Aging is associated with an inability to mount protective antibody responses to vaccines and infectious agents. This decline is associated with acquisition of defects in multiple cellular compartments, including B cells. While peripheral B-cell numbers do not decline with aging, the composition of the compartment appears to change, with loss of naïve follicular B cells, accumulation of antigen-experienced cells, and alteration of the antibody repertoire. The underlying cause of this change is unknown. We tested the hypothesis that aging-associated repertoire changes can be attributed directly to decreased B lymphopoiesis. Using an Ig transgenic model to report changes in the B-cell repertoire, we show that the reduced B-cell generative capacity of ''aged'' longterm reconstituting hematopoietic stem cells (LT-HSCs) alters the representation of antigen specificities in the peripheral B-cell repertoire. Further, we show that reconstitution using suboptimal numbers of fully functional LT-HSCs results in the generation of a similarly altered B-cell repertoire. This may be an important factor to consider when deciding the number of bone marrow cells to transplant in the clinical setting. In conclusion, when B lymphopoiesis is limited peripheral B-cell homeostasis is altered. This is reflected in reduced diversity of the B-cell repertoire, which likely reduces the protective quality of the immune response.F ollicular (FO) lymphocytes are the largest B-cell subpopulation in peripheral lymphoid organs and display the maximum breadth of germline-encoded antibody specificity for foreign antigens. Because of the breadth of this repertoire, these naïve FO cells are uniquely suited to give rise to protective primary immune responses and to be selected for high-affinity antibody production.Aging is associated with decreased efficacy of vaccination in both humans and mice, increased susceptibility to infection, and increased rate of cancer (1-4). In mice, where it can be examined, aging is associated with a reduction in FO B cells, but not total B-cell numbers (5, 6). This reduction in FO B-cell numbers is further associated with a reduction in immunoglobulin (Ig) diversity within the B-cell pool and an increase in the frequency of antigen-experienced cells, including marginal zone, B1, and memory cells. In young animals the sizes of these compartments is independently regulated (7). Thus, it seems plausible that the humoral immune defects seen in aging may be the consequence of reduced frequency of FO B cells bearing high-affinity receptors for offensive pathogens.FO B cells are short-lived (estimates ranging from 40 to 120 d) and nonself-renewing and therefore must be constantly replenished by new B cells produced in hematopoietic organs, e.g., adult bone marrow (BM) (8, 9). For reasons that are unclear, transplanted bone marrow stem cells from older adult human donors often do not give rise to B cells in recipients (10). Similarly, LT-HSCs from aged mice, while more numerous than in young animals, are selectively impaired in...
Summary In the absence of EBF1, B cell development is arrested at an uncommitted progenitor stage that exhibits increased lineage potentials. Previously, we investigated the roles of EBF1 (E) and its DNA binding partner Runx1 (R) by evaluating B lymphopoiesis in single (Ehet and Rhet) and compound haploinsufficent (ERhet) mice. Here, we demonstrate that reduced Ebf1 gene dosage results in the inappropriate expression of NK cell lineage-specific genes in B cell progenitors. Moreover, prolonged expression of Ly6a/Sca-1 suggested the maintenance of a relatively undifferentiated phenotype. These effects were exacerbated by reduced expression of Runx1 and occurred despite expression of Pax5. Repression of inappropriately expressed genes was restored in most pre-B and all immature B cells of ERhet mice. Enforced EBF1 expression repressed promiscuous transcription in pro-B cells of ERhet mice and in Ebf1−/−Pax5−/− fetal liver cells. Together, our studies suggest that normal levels of EBF1 are critical for maintaining B cell identity by directing repression of non-B cell-specific genes.
Allogeneic hematopoietic stem cell transplant (HSCT) represents a potential curative treatment for a number of life-threatening blood malignancies. The utility of this treatment regimen, however, is limited by a number of serious complications including graft versus host disease, which occurs in approximately half of all transplant patients. Standard-of-care for treating acute GvHD has remained unchanged for several decades and consists of high doses of steroids, which are only effective in approximately 35 percent of the cases. Therefore, the reduction of GvHD represents a large unmet medical need, and new approaches are needed to effectively attenuate GvHD. Here we present a fundamentally novel strategy for potentially reducing GVHD - by modulating donor mobilized peripheral blood cells with small molecules prior to HSCT, a programmed mobilized peripheral blood (mPB) allogeneic graft, with reduced T-cell alloreactivity, can be administered as the hematopoietic cell source for HSCT. To this end, we applied our screening platform to identify a combination of small molecule modulators (FT1050, FT4145) that promote the activation of genes implicated in cell cycle, immune tolerance and anti-viral properties of T cells, as well as in the survival, proliferation and engraftment potential of CD34+ cells. Genome-wide expression analysis of the T-cell compartment of mobilized peripheral blood following treatment with FT1050+FT4145 revealed the induction of genes involved in cell cycle (e.g., CCND1, CCNE1), immune tolerance (e.g., ALDH, AREG) and anti-viral properties (e.g., EFNB2). To further assess the therapeutic impact of ex vivo programming with FT1050 and FT4145, a number of T cell assays to assess T cell phenotype and function were conducted on mPB. Overall, ex vivo programming of mPB resulted in reduced allogeneic T cell responses and was accompanied by reduced capacity of modulated T cells to produce Interferon Gamma (IFN-ɣ). Concomitantly, the ability of the modulated T cells to make Interleukin 4 (IL-4) and 10 (IL-10) was enhanced, suggesting a polarization of these cells towards a less inflammatory functional state. This was further evidenced by increased surface expression of an immune-inhibitory molecule, PD1, and reduced expression of the activation markers 41BB and ICOS. We next examined the potential beneficial role of ex vivo programming with FT1050+FT4145 in a major histocompatibility complex (MHC) mis-matched HSCT mouse model. Briefly, lethally irradiated BALB/c mice received bone marrow and splenocytes from C57BL/6 donor mice pulse treated with vehicle or FT1050+FT4145. Significantly less GvHD, as determined by survival, weight loss, GVHD score (diarrhea, inactivity, hunched posture, ruffled fur, eye lesion, snout swelling/skin integrity), cytokine production and histopathology of GvHD target organs was observed in recipients receiving FT1050+FT4145 treated cells as compared to those receiving vehicle treated cells. In addition, we observed increased levels of donor T regulatory cells (Tregs) in secondary lymphoid organs concomitant with decreased levels of circulating IFN-ɣ in recipients receiving FT1050+FT4145 treated cells. Based on the attenuation of alloreactive T-cell responses in these preclinical studies, we believe our findings provide a compelling scientific basis to support the clinical evaluation of ex vivo programmed mobilized peripheral blood in patients undergoing HSCT for the treatment of hematologic malignancies. Disclosures Levin: Fate Therapeutics, Inc: Employment, Equity Ownership. Shoemaker:Fate Therapeutics Inc: Employment.
Umbilical cord blood (UCB) offers many potential advantages as a source of hematopoietic stem cells (HSCs) for allogeneic transplantation, including ease of collection, rapid availability, flexibility of HLA-matching, lower rates of GvHD and potentially lower relapse rates. However, the low HSC content of UCB compared to other graft sources results in a prolonged time to engraftment, and higher rates of graft failure and early mortality. Pulse ex vivo exposure of HSCs to 16,16-dimethyl PGE2 (FT1050) has been demonstrated to enhance HSC engraftment potential, which could benefit clinical UCB transplant. FT1050 modulation promotes multiple mechanisms, including increased proliferation, reduced apoptosis, and improved migration and homing [North 2007&2009; Hoggatt 2009]. Improved HSC homing is mediated by induction of CXCR4 gene expression leading to increased cell surface CXCR4. Further optimization of the UCB modulation process demonstrated that incubation with 10µM FT1050 for 2 hrs at 37C resulted in a maximal biological response of the FT1050-UCB (ProHema®). A Phase 1 trial was performed to evaluate the safety of FT1050-UCB paired with an unmanipulated UCB unit in reduced-intensity double UCBT (dUCBT) [Cutler 2013]. We observed durable, multi-lineage engraftment of FT1050-UCB with acceptable safety. Earlier neutrophil engraftment was observed relative to historical controls (median 17.5 vs. 21 days (historical control), p=0.045), coupled with preferential engraftment of the FT1050-UCB unit in 10 of 12 subjects. A Phase 2 multi-center clinical trial of FT1050-UCB in adult patients undergoing dUCBT for hematologic malignancies was then initiated. Subjects are randomized 2:1 to FT1050-UCB-containing vs. standard dUCBT after high-dose conditioning. The primary endpoint is a categorical analysis of neutrophil engraftment using a pre-specified control median. Data on the initial 11 subjects, of which 8 were randomized to receive FT1050-UCB, continue to demonstrate acceptable safety with adverse events attributed to FT1050-UCB limited primarily to common infusion-related side effects. Of the 8 FT1050-UCB subjects, 1 died prior to neutrophil engraftment, with the remaining 7 subjects engrafting at a median of 28 days vs. 31 days for the 3 control subjects. With median overall follow-up of 16.1 months, 4 of 8 subjects on the FT1050-UCB arm are alive with a median survival not reached (> 11.0 months). 1 of 3 control subjects is alive with median survival of 6.0 months. During the clinical translation process, the media used during FT1050 modulation of UCB was identified as a key variable. Standard UCB washing media, consisting of a nutrient-free saline solution of low molecular weight dextran and human serum albumin (LMD/HSA), is used clinically to stabilize fragile cells post-thaw by reducing lysis. This media was used in the Phase 1 trial and to initiate Phase 2. Early during the Phase 2 trial, we identified a novel cell-stabilizing nutrient-rich formulation (NRM), containing glucose, amino acids and other HSC-supporting nutrients that promoted full FT1050 modulation of UCB and increased cell viability. The expression of key FT1050-pathway genes was significantly higher with NRM compared to intermediate levels observed with LMD/HSA. Modulation of human CD34+ (hCD34+) cells with FT1050 in NRM led to an 8-fold increase over LMD/HSA in induced CXCR4 gene expression (20-fold total), which translated to significantly increased surface CXCR4 protein. In vivo homing models demonstrated that UCB CD34+ cells modulated with FT1050 in NRM resulted in a 2.2-fold homing increase relative to vehicle (p < 0.001) compared to a 1.6-fold increase with LMD/HSA (p = 0.002), with a significant difference between the two media conditions (p = 0.04). A xenotransplantation study in NSG mice with hCD34+ cells modulated with FT1050 in either NRM or LMD/HSA demonstrated a 2-fold increase in circulating hCD45+ cells 12-weeks post-transplant with NRM (p = 0.007; unpaired t-test). These findings supported the incorporation of NRM into the FT1050-UCB manufacturing process in order to further improve its clinical engraftment potential. Enrollment of a 60-patient Phase 2 trial has been initiated that incorporates this manufacturing change. Disclosures Shoemaker: Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics: Employment. Guerrettaz:Fate Therapeutics: Employment. Robbins:Fate Therapeutics: Employment. Medcalf:Fate Therapeutics: Employment. Wolchko:Fate Therapeutics: Employment, Equity Ownership. Ferraro:Fate Therapeutics: Employment. Multani:Fate Therapeutics: Employment.
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