specific Src kinase. TCR-regulating phosphatase (protein tyrosine phosphatase receptor type E [PTPRE]) expression was also measured. Compared with prevaccination findings, LA-YF-Vax recipient PBMCs demonstrated transient reduction in interleukin 2 release after TCR stimulation and PTPRE levels, unlike in control participants who received quadrivalent influenza vaccine. YFV was detected in 8 of 14 participants after LA-YF-Vax. After incubation of healthy donor PBMCs in serum-derived extracellular vesicles prepared from LA-YF-Vax recipients, TCR signaling and PTPRE levels were reduced after vaccination, even in participants without detectable YFV RNA. LA-YF-Vax reduces TCR functions and PTPRE levels after vaccination. Extracellular vesicles from serum recapitulated this effect in healthy cells. This likely contributes to the reduced immunogenicity for heterologous vaccines after LA-YF-Vax administration. Identification of specific immune mechanisms related to vaccines should contribute to understanding of the "off-target," beneficial effects of live vaccines.

The VISICORT project: Lessons learned from solid organ transplantation

AbstractAims of the Presentation: (a) To summarize the rationale and research strategies for the VISICORT project. (b) To compare and contrast research approaches to understanding adverse immune responses to corneal and solid organ allo‐transplants.AbstractCorneal allo‐transplantation has been utilized for over 100 years to restore and improve vision in patients suffering from opacifying diseases of the cornea. Its development preceded that of solid organ transplantation and its worldwide frequency continues to exceed that of commonly‐transplanted organs including kidney, heart, liver and lung. The scope and success rates for corneal transplantation have evolved over time as a result of technical innovations, clinical practice developments and advances in eye banking. However, the impact of immunological complications, in particular acute rejection, on the long‐term functional survival of high‐risk corneal allografts has remained significant and the evidence base for specific immunosuppressive regimens to prevent rejection is limited. Strikingly, the large amount of elegant pre‐clinical research into the immunological response to corneal allografts has not been complemented by extensive studies of adverse immune responses in human recipients. Against this background, the VISICORT project was initiated in 2014 to bring together corneal transplant experts and investigators with expertise in solid organ transplantation, immunological profiling, proteomic analysis, regenerative therapies and biobanking to collaboratively address the challenge of better understanding adverse immune responses in human corneal transplantation.In this presentation, I will first summarize the strategies and designs for cross‐sectional and longitudinal clinical cohort studies of corneal allo‐transplant recipients at 5 European academic centres with collection of extensive clinical data and biological specimens including donor and recipient cornea, aqueous humour, tears, whole blood, serum, plasma and peripheral blood mononuclear cells. I will also describe the development of bespoke resources and protocols for the coordination of patient enrolment, data gathering and biospecimen processing, storage and transfer to a central biobank which underpinned the results to be described by other presenters in this session. Finally, I will discuss the extent to which the fields of corneal and solid organ transplantation have engaged in collaborative investigation prior to and during the course of the VISICORT project and reflect on the potential for such cross‐discipline research to advance towards the shared goal of preventing harmful allo‐immune responses and promoting donor‐specific tolerance.

Activation of the Aryl Hydrocarbon Receptor Ameliorates Acute Rejection of Rat Liver Transplantation by Regulating Treg Proliferation and PD-1 Expression.

Aryl hydrocarbon receptor (AhR) plays important roles in modulating immune responses. However, the role of AhR in rat liver transplantation (LT) has not been explored. Safety and side effects of N -(3,4-dimethoxycinnamonyl) anthranilic acid (3,4-DAA) and 2-methyl-2H-pyrazole-3-carboxylic acid amide (CH223191) were evaluated. We used optimal doses of 2 drugs, 3,4-DAA, a drug used for mediating AhR activation, and CH223191, antagonist of AhR (3,4-DAA, CH223191, and 3,4-DAA + CH223191), intraperitoneally administered to recipients daily to investigate the role of AhR in the rat LT model. The recipient livers were used to observe the pathological changes, the cells infiltrating the graft, and changes of AhR and programmed death-1 (PD-1) by Western blot, real-time polymerase chain reaction, and immunofluorescence assays. The contents of Foxp3 + and PD-1 + T cells in the recipient spleen and peripheral blood mononuclear cells were evaluated by flow cytometry. In vitro, after isolating CD4 + T cells, they were treated with different AhR ligands to observe the differentiation direction and PD-1 expression level. The activation of AhR by 3,4-DAA prolonged survival time and ameliorated graft rejection, which were associated with increased expression of AhR and PD-1 in the livers and increased Foxp3 + T cells and PD-1 + T cells in recipient spleens, livers, and peripheral blood mononuclear cells. In vitro, primary T cells incubated with 3,4-DAA mediated increased proportion of Treg and PD-1 + T cells. However, the suppression of AhR with CH223191 reverses these effects, both in the LT model and in vitro. Our results indicated that AhR activation might reduce the occurrence of rat acute rejection by increasing the proportion of Treg and the expression of PD-1.

Adenosinergic Pathway and Linked Suppression: Two Critical Suppressive Mechanisms of Human Donor Antigen Specific Regulatory T Cell Lines Expanded Post Transplant.

Regulatory T cells are an important component of an immune response shaping the overall behavior to potential antigens including alloantigens. Multiple mechanisms have been shown to contribute towards developing and sustaining a immunological regulatory response. One of the described contact dependent suppressive mechanisms regulatory cells have been shown to utilize is through the production of adenosine from extracellular ATP mediated by CD39 and CD73. In this study we demonstrate that the adenosinergic pathway plays a major role in the suppressive/regulatory effects antigen specific regulatory T cell enriched lines (ASTRLs) that have been of expanded ex vivo from stable kidney transplant patients. We have previously shown that these ASTRL cells are capable of suppressing alloimmune responses in vitro and significantly prolonging allograft survival in an animal model of kidney transplantation. For this study nineteen ASTRLs were expanded from 17 kidney transplant patients by repeated stimulation of recipient peripheral blood mononuclear cells with donor specific HLA-DR peptides. All 19 ASTRLs showed upregulation of numerous markers associated with regulatory cells and were able to inhibit donor antigen specific T cell proliferation in a dose dependent fashion. ASTRLs suppressed indirect and direct alloimmune responses compatible with our previous animal study findings. Upregulation of both CD39 and CD73 was observed post expansion and ASTRLs demonstrated extracellular hydrolysis of ATP, indicating functionality of the upregulated proteins. We also showed that inhibition of the adenosinergic pathway using inhibitors of CD39 resulted in abrogation of suppression and increased antigen specific T cell proliferation. This demonstrates that the main mechanism of action of the suppressive activity donor peptide driven ASTRLs generated from kidney transplant patients is the adenosinergic pathway. Furthermore this suggests the possibility that combining infusion of Tregs with other treatments, such as adenosine receptor agonists or increasing CD39 expression in the grafts may further enhance a regulatory response to the allograft and possibly achieve transplantation tolerance.

Early reduction of regulatory T cells is associated with acute rejection in liver transplantation under tacrolimus-based immunosuppression with basiliximab induction.

Regulatory T (Treg) cells are important in preventing acute rejection (AR) in solid organ transplantation, but the clinical relevance of the different kinetics early after liver transplantation (LT) in acute rejectors and non-rejectors is unclear. We analyzed peripheral blood samples of 128 LT recipients receiving basiliximab induction plus tacrolimus immunosuppression. Samples were obtained at pretransplant, D7, and D30 after LT. Frequency and phenotype of Tregs were analyzed by flow cytometry. The predictive value of Treg frequency at D7 was assessed for suspected acute rejection (SAR) and was validated for biopsy-proven AR (BPAR). We found that the frequencies of total and activated Tregs at D7 were significantly lower in recipients with SAR and BPAR. Treg was more reduced in BPARs by in vitro tacrolimus treatment in the presence of basiliximab. Moreover, an early reduction of Treg frequency in rejectors was associated with a greater increase in Treg apoptosis and further attenuated IL-2 signaling. D7 Treg frequency was an independent risk factor for SAR, which was also validated for BPAR. In conclusion, first-week peripheral blood Treg frequency correlates with AR after LT under tacrolimus-based immunosuppression, which needs to be proven in larger, geographically and clinically diverse populations.

Differential MicroRNA Expressions in Human Peripheral Blood Mononuclear Cells Are Predictive of Renal Allograft Function

BackgroundThe present diagnostic methods for detecting graft damage after kidney transplantation are either invasive or not available early enough. The microRNAs (miRNAs) in peripheral blood mononuclear cells (PBMCs) have been suggested as promising biomarkers. MethodsUsing quantitative real-time polymerase chain reaction, we identified 9 miRNAs (miR-142-5p, miR-142-3p, miR-223, miR-211, miR-486, miR-155, miR-10b, miR-30a, and let-7c) related to the human renal allograft status in PBMCs from 104 kidney transplant recipients. ResultsThe miR-142-5p, miR-142-3p, and miR-223 were significantly upregulated and miR-10b was significantly downregulated in recipients with abnormal levels of serum creatinine 3 to 4 weeks after initial sample collection. Moreover, the miR-142-5p and miR-142-3p were also found to be significantly upregulated in recipients with abnormal levels of cystatin C. Through a combination of the validated miRNAs, receiver operating characteristic analyses yielded the highest area under the curve value of 0.7913 and 0.7063 in predicting the levels of serum creatinine and cystatin C, respectively. In the testing stage, the developed models correctly predicted allograft function in 16 to 17 of 22 recipients (false rate, 22.7%-27.2%). ConclusionsmiRNAs in PBMCs of recipients hold great promise to be used as predictive and noninvasive biomarkers after transplantation.

Recipient single nucleotide polymorphisms in Paneth cell antimicrobial peptide genes and acute graft-versus-host disease: analysis of BMT CTN-0201 and -0901 samples.

Host genetics shape the gut microbiota, and gut dysbiosis increases the risk of acute graft-versus-host disease (aGVHD). Paneth cells and microbiota have interactions that contribute to immune regulation. α-defensin-5 (HD5) and regenerating islet-derived protein 3 alpha (Reg3A) are the most abundant Paneth cell antimicrobial peptides (AMPs). We hypothesized that single nucleotide polymorphisms (SNPs) in the genes for HD5 (DEFA5) and Reg3A (REG3A) predict aGVHD risk. We analysed pre-transplant recipient peripheral blood mononuclear cell samples from randomized Blood and Marrow Transplant Clinical Trials Network (BMT CTN) studies 0201 (94 patients with bone marrow and 93 with peripheral blood grafts) and 0901 (86 patients with myeloablative and 77 with reduced-intensity conditioning; all using peripheral blood grafts). In multivariable analysis (with a SNP×graft source interaction term in CTN-0201 and a SNP×conditioning intensity term in CTN-0901), DEFA5 rs4415345 and rs4610776 were associated with altered incidence of aGVHD grade II-IV [rs4415345 G vs. C: hazard ratio (HR) 0·58, 95% confidence interval (95% CI) 0·37-0·92, P=0·02; rs4610776 T vs. A: HR 1·53, 95% CI 1·01-2·32, P=0·05] in CTN-0201, but not CTN-0901, suggesting a stronger effect in bone marrow allografts. REG3A SNP was not associated with aGVHD. Host genetics may influence aGVHD risk by modulating Paneth cell function.

Ex-Vivo Generation of Alloantigen-Specific Immunomodulatory Cells

Introduction The efficacy of adoptive transfer of ex-vivo generated Tregs has been attempted to induce operational tolerance in preclinical models and early clinical trials. However, Tregs purification strategies require highly specialized facilities using complicated techniques. Recently, Todo et al. reported that ex-vivo generated immunomodulatory cells without any purification processes induced operational tolerance in 7/10 liver transplanted recipients. In this trial, immunomodulatory cells were ex-vivo generated by coculture of recipient PBMCs with irradiated donor cells in the presence of CD28/B7 co-stimulatory blockade. In terms of the wide spread application of tolerance induction strategies, it is crucial to address which cells are responsible for the donor-antigen specific immunomodulatory effects. Material and Methods Immunomodulatory cells were generated by 14 days co-culture of recipient peripheral blood mononuclear cells (PBMCs) (50 x 106 cells) with irradiated HLA-mismatched donor PBMCs (20 x 106 cells) with CTLA4-Ig (40 mg/106 recipient PBMCs). At day 14, the generated cells were collected and enriched for CD4+ CD25+ T cells (Tregs) using MACS, or depleted of CD4+ T cells, CD8+ T cells (MACS), CD19+ (B cells), or (CD25+CD127+lo) Tregs using FACS. The suppressive functions of the generated cells (non-sorted), and each enriched or depleted cells were measured in vitro, i.e. the freshly isolated recipient PBMCs were stimulated by irradiated donor or 3rd party PBMCs, with or without varying numbers of generated cells. The cell proliferation and cytokine production was compared. Results and Discussion The addition of non-sorted generated cells significantly inhibited proliferation of responder cells against donor antigen in a generated cell-number dependent fashion (Fig.1). Interestingly, non-sorted cells showed significantly stronger donor antigen-specific immunomodulatory effects than Tregs enriched cells (Fig. 2), indicating that non-T regs in generated cells also play important roles for the immunomodulatory effects. In order to examine the hypothesis, we further depleted specific population from the generated cells. The results indicate that Treg or B cell depleted generated cells show immunomodulatory effects against both donor and 3rd party stimulations (Fig. 3-4) . Interestingly, the depletion of CD4+ or CD8+ T cells from the generated cells maintained the donor-antigen specific inhibitory effects.IFN-g was mainly produced by CD4+ T cells in the generated cells. Depletion of B cells or Treg from the generated cells lower the production of IL-10, indicating these cells are major producer of IL-10.Conclusion The ex-vivo generated cells without any purification showed stronger donor antigen-specific immunomodulatory effect. In this ex-vivo generation protocol, Tregs don’t need to be purified. The Tregs and B cells seems to be important in generating the donor antigen-specific immunomodulatory effects.

Myeloid-derived suppressor cells increase and inhibit donor-reactive T cell responses to graft intestinal epithelium in intestinal transplant patients.

Recent advances in immunosuppressive regimens have decreased acute cellular rejection (ACR) rates and improved intestinal and multivisceral transplant (ITx) recipient survival. We investigated the role of myeloid-derived suppressor cells (MDSCs) in ITx. We identified MDSCs as CD33+ CD11b+ lineage(CD3/CD56/CD19)- HLA-DR-/low cells with 3 subsets, CD14- CD15- (e-MDSCs), CD14+ CD15- (M-MDSCs), and CD14- CD15+ (PMN-MDSCs), in peripheral blood mononuclear cells (PBMCs) and mononuclear cells in the grafted intestinal mucosa. Total MDSC numbers increased in PBMCs after ITx; among MDSC subsets, M-MDSC numbers were maintained at a high level after 2months post ITx. The MDSC numbers decreased in ITx recipients with ACR. MDSC numbers were positively correlated with serum interleukin (IL)-6 levels and the glucocorticoid administration index. IL-6 and methylprednisolone enhanced the differentiation of bone marrow cells to MDSCs in vitro. M-MDSCs and e-MDSCs expressed CCR1, -2, and -3; e-MDSCs and PMN-MDSCs expressed CXCR2; and intestinal grafts expressed the corresponding chemokine ligands after ITx. Of note, the percentage of MDSCs among intestinal mucosal CD45+ cells increased after ITx. A novel in vitro assay demonstrated that MDSCs suppressed donor-reactive T cell-mediated destruction of donor intestinal epithelial organoids. Taken together, our results suggest that MDSCs accumulate in the recipient PBMCs and the grafted intestinal mucosa in ITx, and may regulate ACR.

Linking Early Humoral Sensitization in Lung Transplant Recipients with Even Earlier Cellular Alloreactivity in Humanized Mice

Early donor-specific anti-HLA antibodies (DSA) after lung transplantation are correlated with the development of chronic lung allograft dysfunction (CLAD). It remains unknown, however, whether this is a primary antibody-mediated effect, or an epiphenomenon of other immune mechanisms. Here, we studied lung recipient peripheral blood mononuclear cells (PBMC) drawn at the time of transplantation in a humanized mouse model and correlated the results to the later development of early DSA.

Optimization of In vitro Culture Conditions of Interleukin-10 Secreting B Regulatory Cells of Human Origin

AbstractAim: B-regulatory cells (B-regs), commonly characterized as CD19+/38hi/24hi, comprise of a major subset of IL-10 secreting B-cells. We report optimization of culture conditions for B-regs of human origin, obtained from in-vitro co-culture of donor adipose tissue derived mesenchymal stem cells (AD-MSC) and renal allograft recipient (RAR) peripheral blood mononuclear cells (PBMC) for potential cell therapy.Material and methodology: Five sets of 6-well plates were filled with proliferation media. Each plate was seeded with 5 × 104 ADMSC and 1 × 106 cells of responder and stimulator peripheral blood mononuclear cells (PBMC) and incubated at 37˚C, 5% CO2 after addition of lipopolysaccharide–E.coli-K12 strain (LPSEK) in varying concentrations. At each time point, one plate was withdrawn and contents centrifuged. IL-10 concentration of cell supernatant was estimated by quantitative ELISA. Pellet was resuspended to determine morphology, sterility, total count, viability and immunophenotype of cells.Results: The highest B-reg population (0.19×106 cells/ml) was noted at 24 hours with 400 ng/ml LPS-EK, which also correlated with optimum secretion of IL-10 (1.58 pg/ml), for the same time interval.Conclusion: The present study provides valuable information about the dynamics of IL-10 secretion by in vitro generated human B-regs, obtained from donor AD-MSC and recipient PBMC.

In-vitro generation of interleukin-10 secreting B-regulatory cells from donor adipose tissue derived mesenchymal stem cells and recipient peripheral blood mononuclear cells for potential cell therapy

BackgroundInterleukin-10 secreting B-cells are a major subset of B-regulatory cells (B-regs), commonly recognized as CD19+/38hi/24hi/IL10+. They carry out immunomodulation by release of specific cytokines and/or cell-to-cell contact. We have generated B-regs in-vitro from donor adipose tissue derived mesenchymal stem cells (AD-MSC) and renal allograft recipient (RAR) peripheral blood mononuclear cells (PBMC) for potential cell therapy. Material and methodsMononuclear cells separated by density gradient centrifugation from 50 ml anti-coagulated blood of 15-RAR and respective donors were analysed for baseline B-regs using appropriate antibodies. Equal amount (20 × 106 cells/ml) of stimulator (irradiated at 7.45 Gy/min for 10 min) and responder (non-irradiated) cells were co-cultured with in-vitro generated AD-MSC (1 × 106 cells/ml) in proliferation medium containing lipopolysaccharide from E. coli K12 strain at 37 °C with 5% CO2. Cells were harvested on day-7 and analyzed for viability, sterility, quantity, morphology and phenotyping. In-vitro generated B-reg levels were compared with baseline B-regs. ResultsIn-vitro generated B-reg count increased to 16.75% from baseline count of 3.35%. ConclusionB-regs can be successfully generated in-vitro from donor AD-MSC and RAR PBMC for potential cell therapy.

Ex Vivo Costimulatory Blockade to Generate Regulatory T Cells From Patients Awaiting Kidney Transplantation.

Short-term outcomes of kidney transplantation have improved dramatically, but chronic rejection and regimen-related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless, the immunomodulatory effects of end-stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study, we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of exvivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover, these Tregs maintain a CD3(+) CD4(+) CD25(+) CD127(lo) surface phenotype, high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg-specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple, brief Treg production strategy in clinical trials of mismatched kidney transplantation.

Donor Cell Composition and Reactivity Predict Risk of Acute Graft-versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation.

Background. Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (HSCT). We designed a functional assay for assessment of individual risk for acute GVHD. Study Design and Methods. Blood samples were collected from patients and donors before HSCT. Two groups of seven patients each were selected, one in which individuals developed acute GVHD grades II–IV and one in which none showed any clinical signs of GVHD. Peripheral blood mononuclear cells (PBMCs) isolated from donors were incubated in mixed lymphocyte cultures (MLCs) with recipient PBMCs. The cells were characterized by flow cytometry before and after MLC. Results. Samples from donors in the GVHD group contained significantly lower frequencies of naïve γδ T-cells and T-cells expressing NK-cell markers CD56 and CD94. Donor samples in this group also exhibited lower frequencies of naïve CD95+ T-cells compared to controls. After MLC, there were dissimilarities in the CD4/CD8 T-cell ratio and frequency of CD69+ T-cells between the two patient groups, with the non-GVHD group showing higher frequencies of CD8+ and CD69+ T-cells. Conclusion. We conclude that a thorough flow cytometric analysis of donor cells for phenotype and allogeneic reactivity may be of value when assessing pretransplant risk for severe acute GVHD.

Elevated Placental Growth Factor Levels after Allogeneic Hematopoietic Cell Transplantation: Clinical and Peripheral Blood Mononuclear Cell Receptor Expression Correlates

Background: Placental growth factor-1 (PlGF) is a member of the vascular endothelial growth factor (VEGF) family of angiogenic factors possessing inflammatory properties as well as a chemotactic effect for monocytes. Additionally, circulating PlGF levels are associated with disease severity in hypoxic conditions including sickle cell disease and coronary artery disease. We have recently identified that circulating levels of PlGF are significantly elevated in recipients of allogeneic hematopoietic cell transplantation (HCT), and most significantly at the onset of acute graft versus host disease [(aGVHD), Holtan, SG et al, ASH 2014]. It is currently unknown whether PlGF levels are associated with an alteration of expression levels of angiogenic factor receptors on mononuclear cells subsets or other clinical factors post-allogeneic HCT. Thus, as PlGF could play an immunomodulatory role on the developing donor-derived immune system, we sought to characterize peripheral blood mononuclear cell (PBMC) subset expression of the PlGF receptor (VEGFR1) using HCT donor cells collected prior to stem cell collection compared to HCT recipient PBMC collected at 3 months post-HCT. We also correlated clinical factors with PlGF levels and VEGF receptor expression on PBMCs at 3 months post-HCT.Patients and methods: Plasma and PBMCs from a cohort of 14 adult patients undergoing allogeneic HCT at Oregon Health & Science University were collected at 3 months post-HCT and compared to samples obtained from their matched sibling donors (MSD) prior to stem cell donation. Plasma levels of PlGF were quantified by ELISA. Lymphocyte and monocyte expression of VEGFR1 was determined by multiparameter flow cytometry. Comparison of donor and recipient plasma PlGF levels and PBMC phenotypes were performed by Wilcoxon signed rank. Correlations were determined using Spearman’s rho. Survival curves generated using Kaplan Meier estimates with differences between curves determined by log rank.Results: PlGF and clinical correlates: PlGF levels were 3.5-fold higher (25.1 versus 7.1 pg/mL, p=0.006) in recipients at 3 months post-HCT compared to their MSD. Donor PlGF levels were not associated with donor age or donor sex. Allogeneic HCT recipient PlGF levels were not associated with donor age, donor sex, recipient age, recipient sex, sex mismatch, conditioning intensity, disease risk index, or disease type. PlGF levels were not associated with the development of acute GVHD prior to the 3-month post-HCT blood draw (p=0.4); however, PlGF levels were negatively associated with steroid dose as a continuous variable (Spearman’s rho -0.6, p=0.02). Recipient PlGF levels >25.1 pg/ml at 3 months post-HCT were associated with improved overall survival. PBMC subsets: Allogeneic HCT recipients had significantly higher levels of circulating monocytes (15.4 vs 8.1%, p=0.05) and approximately double the percentage of CD8+ T-cell (1.5 vs 0.6%, p=0.009) and CD3-CD11b+DRlo/- subsets (6.0 vs 3.3%, p=0.01) expressing VEGFR1 as compared to their donors. CD16+ NK cells (r=0.62, p=0.02), CD14+16+ monocytes (r=0.64, p=0.01), and CD14+16+VEGFR1+ monocytes (r=0.61, p=0.02) correlated with PlGF levels in recipients.Conclusion: Elevated PlGF in HCT recipients is associated with an increase in monocyte percentage and an approximate doubling of VEGFR1-expressing lymphoid and myeloid cells compared to their MSD. Despite published association of elevated PlGF levels and chronic inflammatory states, in our small series, higher PlGF levels were associated with improved early survival. Studies are ongoing to correlate PlGF levels with later time points. Further research into the direct angiogenic and immune regulatory effects of PlGF in allogeneic HCT recipients, as well as larger scale studies of the impact of PlGF in late complications of HCT, are indicated. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 1658: Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells: Potential effects on the tumor microenvironment

Abstract The gene encoding the cytoskeletal regulator DIAPH3 is lost at high frequency in metastatic prostate cancer, and silencing of DIAPH3 evokes a transition to an amoeboid phenotype in multiple tumor cell backgrounds. This amoeboid transformation is accompanied by increases in plasma membrane deformations (blebbing), cell migration, invasion, and metastasis. DIAPH3 silencing also promotes the shedding of atypically large (>1μm) extracellular vesicles (EV) containing bioactive cargo. Whether loss of DIAPH3 also stimulates the release of bioactive nano-sized EV (exosomes) is not established. Here we examined the mechanism of release and potential biological functions of EV shed from DIAPH3-silenced and other prostate cancer cells. We observed that stimulation of LNCaP cells with the prostate stroma-derived growth factor heparin-binding EGF-like growth factor (HB-EGF), combined with p38MAPK inhibition caused exosome shedding, a process mediated by ERK1/2 hyperactivation. DIAPH3 silencing in DU145 cells also increased rates of exosome production. EV isolated from DIAPH3-silenced cells activated AKT1 and androgen signaling, increased proliferation of recipient tumor cells, and suppressed proliferation of human macrophages and peripheral blood mononuclear cells (PBMC). DU145-derived EV contained miR-125a, which suppressed AKT1 expression and proliferation in recipient human peripheral blood mononuclear cells and macrophages. Our findings suggest that EV across a wide size range (∼70nm to >1um) are produced by amoeboid prostate cancer cells as a result of DIAPH3 loss and/or growth factor stimulation. These EV may condition the tumor microenvironment through multiple mechanisms, including promoting the growth of cancer cells and suppression of tumor-infiltrating immune cells. Citation Format: Jayoung Kim, Samantha Morley, Minh Le, Denis Bedoret, Dale Umetsu, Dolores Di Vizio, Michael Freeman. Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells: Potential effects on the tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1658. doi:10.1158/1538-7445.AM2014-1658

Mesenchymal Stem Cells Induced In Vitro Generation of Antigen Specific Regulatory T-Cells Containing CD4+CD8+ Population: A Cell-Based Therapy To Promote Transplantation Tolerance.

Introduction:Harnessing regulatory T-cells (Tregs), for adoptive cell therapy to promote transplantation tolerance is promising. We present in-vitro model of induction of Tregs using donor adipose tissue derived mesenchymal stem cells (AD-MSC) and recipient peripheral blood mononuclear cells (PBMC) and their application in living donor renal transplantation. Material and Methodology:Subcutaneous adipose tissue was collected from each of 50 renal allograft donors following nephrectomy, cultured in-vitro and AD-MSC were generated. PBMC were derived from immunosuppressed recipients, divided in two parts, one as responder-PBMC (R.PBMC) and other subjected to irradiation served as stimulator-PBMC (S.PBMC). R.PBMC+S.PBMC were layered on AD-MSC, supplemented with IL-2. On 19th day Tregs were harvested, analyzed for characterization [CD127-/lowCD4+CD25high,FoxP3+, CTLA-4+](FACScan) and infused in thymus of recipients 3 weeks post-transplant. Results: Mean CD127-/lowCD4+CD25high increased from 3.1% to 10.5%, FoxP3+ increased from 12.50% to 33.9% and CTLA-4+ increased from 14.9% to 33.8% at the end. Interestingly there was rise in CD4+CD8+ cell population from mean 1.1% to 38.8%. Mean Tregs infused were 34.5 x104 cells/kg BW. Over a mean follow-up of 7.2 months, all patients are doing well on immunosuppression of Tacrolimus,0.05mg/kgBW/day and Prednisone, 10 mg/day with stable mean serum creatinine of 1.2 mg/dL and have no immune injury. Conclusion: We have successfully induced Tregs comprising of CD4+CD8+ population, using donor AD-MSC from immunosuppressed recipient PBMC. This novel model will open up the gateway to tolerance in organ transplantation.

Substantial Proliferation of Human Renal Tubular Epithelial Cell–Reactive CD4+CD28null Memory T Cells, Which Is Resistant to Tacrolimus and Everolimus

In spite of maintenance treatment with immunosuppressive drugs, tubulitis still occurs and can lead to structural kidney graft damage. We hypothesize that human renal tubular epithelial cells (TECs) trigger selective proliferation of recipient T-cell subsets with variable sensitivity to immunosuppressive drugs. Recipient peripheral blood mononuclear cells were cocultured with donor-derived TECs for 7 days. The proliferation of the total CD4 T-cell pool was assessed. Next, we analyzed which CD4 T-cell subset proliferated and how this response was affected by tacrolimus, everolimus, prednisolone, and mycophenolic acid (MPA) in clinically relevant concentrations. CD4 T-cell proliferation upon TEC encounter was mainly executed by memory T cells. Interestingly, 38%±7% of the proliferating CD4 T-cell pool showed a CD28 phenotype. These proliferating CD4CD28 memory T cells produced high levels of interferon-γ, tumor necrosis factor-α, and the cytolitic protease granzyme B. TEC-reactive CD4 T-cell proliferation was significantly suppressed by tacrolimus, everolimus, prednisolone, and MPA (P<0.05). Surprisingly and in contrast to prednisolone and MPA, neither tacrolimus nor everolimus could inhibit the CD4CD28 T-cell proliferative response. Our data show substantial proliferation of TEC-reactive CD4CD28 memory T cells, which are resistant to tacrolimus and everolimus. This phenomenon might play an important mechanistic role during cellular rejection under full immunosuppression.