Cytokine Deprivation Research Articles

Ubiquitin Ligase SCF-FBXO11 Controls a Network of RNA-Binding Proteins and Splicing in MDS

The F-Box protein FBXO11 is a substrate receptor of Skp-Cullin-F-Box (SCF) ubiquitin ligase complexes. Loss-of-function mutations in FBXO11 are associated with diffuse large B-cell lymphoma, and FBXO11 has been shown to regulate epigenetic drivers of erythroid cell maturation. In our prior study, we found that deletion of FBXO11 improved cell survival under cytokine deprivation in an MDS cell line. Furthermore, we had observed a striking decrease in FBXO11 protein levels in AML patient samples. However, how FBXO11 contributes to myeloid malignancies is unclear. Here, using RNA-sequencing, proteomics, and CRISPR-Cas9 screening, we reveal broad ubiquitylation of RNA-binding proteins (RBPs) and alternative splicing as a mechanism by which deregulation of FBXO11 enhances malignant cell function. In addition, we report the presence of mutations in the uncharacterized N-terminal extension of FBXO11 in both lymphoid and myeloid malignancies. We first observed that heterozygous deletion of FBXO11 increases colony-forming ability of MDS-AML cell lines in vitro whilea2-fold increase in expression results in decreased cell growth and apoptosis. In a transplant model with a retrovirally-driven Runx1 mutation, heterozygous deletion of Fbxo11 exacerbated neutropenia and shortened overall survival. However, depletion of FBXO11 in healthy human and murine HSPCs significantly impaired myeloid progenitor colony-formation, indicating a unique requirement for FBXO11 in myelopoiesis. Based on its tumor suppressive activity, we evaluated whole-exome sequencing data from patient samples of both lymphoid and myeloid malignancies for FBXO11 mutations. This analysis uncovered rare mutations in FBXO11 in myeloid malignancies and revealed a substantial cluster of previously unidentified mutations in the Q-rich, N-terminal extension of FBXO11. We found that this uncharacterized, extended protein isoform is predominantly expressed in both normal and malignant hematopoietic cells. In colony-forming assays, expression of the long isoform and several of the N-terminal mutations inhibited CD34+ progenitor function. On a cellular level, lack of this N-terminal extension in the short isoform resulted in aberrant subcellular localization of FBXO11 within the nucleus. To elucidate the mechanism of FBXO11 in myeloid malignancy, we performed immunoprecipitation of FBXO11 followed by proteomics to identify endogenous FBXO11 complexes in MDS-AML cells. We integrated these data with ubiquitin proteomics of WT and FBXO11-KO MDS cells to identify candidate substrates of SCF-FBXO11-mediated ubiquitylation. FBXO11 complexes are significantly enriched for RNA-binding proteins affecting mRNA splicing, a process commonly deregulated in MDS. FBXO11-KO MDS cells displayed broad depletion of ubiquitylation in a network of RBPs. As expected, targeting FBXO11 did not affect the level of transcripts of interacting proteins; however, there were variable changes in total protein levels indicative of post-transcriptional regulation. We then applied a focused CRISPR-Cas9-KO screen of candidate SCF-FBXO11 substrates in MDS-AML cells depleted of FBXO11. We observed significant bi-directional effects in colony-forming ability mediated by several of the FBXO11-regulated RBPs including TRIM28, HNRNPU, NPM1, and SYNCRIP. Though FBXO11 is significantly decreased in MDS compared to healthy controls, we saw no difference in FBXO11 expression between splicing factor WT and mutant samples, suggesting that the function of FBXO11 in regulating mRNA splicing in MDS is independent of these mutations. Compared to healthy controls, FBXO11-low MDS samples exhibited a greater number of alternative splicing events, predominantly in skipped or mutually-exclusive exons. To experimentally test whether FBXO11 indeed affects RNA splicing, we utilized a bichromatic splicing reporter assay in MDS-AML cells which showed that depletion of FBXO11 is sufficient to alter splicing of the reading frame in the cassette exon, read out as a shift from GFP to RFP. Finally, STRING protein network analysis revealed that the alternative splicing events occurring in FBXO11-low MDS patient samples affect key nodes in protein translation and metabolism. Collectively, our data support a model whereby the change in FBXO11-mediated ubiquitylation of RBPs drives alternative exon usage that exacerbates malignant cell function in MDS.

Allogeneic Veto-CAR CD8 T Cells Enabling Prolonged Graft Survival, Anti-Tumor Specificity, and Low Risk of Graft Versus Host Disease

¥ Equal contribution Studies in mice have demonstrated that CD8 T cells exhibit marked veto activity against host T cells with anti-graft specificity, enhancing engraftment after non-myeloablative T cell depleted mismatched bone marrow transplantation. To reduce the risk of GVHD associated with allogeneic CD8 veto T cells, these studies used naïve CD8 T cells stimulated against 3rd party stimulators under cytokine deprivation and subsequent expansion in the presence of IL-15. Recently, we showed that mouse CD8 veto T cells can be generated by stimulating CD8 memory T cells from ovalbumin-immunized mice under cytokine deprivation, using ovalbumin as a third-party antigen (Or-Geva et al., Leukemia, 2019). Based on these insights, human anti-viral CD8 central memory veto cells were successfully generated from CMV and EBV positive donors. Donor PBMC collected by leukapheresis were initially depleted of CD45RA+, CD4+ and CD56+ cells, and co-cultured with dendritic cells from the same donor, pulsed with a mixture of CMV, EBV, Adeno and BKV viral peptides. Within 12 days of culture, starting with 3 days of cytokine deprivation, more than 3x10 9 highly homogenous CD8 T cells, predominantly expressing a central memory phenotype could be routinely harvested. These cells exhibited marked veto activity in-vitro with more than a 3 log-depletion of alloreactivity as measured by limiting dilution analysis. Based on these results, a first in human phase 1-2 clinical trial is currently in progress, testing the safety of such veto cells in the context of non-myeloablative haploidentical T cell-depleted hematopoietic stem cell transplantation (HSCT). Preliminary results indicate that infusion of 5-10x10 6 veto cells / kg supports engraftment with a low risk of GVHD (Champlin et al. Blood, 2022). Most attempts to use allogenic chimeric antigen receptor (CAR) T cells involve gene editing, knocking down the TCR to avoid GVHD, and eliminating MHC expression to avoid graft rejection. Our veto cell preparation could potentially offer an attractive platform for attaining allogeneic CAR T cells capable of deleting host anti-donor T cell clones by virtue of their veto activity, with a very low risk for GVHD and without the need of gene editing. Using our current protocol for production of anti-viral central memory veto CD8 T cells, we first evaluated transduction efficacy at different time points during the veto production using a retroviral CAR directed against the Her-2 antigen (N29). The scFv N29 was attached to a costimulatory determinant of CD28 and a T cell activating domain followed by a GFP reporter gene. Our results suggested that optimal CAR transduction was attained on day 5 of culture. The veto cell product harvested on day 12 exhibited 92% CD8 T cells, of which 72% expressed the transduced N29 CAR. Notably, the transduced cells continued to exhibit veto activity and functional assays showed killing of target cells in a dose dependent manner. Next, we performed similar experiments transducing veto Tcm with a clinical grade gammaretroviral vector encoding a second-generation 4-1BB co-stimulated CD19-specific CAR. A typical experiment, shown in Fig.1, demonstrates a transduction level of 66.7 % (Fig. 1B,) and confirms the central memory phenotype of the transduced cells (Fig. 1C). Notably, upon infusion of 3x10 6 veto-CD19CAR into NSG mice implanted 3 days earlier with 0.25x10 6 Nalm-6-Luc+ tumor cells (CD19+), a marked eradication of tumor cells (Fig.1D) and survival benefit (Fig.1E) were found, while infusion of 3x10 6 non-transduced veto cells were ineffective. Based on these results, we plan to initiate clinical trials using haploidentical VETO-CD19CAR for the treatment of B-cell malignancies, in combination with hematopoietic transplantation and as off the shelf cellular therapy. .

Generation of Non-Alloreactive Antiviral Central Memory CD8 Human Veto T Cells for Cell Therapy

Previous studies in mice demonstrated that CD8 T cells exhibit marked veto activity enhancing engraftment in several models for T cell-depleted bone marrow (TDBM) allografting. To reduce the risk of graft-versus-host disease (GVHD) associated with allogeneic CD8 veto T cells, these studies made use of naive CD8 T cells stimulated against third-party stimulators under cytokine deprivation and subsequent expansion in the presence of IL-15. More recently, it was shown that mouse CD8 veto T cells can be generated by stimulating CD8 memory T cells from ovalbumin immunized mice under cytokine deprivation, using ovalbumin as a third-party antigen. These cells also exhibited substantial enhancement of BM allografting without GVHD. In this study, we tested the hypothesis that stimulation and expansion of human CD8 memory T cells under IL-15 and IL-7 deprivation during the early phase of activation against recall viral antigens can lead to substantial loss of alloreactive T clones while retaining marked veto activity. Memory CD8 T cells were enriched by removal of CD45RA+, CD4+, and CD56+ cells from peripheral blood of cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-positive donors. In parallel, CD14+ monocytes were isolated; differentiated into mature dendritic cells (mDCs); pulsed with a library of CMV, EBV, adenovirus, and BK virus peptides; and irradiated. The CD8 T cell-enriched fraction was then cultured with the pulsed mDCs in the presence of IL-21 for 3 days, after which IL-15 and IL-7 were added. After 12 days of culture, the cells were tested by limiting dilution analysis for the frequency of alloreactive T cell clones and their veto activity. In preclinical runs using GMP reagents, we established that within 12 days of culture, a large number of highly homogenous CD8 T cells, predominantly expressing a central memory phenotype, could be harvested. These cells exhibited marked veto activity in vitro and >3-log depletion of alloreactivity. Based on these preclinical data, a phase 1-2 clinical trial was initiated to test the safety and efficacy of these antiviral CD8 central memory veto cells in the context of nonmyeloablative (NMA) T cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT). In 2 validation runs and 11 clinical runs using GMP reagents, >1×1010 cells were generated from a single leukapheresis in 12 out of 13 experiments. At the end of 12 days of culture, there were 97 ± 2.5% CD3+CD8+ T cells, of which 84 ± 9.0% (range, 71.5% to 95.1%) exhibited the CD45RO+CD62L+ CM phenotype. Antiviral activity tested by intracellular expression of INF-γ and TNF-α and showed an average of 38.8 ± 19.6% positive cells on 6 hours of stimulation against the viral peptide mixture. Our results demonstrate a novel approach for depleting alloreactive T cell clones from preparations of antiviral CD8 veto cells. Based on these results, a phase 1-2 clinical trial is currently in progress to test the safety and efficacy of these veto cells in the context of NMA haploidentical T cell-depleted HSCT. Studies testing the hypothesis that these non-alloreactive CD8 T cells could potentially offer a platform for off-the-shelf veto chimeric antigen receptor T cell therapy in allogenic recipients, are warranted.

The potential role of infusions of T regulatory cells in inducing and maintaining liver allograft tolerance.

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Nfix  Promotes Survival of Immature Hematopoietic Cells Via Regulation of C-Mpl

Hematopoietic stem and progenitor cells (HSPC) are necessary for life-long blood production and replenishment of the hematopoietic system during stress. We recently identified the transcription factor nuclear factor I/X (Nfix) as a critical regulator of hematopoietic stem and progenitor cell (HSPC) survival post-transplantation. Nfix -depleted HSPC are lost from the marrow between 7-10 days post-transplant and residual Nfix -depleted HSPC display elevated levels of apoptosis at these time-points. To further characterize the anti-apoptotic role of Nfix in HSPCs, we overexpressed Nfix in Lineage- Sca-1+ c-Kit+ (LSK) cells that were then maintained ex vivo under serum-free conditions with supportive cytokines and monitored the cultures for cell surface phenotype, growth, vector selection, and apoptosis. Ectopic expression of Nfix in primary mouse HSPCs had no significant effect on the expression of lineage markers compared to controls, but did appear to accelerate the loss of immunophenotypic LSK cells, as well as colony forming units (p = 0.023). Nfix -overexpressing HSPCs were extended in their ex vivo culture lifespan from about 20 days to 40 days, and were steadily selected for in culture. HSPCs overexpressing Nfix also displayed hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation compared to controls (p = 0.032). NFIX has been shown to potentially influence transcription of several known regulators of HSC biology. Here, we show that ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPCs as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL. Consequently, Bcl-XL, an anti-apoptotic factor induced by STAT5, was also significantly upregulated in Nfix -overexpressing cells by two weeks of culture compared to controls (p = 0.0038). Blocking c-MPL signaling by removal of thrombopoietin or addition of a c-MPL neutralizing antibody significantly negated the anti-apoptotic effect of Nfix overexpression on cultured HSPCs (p = 0.045, p = 0.0098, respectively). Further, NFIX was capable of transcriptional activation of a proximal c-Mpl promoter fragment (p = 0.0056). In sum, these data suggest that NFIX-mediated up-regulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo . DisclosuresNo relevant conflicts of interest to declare.

Maternal Deprivation and Sex Alter Central Levels of Neurotrophins and Inflammatory Cytokines in Rats Exposed to Palatable Food in Adolescence

Maternal deprivation (MD) in rodents is used to simulate human-infant early life stress, which leads to neural, hormonal, and behavioral alterations. Palatable food (PF) can reduce the stress response, and individuals use it as a self-applied stress relief method. Thus, the present study aimed to evaluate the effect of the association between MD in the early life (P1–P10) and PF consumption (condensed milk, P21–P44) in the central neuroplasticity (BDNF/NGF levels) and central neuroinflammatory parameters (TNF-α, IL-6, and IL-10 levels) in male and female Wistar rats in the adolescence. In addition, weight-related parameters (weight gain, Lee Index, and relative adipose tissue weight) were evaluated. PF exposure increased relative adipose tissue weight; however, it did not lead to a change in animals’ body weight. MD reduced hypothalamic BDNF and NGF levels, and hippocampal TNF-α levels in male and female rats. Animals of both sexes that received PF, exhibited reduced hypothalamic NGF levels. Neuroinflammatory marker evaluations showed that male rats were more susceptible to the interventions than female rats, since MD reduced their cortical IL-10 levels and PF increased their IL-6 levels. Differences in the Lee index, central BDNF, TNF-α, and IL-6levels were observed between sexes. Male animals per se presented greater Lee index. Female rats had higher BDNF and IL-6 levels in the hippocampus and hypothalamus and higher hypothalamic TNF-α levels than those observed in males. In conclusion, there were more noticeable effects of MD than PF on the variables measured in this study. Sex effect was identified as an important factor and influenced most of the neurochemical measures in this study. In this way, we suggest including both female and male animals in researches to improve the quality of translational studies.

Towards Safer Allogeneic HSCT in Autoimmunity: Proof of Concept in Diabetes-Prone Mice

R.S.K and B.N.L contributed equally to this study. The induction of tolerance towards pancreas autoantigens is a promising approach in the treatment of type 1 diabetes mellitus (T1DM). This goal can be partially attained by immuno-ablation followed by autologous HSCT which mitigates the immune reaction leading to diabetes, at least temporarily, if performed soon after diagnosis. However, most T1DM patients transplanted with autologous HSCT eventually relapse (E Snarski et al., Bone marrow Transplantation; 2016; 51, 398-402). Thus, developing methodologies for allogeneic HSCT, to provide a durable non-autoimmune TCR repertoire, could be a promising treatment approach provided that such protocols can safely achieve donor type chimerism. Accordingly, transplantation of T cell-depleted allogeneic HSCT (TD-HSCT) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option. However, overcoming rejection after reduced conditioning in T1DM represents a major challenge. We previously demonstrated in wild type mice that rejection of TD-HSCT can be prevented using donor-derived veto cells. Here, we show proof of concept of the safety and efficacy of veto cell mediated non-myeloablative mismatched allogeneic TD-HSCT, in the established NOD mouse T1DM model. Veto activity, first defined by Miller (Miller, R. G; Nature; 1980; 287; 544-54), is based on the ability of specific cell populations to attack host CTL-precursors (CTLp) that are directed against the antigens presented by the veto cells themselves. This response spares cells that are not targeted against the veto cells, including those recognizing pathogens. Among different veto cell populations described in the literature, central memory CD8 T cells exhibit the most robust veto activity upon transplantation, but are also endowed with marked GvH activity. We overcame GvH reactivity by expanding naïve or memory CD8 T cells against 3rd party MHC or viral antigens, under culture conditions favoring expression of the central memory phenotype. Such anti-3rd party central memory CD8 T cells (Tcm) are endowed with marked veto activity, while effectively depleted of GVH reactivity in fully mis-matched recipients (Reviewed in Reisner Y, Or-Geva N. Semin. Hematol. 2019; 56(3): 173-182). In this study, Tcm veto cells were generated from splenocytes obtained from Balb/c donors (H2d) cultured against irradiated third-party splenocytes (FVB; H2q), under cytokine deprivation. The selective expansion of CD8 mouse T cells against 3rd party stimulators under these conditions leads to selective 'death by neglect' of bystander anti-host T cell clones that could mediate GvHD; such cells are further diluted out by subsequent expansion of anti-3rd party T clones during further culture in the presence of IL15. In addition to causing selective loss of GvH reactive T cells, these culture conditions induce a central memory phenotype shown to be crucial for robust veto activity in vivo. To evaluate the safety and efficacy of such a transplant for diabetes therapy, 8 week old NOD mice (before diabetes onset) were treated with 4.5 Gy TBI conditioning at day -1, anti-3rd party veto Tcm and megadose nude bone marrow (NuBM) on day 0, and Rapamycin treatment from day -1 to day +4 (Scheme 1). Controls were untreated or received conditioning with no transplant. In four independent experiments (n= 35 NOD mice in the transplanted group), high chimerism from 83.5% to 99.6 % was found in all transplanted mice at 6 months post-transplant. Notably, with a follow-up of 200 days, 72.4% mice in the untreated group, and 96% in the conditioned group died of diabetes, while only 8.5% diabetes-associated mortality (Fig.1A) and no GvHD or other transplant-related mortality was observed in the transplanted mice (Fig.1B). Our results demonstrate a proof of concept for the safety and efficacy of non-myeloablative allogeneic TD-HSCT in type 1 diabetes. A clinical protocol testing the safety and efficacy of anti-3rd party veto cells in the context of low toxicity non-myeloablative TD-HSCT in hematological malignancies is commencing at MD Anderson Cancer Center. If successful, our results support further extension of this platform to autoimmune diabetes and other autoimmune diseases. Disclosures Lustig: Yeda Ltd.: Patents & Royalties. Reisner:Cell Source, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Proof of Concept for Repair of Sickle Cell Disease By Non-Myeloablative MHC Disparate T Cell Depleted HSCT Combined with Donor-Derived Veto Cells

Although life extending medical treatments are available for sickle cell disease (SCD), allogenic hematopoietic stem cell transplantation (HSCT) is considered a treatment of choice (Ozdogu et al., Bone Marrow Transplant 2018; 53(7): 880-890). However, HSCT is associated with several limitations, including conditioning-related toxicity and graft-versus-host disease (GvHD), especially when using MHC disparate transplants. Thus, the development of safe transplantation protocols for MHC disparate HSCT in sickle cell disease is vital. While the risk of GvHD and conditioning toxicity can be effectively reduced by the use of T-cell-depleted HSCT (TD-HSCT) under reduced intensity conditioning (RIC), rejection of TD-HSCT remains a challenge. We previously demonstrated in wild type mice that this barrier can be overcome using donor-derived veto cells. Here, we demonstrate the safety and efficacy of this approach in a well-defined murine model for sickle cells disease. Veto activity is based on the ability of certain cells to attack host CTL-precursors (CTLp) which are directed against antigens expressed on the veto cells themselves, sparing cells that are not targeted against the veto cells including T cells needed for defense against pathogens. Central memory CD8 T cells exhibit the most robust veto activity upon transplantation; however, these cells are also endowed with marked GvH activity. We overcame this issue by expanding naïve or memory CD8 T cells against 3rd party MHC or viral antigens, respectively, under culture conditions favoring expression of central memory phenotype. Such anti-3rd party central memoryCD8 T cells (Tcm), which are endowed with marked veto activity, also exhibit reduced risk for GvHD in fully mis-matched recipients (Reviewed in Reisner Y, Or-Geva N. Semin Hematol. 2019; 56(3): 173-182.) To generate Tcm veto cells, splenocytes obtained from Balb/c donors (H2d) were cultured against irradiated third-party splenocytes (FVB; H2q) under cytokine deprivation. The selective expansion of CD8 mouse T cells against 3rd party stimulators leads to selective 'death by neglect' of bystander anti-host T cell clones potentialy mediating GvHD, and these are further diluted out by subsequent expansion of anti-3rd party T clones during continued culture in the presence of IL-15. Apart from selective loss of GvH reactive T cells, these culture conditions induce a central memory phenotype shown to be important for attaining robust veto activity in vivo (Ophir et al., Blood. 2013; 121(7): 1220-8). We first calibrated the optimal irradiation dose for sickle mice (Berkeley model, H2b), comparing 4.5 Gy versus 5 Gy TBI in a conditioning protocol also including short term rapmycin treatment (Scheme 1). Higher levels of engraftment and chimerism were found in the group receiving 5Gy TBI (Fig. 1). All mice of both treatment groups survived >140 days with no evidence of GvHD. To further evaluate this treatment approach, 8 week old sickle mice (N=7) were given bone marrow transplants using the protocol described in Scheme 1, including conditioning with 5 Gy TBI (day -1), rapamycin treatment (day -1 to day 4), and transplantation of NuBM (day 0) plus veto cells (day 7; Scheme 1). Notably, at 44 days post-transplant, 6 out of 7 mice receiving NuBM + TCM + Rapa (85.7%) showed donor chimerism in the peripheral blood, ranging between 77-94% (Fig.2A-B), while no chimerism was detected in mice receiving conditioning alone, or conditioning and transplantation with only NuBM or only veto cells . All mice in all groups survived (N=26) , and no GvHD was detected with a follow up of 77 days, even in the transplanted group which exhibited high donor-derived chimerism. Furthermore, reversal of sickle disease symptoms was observed, including reticulocyte levels (p=0.001;Fig.2C) and expression of wild type hemoglobin ( Fig.2D) in all engrafted mice. Our results offer a proof of concept for the treatment of sickle disease by MHC disparate non-myeloablative T cell depleted HSCT in conjunction with anti-3rd party central memory veto CD8 T cells. A clinical trial testing the safety and efficacy of anti-3rd party veto cells in the context of low toxicity non-myeloablative TD-HSCT in hematological malignancies is currently ongoing at MD Anderson Cancer Center. If successful, our present results support further evaluation of this platform for sickle cell disease. Disclosures Lustig: Yeda Ltd.: Patents & Royalties. Reisner:Cell Source, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Fibroblastic reticular cells at the nexus of innate and adaptive immune responses.

SummaryLymphoid organs guarantee productive immune cell interactions through the establishment of distinct microenvironmental niches that are built by fibroblastic reticular cells (FRC). These specialized immune‐interacting fibroblasts coordinate the migration and positioning of lymphoid and myeloid cells in lymphoid organs and provide essential survival and differentiation factors during homeostasis and immune activation. In this review, we will outline the current knowledge on FRC functions in secondary lymphoid organs such as lymph nodes, spleen and Peyer's patches and will discuss how FRCs contribute to the regulation of immune processes in fat‐associated lymphoid clusters. Moreover, recent evidence indicates that FRC critically impact immune regulatory processes, for example, through cytokine deprivation during immune activation or through fostering the induction of regulatory T cells. Finally, we highlight how different FRC subsets integrate innate immunological signals and molecular cues from immune cells to fulfill their function as nexus between innate and adaptive immune responses.

Role of Exosomes in the Regulation of T-cell Mediated Immune Responses and in Autoimmune Disease.

T-cell mediated immune responses should be regulated to avoid the development of autoimmune or chronic inflammatory diseases. Several mechanisms have been described to regulate this process, namely death of overactivated T cells by cytokine deprivation, suppression by T regulatory cells (Treg), induction of expression of immune checkpoint molecules such as CTLA-4 and PD-1, or activation-induced cell death (AICD). In addition, activated T cells release membrane microvesicles called exosomes during these regulatory processes. In this review, we revise the role of exosome secretion in the different pathways of immune regulation described to date and its importance in the prevention or development of autoimmune disease. The expression of membrane-bound death ligands on the surface of exosomes during AICD or the more recently described transfer of miRNA or even DNA inside T-cell exosomes is a molecular mechanism that will be analyzed.

Graft-Vs-Host Reactivity Against the Bone Marrow Is Directed Against the Hematopoietic and Non-Hematopoietic Compartments in Mice

Hematopoietic dysfunction is frequently observed following allogeneic hematopoietic cell transplantation (allo-HCT) and contributes to substantial morbidity. Various factors, e.g. viral infections, poor graft-function, and certain drugs can cause cytopenias, but usually, graft-vs-host disease (GVHD) of the bone marrow (BM) is not taken into account as a major cause of hematopoietic dysfunction, particularly when no other signs of GVHD in classical target organs are present.Here, we studied in an MHC-matched, minor antigen mismatched HCT model (C3H.SWàC57BL/6 (B6)) the effects of lethal irradiation and allo-HCT of HSC (cKIT+Sca1+Lin-) alone or in combination with T cells (TC) on the hematopoietic compartment (HC) and non-hematopoietic compartment (NHC) of the BM. FACS analysis was used to assess relative and absolute cell counts of hematopoietic and non-hematopoietic populations. Confocal microscopy was used to establish 3D images of structural integrity vs. disruption, and qRT-PCR was used to examine functional alterations of gene expression within NHC populations.In this HCT model mice did not develop overt signs of acute GVHD. In recipients of HSC+TC recovery of hemoglobin (Hb) and neutrophils (Nph) was significantly delayed beyond d+56, while Hb and Nph were normalized by d+21 in recipients of pure HSC. Recovery of BM cell counts was also faster in HSC recipients, whereas addition of TC to the graft delayed reconstitution of total BM counts, BM B cells and BM neutrophils significantly. Not only the HC was affected by alloreactive TC, but also non-hematopoietic populations, including CXCL12-abundant reticular (CAR) cells (CD45-Ter119-CD31-CD140b+) and endothelial cells (EC; CD45-Ter119-CD31+) declined post-HCT, and showed a significantly slower recovery in mice given allogeneic TC within their HCT. 3D-confocal microscopy confirmed the alloreactive damage to the NHC, as imaging revealed rapid recovery of CAR cells in B6.CXCL12-GFP recipients of pure HSC, whereas regeneration of CAR cells was impaired in mice given HSC+TC. Likewise, the extracellular matrix and sinusoidal vascular structures recovered promptly in the HSC group, whereas HSC+TC recipients displayed severe disruption of the structural integrity with impaired recovery of the BM microvessels. Increased microvascular permeability was shown in all transplanted (and irradiated) mice on d+7 post-HCT by Evans blue dye extravasation assay. Recovery from leakiness was faster in mice transplanted with HSC alone compared with those given alloreactive TC. Of note, confocal microscopy revealed that in recipients of alloreactive TC hematopoietic cell content was replaced by an abundance of adipocytes for a prolonged period of time (Figure 1A). To further elucidate functional alterations of the NHC in the context of HCT with/without TC we FACS-isolated CAR cells and EC from HSC and HSC+TC recipients on d+14 and d+28 post-HCT and performed qRT-PCR of candidate genes in these populations. In HSC+TC recipients both CAR cells and EC showed significantly lower relative expression levels of SCF (stem cell factor, ligand to cKIT), CXCL12 (C-X-C-motif chemokine 12, = SDF-1, a cytokine with various functions in hematopoiesis and lymphopoiesis), and IL-7 (a growth factor that stimulates the differentiation of pluripotent HSC into lymphoid progenitors) compared with mice transplanted with HSC only. Rather, in CAR cells of HSC+TC recipients adiponectin and PPARγ (peroxisome proliferator-activated receptor γ, stimulates lipid uptake and adipogenesis) were significantly higher compared with HSC only recipients (Figure 1B). These findings suggest that alloreactive TC promote adipogenesis while at the same time suppressing hematopoiesis via deprivation of essential cytokines and growth factors required for HSC function and maturation.Delayed hematopoietic regeneration post allo-HCT represents a major problem in clinical patient care. GVH reactions of the BM have been poorly characterized. Our experiments help to further characterize “marrow GVHD” and its impact on hematopoietic output. We illustrate the damage that alloreactive TC can have on both hematopoiesis directly but also on the nonhematopoietic compartment and its critical functions in stimulating and supporting hematopoietic regeneration. DisclosuresNo relevant conflicts of interest to declare.

Nfix Promotes Survival of Immature Hematopoietic Cells via Regulation of c-Mpl.

Hematopoietic stem and progenitor cells (HSPCs) are necessary for life-long blood production and replenishment of the hematopoietic system during stress. We recently reported that nuclear factor I/X (Nfix) promotes HSPC survival post-transplant. Here, we report that ectopic expression of Nfix in primary mouse HSPCs extends their ex vivo culture from about 20 to 40 days. HSPCs overexpressing Nfix display hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation relative to controls. Ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPCs as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL. Blocking c-MPL signaling by removal of thrombopoietin or addition of a c-MPL neutralizing antibody negated the antiapoptotic effect of Nfix overexpression on cultured HSPCs. Furthermore, NFIX was capable of binding to and transcriptionally activating a proximal c-Mpl promoter fragment. In sum, these data suggest that NFIX-mediated upregulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo. Stem Cells 2018;36:943-950.

A Flow Cytometry Analysis of Apoptosis Reversibility in Mammalian Lymphocytes Following Cytokine Deprivation

Apoptosis is a form of programmed cell death that occurs in multicellular organisms in which cells are dismantled and removed in a controlled manner, avoiding harm to surrounding tissues. Apoptotic cell death involves a series of characteristic morphological and biochemical changes that occur in specific temporal fashion, and include early/intermediate (i.e.: caspase activation and phosphatidylserine externalization) and late (i.e.: internucleosomal DNA fragmentation) events. Previous studies carried out in cancer cell lines treated with various apoptotic inducers revealed that surprisingly, apoptosis was reversible and that cells could recover and resume growth following removal of the inducer. In this study, we have developed a mammalian model of apoptosis rescuability and reversibility using cultured mouse HT‐2 T lymphocytes deprived of the essential growth and survival factor interleukin 2 (IL2). Using quantitative flow cytometry analysis, we demonstrate that HT‐2 cells accumulate in the G1 phase of the cell cycle by 24 hours following IL2 deprivation prior to undergoing DNA fragmentation and death within 36–48 hours. Our findings further indicate that IL2‐deprived HT‐2 lymphocytes can be rescued from apoptosis by re‐addition of IL2 to the culture medium. We show that cellular morphology and cell cycle re‐entry is restored within 24–36 hours. Experiments are currently underway to determine which events along the apoptotic pathway (caspase activity, internucleosomal DNA fragmentation/TUNEL assay, and phosphatidylserine externalization/Annexin V binding) are congruent with reversibility.Support or Funding InformationAll funding provided by the Internal Education Fund (Union College).

Saponins from Sanguisorba officinalis Improve Hematopoiesis by Promoting Survival through FAK and Erk1/2 Activation and Modulating Cytokine Production in Bone Marrow.

Radix Sanguisorbae, the root of Sanguisorba officinalis L. is used as traditional Chinese medicine. In recent decades, it has been reported to be clinically effective against myelosuppression induced by chemotherapy and/ or radiotherapy. However, the underlining mechanism has not been well studied. In this work, we evaluated the hematopoietic effect of total saponins from S. officinalis L. on myelosuppressive mice induced by cyclophosphamide and by60Co-γ-irradiation and confirmed the therapeutic effect. Then, we found total saponins and their characteristic constituents Ziyuglycoside I and Ziyuglycoside II can inhibit apoptosis of TF-1 cells caused by cytokine deprivation, and promote survival of mouse bone marrow nuclear cells through focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (Erk1/2) activation in vitro. In addition, they can down-regulate macrophage inflammatory protein 2 (MIP-2), platelet factor 4 (PF4) and P-selectin secretion, which are reported to be suppressive to hematopoiesis, both in vitro and in vivo. These results suggest that promotion of survival through FAK and Erk1/2 activation and inhibition of suppressive cytokines in the bone marrow is likely to be the pharmacological mechanism underlying the hematopoietic effect of saponins from S. officinalis L.

Next Generation Veto Cells for Non-Myeloablative Haploidentical HSCT: Combining Anti-Viral and Graft Facilitating Activity

Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders or can be used to induce immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. We previously demonstrated that this barrier can be overcome using donor-derived naïve CD8 T cells if they are expanded against third-party alloantigens under culture conditions inducing a central memory phenotype. Thus, such anti-3rd party CD8 T cells (Tcm) exhibit marked veto activity and reduced risk for GVHD due to the lower frequency of anti-host clones achieved during the culture period. In the present study, we tested the feasibility of generating veto Tcm by stimulation against well-defined peptides including viral antigens. For proof of concept, we used OT1 mice that express a transgenic (Tg) TCR designed to recognize ovalbumin (OVA) residues 257-264 in the context of H2Kb MHC-I. Prior to harvest of OT1 CD8+ T cells, mice were immunized twice with OVA-peptide. Mice were sacrificed 7 to 14 days after immunization, their spleens and lymph nodes removed and crushed, and magnetic bead sorting utilized to isolate the memory cells (CD8+CD44+). The resulting population was subjected to third-party stimulation by co-culture with irradiated splenocytes generated from spleens of OVA-expressing mice, under cytokine deprivation. hIL-15 (10ng/ml) was added to the culture 60 hrs after culture initiation to induce the cells to express a Tcm like phenotype, as previously described for anti-3rd party Tcm generated from WT naïve CD8 T cells (WT Tcm).When tested in vivo, the OT-1 Tcm (H2Kb) were able to enhance engraftment of allogeneic T cell depleted BMT (H2Kb) in sub-lethally irradiated (5.5 Gy TBI) Balb/c recipients (H2Kd), in analogy to the chimerism induced by WT Tcm. This initial successful experiment was then followed by an experiment, which more closely resembled the human setting, in which the CD8+CD44+ cells were isolated from WT C57BL/6 OVA-immunized mice and subsequently introduced to co-culture with irradiated splenocytes generated from spleens of OVA-expressing mice. Results showed that Tcm grown from such a starting population of CD8+CD44+ cells were also able to achieve marked donor chimerism when administered with megadose of T cell depleted (TCD) alloSCT in sublethally irradiated Balb/c recipients (Fig.1A). Notably, infusion of 5x106 Tcm into sublethally irradiated mice (5.5. GY TBI) without BMT, did not cause any GVHD as measured by weight loss, in contrast to CD8+CD44+ T cells used to generate the Tcm (Fig.1B). These results demonstrate that CD8+CD44+ derived from memory CD8 T cells by expansion against cognate peptides exhibit markedly reduced risk for GVHD compared to freshly isolated memory cells, while retaining their veto activity and inducing tolerance.Finally, based on these proof of concept studies, we were able to translate this approach and generate human anti-viral CD8 veto cells with central memory phenotype. Thus, CD8+CD45RO+ memory T cells were selected by depletion of CD4+CD56+CD45RA+ cells from PBMC of normal donors and then co-cultured with donor DCs pulsed with a viral peptide mixture of three prominent viruses (EBV, CMV and Adenovirus). In three large scale experiments using leukapheresis preparations of normal CMV and EBV positive donors, with GMP grade reagents, more than 1x109 Tcm could be attained by the end of 9 days of culture (average expansion of Tcm=13.5±4 fold) with greater than 90% purity of CD45RO+CD62L+CD8+ T cells (Fig 2).The harvested anti-viral Tcm exhibited more than 3 log depletion of alloreactivity, compared to fresh CD8 T cells, as measured by limit dilution analysis of cytotoxic T cell precursors against host type target cells (Fig. 3).In conclusion, our results suggest that potent veto CD8 Tcm can be generated from the memory pool of donors positive for viral reactivity by stimulation against viral antigens. Such veto Tcm could be most attractive for haploidenitcal transplantation of TCDBM, as they can enable engraftment following non-myeloablative conditioning and at the same time provide anti-viral protection.*E.B.L and N.O.G contributed equally [Display omitted] [Display omitted] [Display omitted] DisclosuresBachar Lustig:Yeda LTD: Patents & Royalties. Or Geva:Yeda LTD: Patents & Royalties. Lask:Yeda LTD: Patents & Royalties. Gidron:Yeda LTD: Patents & Royalties. Kagan:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

USP10 Is an Essential Deubiquitinase for Hematopoiesis and Inhibits Apoptosis of Long-Term Hematopoietic Stem Cells.

SummarySelf-renewal, replication, and differentiation of hematopoietic stem cells (HSCs) are regulated by cytokines produced by niche cells in fetal liver and bone marrow. HSCs must overcome stresses induced by cytokine deprivation during normal development. In this study, we found that ubiquitin-specific peptidase 10 (USP10) is a crucial deubiquitinase for mouse hematopoiesis. All USP10 knockout (KO) mice died within 1 year because of bone marrow failure with pancytopenia. Bone marrow failure in these USP10-KO mice was associated with remarkable reductions of long-term HSCs (LT-HSCs) in bone marrow and fetal liver. Such USP10-KO fetal liver exhibited enhanced apoptosis of hematopoietic stem/progenitor cells (HSPCs) including LT-HSCs but not of lineage-committed progenitor cells. Transplantation of USP10-competent bone marrow cells into USP10-KO mice reconstituted multilineage hematopoiesis. These results suggest that USP10 is an essential deubiquitinase in hematopoiesis and functions by inhibiting apoptosis of HSPCs including LT-HSCs.

Abstract 1291: Tumor-infiltrating FOXP3+ T-regulatory (Treg) cells in early-stage human lung cancer exhibit enhanced suppressive function when compared to blood or lymph node (LN) Treg cells

Abstract Aim: Advances in immunotherapy require a detailed understanding of host immunity. Data from murine models indicate solid tumors of various types can recruit Tregs and/or promote iTreg development, and thereby curtail antitumor responses. However, clinical data have been restricted to either evaluation of Treg numbers in tumors, or studies of PBMC Treg in patients with cancers. Surprisingly, no studies of Treg function have ever been reported using Tregs isolated from tumors themselves. Methods: Using our recently reported technique, we isolated Tregs within 20 mins of resection from 13 lung cancer patients, stages Ia-IIIa, and 2 control non-cancer patients (diaphragmatic hernia and lung granuloma). The mean age of the cancer patients (10 males, 3 females) was 65.4±2.2 years; 7 patients had adenocarcinoma, 3 had squamous cell carcinoma, and the remainder had large cell neuroendocrine carcinoma, colon adenocarcinoma and high-grade carcinoma. The mean size of their tumors was 4.0±0.8 cm, and 1 of the 13 patients developed metastases during the follow-up period. We compared the suppressive function of Tregs isolated from each patient's freshly resected tumor, LN and blood, using cryopreserved healthy donor PBMC aliquots as responders, and area-under-curve (AUC) analysis. We controlled for comparable CD4+ and FOXP3+ purity in all isolated Treg samples, and cell viability, excluding from further analysis all data from non-comparable FOXP3+ subsets. Thus, we were able to avoid artifacts arising from different cell conditions (e.g. use of autologous responder cells) or from unequal FOXP3+ content in Treg isolates. Results: Tumor-infiltrating Tregs had significantly enhanced suppressive function when compared with Tregs isolated from other locations in the same patient. Thus, tumor Tregs were significantly more suppressive than lung LN Tregs (p = 0.0003), and significantly more suppressive than blood Tregs (p = 0.03). Tregs isolated from LN in close proximity to tumors had comparable suppressive function to Tregs isolated from distant LN (p>0.05), and blood Tregs had comparable suppressive function to that of LN Tregs (p>0.05). Additional analyses showed that the enhanced suppressive function of tumor Treg was not due to Treg-induced killing/apoptosis of responder cells, cytokine and/or growth factor deprivation, or soluble factors produced by tumor Treg cells. Conclusions: We demonstrate for the first time that tumor-infiltrating human lung cancer Treg have significantly increased suppressive function. This finding is important for the development of effective anti-tumor therapy. While we are continuing to study the basis for such differences, our data suggest that studies of peripheral blood Tregs in cancer patients may be inadequate and inefficient approaches for understanding the fundamental mechanisms of limited host antitumor immune responses. Citation Format: Tatiana Akimova, Evgeniy B. Eruslanov, Pratik S. Bhojnagarwala, Jon G. Quatromoni, Jacqueline Morgen, Sunil Singhal, Steven M. Albelda, Wayne W. Hancock. Tumor-infiltrating FOXP3+ T-regulatory (Treg) cells in early-stage human lung cancer exhibit enhanced suppressive function when compared to blood or lymph node (LN) Treg cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1291. doi:10.1158/1538-7445.AM2015-1291

Flow Cytometry Cell Cycle Analysis of Apoptotic Mouse HT‐2 T Lymphocytes

Apoptosis is an essential form of programmed cell death (PCD) in multicellular animals, in which cells are dismantled and disposed of in a controlled fashion. In the vertebrate immune system, apoptosis prevents the emergence of autoreactive lymphocytes, and is essential for self‐tolerance. In mouse HT‐2 T lymphocytes, apoptosis and internucleosomal DNA fragmentation can be initiated by cytokine deprivation (interleukin‐2 or IL‐2), addition of the protein kinase inhibitor staurosporine, or addition of the topoisomerase inhibitor camptothecin. We used flow cytometry to determine if apoptosis in HT‐2 cells involved accumulation in a specific phase of the cell cycle, and whether the mode of induction affected cell cycle distribution. Our findings indicate that IL‐2 deprivation induces cell cycle arrest in the G1 phase, prior to the accumulation of cells with sub‐G1 DNA content. In contrast, HT‐2 cells treated with staurosporine and camptothecin accumulated in G2/M and S phase, respectively. We used western blotting and quantitative chemiluminescence to demonstrate that Kip‐1 (p‐27), a CDK‐inhibitor that regulates the progression of cells from G1 to S phase, is up‐regulated in a time‐dependent fashion following IL‐2 deprivation and staurosporine. When compared to HT‐2 control cultures (+IL‐2) and Beta‐tubulin protein levels, Kip‐1 levels were significantly increased 24 hours following IL‐2 deprivation, and 6 hours post‐staurosporine addition. These findings suggest that Kip‐1 up‐regulation may be involved in multiple apoptotic pathways of T lymphocytes. Studies are currently underway to quantitatively determine protein levels of S (PCNA) and M (phospho‐histone H3) phase‐specific markers in HT‐2 cells in response to apoptotic stimuli.

Transient Bcl-XL Overexpression in Donor Stem Cells Increases Efficacy of Hematopoietic Stem Cell Transplantation without Increasing the Risk of Leukemogenesis

Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for many hematological and immunological diseases but is hampered by the risk of graft failure or delayed engraftment. Clinical experience has shown that these problems can be overcome by transplantation of higher numbers of donor stem cells. This can be achieved by more efficient collection strategies (i.e. mobilization regimens) or by ex vivoexpansion. Here, we aim to transiently inhibit apoptosis in donor hematopoietic stem and progenitor cells (HSPCs) prior to transplantation in order to increase their numbers and their fitness.In previous studies we have identified two Bcl-2 proteins from the pro-apoptotic BH3-only subgroup, Bim and Bmf, to be central players in apoptosis induction of HSPCs during transplantation. Both proteins are efficiently repressed by the cytokines Flt3L and SCF and upregulated under cytokine deprivation in vitro. Lack of either protein or overexpression of their anti-apoptotic antagonists Bcl-2 or Bcl-xL strongly increased HSPC competitiveness during transplantation, both in murine transplantation and human xenotransplantation models. Moreover, less donor HSPCs were required for successful engraftment when Bim-mediated apoptosis was inhibited. In sum, our data indicated that modulation of Bim or Bmf levels inhibits apoptosis in murine and human HSPCs and that the resulting extended life span is beneficial during HSCT (Labi et al, 2013).Inhibition of the intrinsic apoptosis pathway could serve as a novel therapeutic approach to increase resistance of human HSPCs to factor deprivation and other types of stress caused during HSCT. However, permanent apoptosis inhibition in HSPCs can trigger their malignant transformation over time, especially when occurring together with activation of oncogenes promoting cell proliferation. Thus, such apoptosis inhibition needs to be transient when used therapeutically.Here we analyze whether transient apoptosis resistance lasting for only a limited time span is sufficient to increase competitiveness of HSPCs during HSCT. For overexpression of Bcl-xL we used adenoviral vectors known to act transiently. In proliferating murine HSPCs, adenoviral Bcl-xL persisted for 5-7 days, and for this period cells were protected from different stress stimuli engaging the intrinsic apoptosis pathway. Most importantly, adenoviral Bcl-xL overexpression increased the reconstitution potential of murine HSPCs in competitive transplantation experiments. Persistence of adenoviruses was excluded. As expected, transient Bcl-xL overexpression did not accelerate lymphomagenesis, neither on a wildtype nor on a premalignant murine background.However, adenoviral infection was associated with a relevant amount of toxicity to murine HSPCs. We thus performed transfection of full length Bcl-xL protein coupled to a protein transduction domain. Fluorescence microscopy indicated its mitochondrial localization, and functional tests revealed protection from apoptosis. However, when compared to adenoviral overexpression, Bcl-xL protein transduction proved to be less efficient due to the short protein half-life. In vivoexperiments are ongoing and will show whether such short-term apoptosis inhibition is sufficient to increase efficacy of transplantation.In sum, our studies will evaluate the benefit of therapeutic apoptosis inhibition in donor HSPCs during HSCT and contribute to on-going efforts aiming to improve transplantation medicine. DisclosuresNo relevant conflicts of interest to declare.

Impact of conditional deletion of the pro-apoptotic BCL-2 family member BIM in mice.

The pro-apoptotic BH3-only BCL-2 family member BIM is a critical determinant of hematopoietic cell development and homeostasis. It has been argued that the striking hematopoietic abnormalities of BIM-deficient mice (accumulation of lymphocytes and granulocytes) may be the result of the loss of the protein throughout the whole animal rather than a consequence intrinsic to the loss of BIM in hematopoietic cells. To address this issue and allow the deletion of BIM in specific cell types in future studies, we have developed a mouse strain with a conditional Bim allele as well as a new Cre transgenic strain, Vav-CreER, in which the tamoxifen-inducible CreER recombinase (fusion protein) is predominantly expressed in the hematopoietic system. We show that acute loss of BIM in the adult mouse rapidly results in the hematopoietic phenotypes previously observed in mice lacking BIM in all tissues. This includes changes in thymocyte subpopulations, increased white blood cell counts and resistance of lymphocytes to BIM-dependent apoptotic stimuli, such as cytokine deprivation. We have validated this novel conditional Bim knockout mouse model using established and newly developed CreER strains (Rosa26-CreER and Vav-CreER) and will make these exciting new tools for studies on cell death and cancer available.