ALBERT

Description: Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte-colony stimulating factor (G-CSF) is often induced with infection and plays a key role in the stress granulopoiesis response. However, its effects on HSCs are unclear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect. RNA profiling and flow cytometry studies of HSCs from G-CSF treated mice show that multiple toll- like receptors (TLRs) are upregulated in HSCs upon G-CSF treatment, and gene set enrichment analysis shows enhancement of TLR signaling in G-CSF-treated HSCs. G-CSF-induced expansion of phenotypic HSCs is reduced in mice lacking the TLR signaling adaptors MyD88 or Trif, and the induction of quiescence is abrogated in mice lacking these adaptors. Furthermore, loss of TLR4 mitigates the G-CSF-mediated HSC repopulating defect. Interestingly, baseline HSC function is also dependent on TLR signaling. We show that HSC long-term repopulating activity is enhanced in Tlr4-/- and MyD88-/- mice, but not Trif-/- mice. One potential source of TLR ligands affecting HSC function in the bone marrow is the gut microbiota. Indeed, we show that in mice treated with antibiotics to suppress intestinal flora, G-CSF induced HSC quiescence and hematopoietic progenitor mobilization are attenuated. Moreover, in germ free mice, HSC long-term repopulating activity is enhanced. Collectively these data suggest that low level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling. Our finding of enhanced TLR signaling upon G-CSF treatment, and the mitigation of G-CSF’s effects in mice deficient for TLR signaling or commensal organisms, suggest that TLR antagonists and/or agonists may ultimately be used clinically to enhance engraftment following bone marrow transplantation or applied toward the treatment of patients with bone marrow failure. Disclosures: No relevant conflicts of interest to declare.

Description: Although approximately 70% of patients with acute promyelocytic leukemia (APL) are cured after treatment with all-trans retinoic acid (ATRA) and anthracycline based chemotherapy, those who relapse cannot be cured even with chemotherapy or arsenic trioxide salvage therapy. In order to study the molecular mechanisms of resistance to ATRA, liposomal ATRA, and arsenic trioxide, we have utilized a murine model of APL generated by “knocking in” the PML/RARα gene into the murine cathepsin G locus (Westervelt et al. Blood. 102(5):1857). Of the 76 single knock-in (SKI) mice followed for a median time of 368 days, 30 (39%) suffered from rapid-onset leukocytosis, anemia, thrombocytopenia, and hepato-splenomegaly. Spleens from these leukemic animals were removed and the tumors cells (〉 90% CD34+/Gr-1+ APL cells) were frozen in aliquots and banked for future passive transfer into genetically compatible recipients. The SKI mice exhibited the expected median time to death of 222 days. Attempts to expand these tumors in vitro failed, thus precluding the use of in vitro methods to generate ATRA and arsenic resistant SKI APL tumor cells. An alternate in vitro model of identifying de novo resistant SKI APL tumors to the drugs was established by incubating individual SKI APL tumors with ATRA, liposomal ATRA, and arsenic in vitro for only 3 days and using ELISA to measure upregulation of MMP-9 (gelatinase B), a surrogate marker for neutrophil differentiation. ATRA, liposomal ATRA, and arsenic induced 6.2 ± 4.4, 5.7 ± 3.5, 1.7 ± 1.0 fold increase in accumulated levels of MMP-9, respectively. These data suggest that ATRA and liposomal ATRA are more potent inducers of terminal differentiation than arsenic in these APL tumors. More importantly, the SKI APL tumors were also assessed for in vivo resistance to these agents by injecting 1 x 106 banked APL tumor cells into the peritoneal cavity of wild-type genetically compatible mice. The mice were then treated i.p. with either arsenic trioxide (0.4 mg q.d.), liposomal ATRA (0.4 mg q.d.), or diluent control. Doses were administered daily starting on day +5 until death. Tumors in the recipient mice were tracked by PCR for the transgene and FACS. The tumors proliferated in the peritoneal cavity (days 1–14) followed by migration to the bone marrow and spleen (days 14–28). By the eighth week post-injection, all mice suffered from leukocytosis and eventually died. Of the 22 banked tumors studied, only two tumors showed de novo resistance to ATRA both in vitro and in vivo. We will present data on preliminary RNA profiling and DNA sequencing of the PML-RARα transgenes in these APL tumors and attempt to correlate these results and the results of in vitro induction of MMP-9. The SKI APL tumors will provide us with unique reagents to study the biology of resistance to ATRA and arsenic trioxide.

Description: Abstract 1246 Infectious stress is associated with a shift in the bone marrow from lymphopoiesis to granulopoiesis. Expression of granulocyte colony-stimulating factor (G-CSF), the principal cytokine regulating granulopoiesis, is often induced during infection. We previously reported that G-CSF treatment is associated with marked suppression of B lymphopoiesis in murine bone marrow. After 5 days of G-CSF treatment (250 μg/kg), total B cells in the bone marrow were reduced 8.1 ± 0.9-fold. Pre-pro-B cells were reduced 1.6 ± 0.3-fold, pro-B cells 12.4 ± 1.9-fold, pre-B cells 5.6 ± 0.8-fold, immature B cells 7.5 ± 1.2-fold, and mature naïve B cells 83 ± 7.6-fold. B-committed lymphoid progenitors (BLP) were modestly but significantly decreased (1.4 ± 0.2-fold), while common lymphoid progenitors (CLP) were not affected by G-CSF treatment. Increased apoptosis of mature naïve B cells in the bone marrow was observed. Studies of G-CSF receptor deficient (Csf3r−/−) bone marrow chimeras show that G-CSF acts in a non-cell intrinsic fashion to suppress B lymphopoiesis. Consistent with this observation, we show that G-CSF treatment results in decreased expression in the bone marrow microenvironment of multiple B-supportive factors including CXCL12, interleukin-6, interleukin-7, and B cell activating factor (BAFF). Prior studies have established that CXCL12-abundant reticular (CAR) cells in the bone marrow play a key role in B cell development. CAR cells are perivascular stromal cells that express very high levels of CXCL12 and are in direct contact with pre-pro-B cells. G-CSF treatment did not affect CAR cell number. However, RNA expression profiling of sorted CAR cells showed that expression of several genes associated with B cell development are significantly decreased by G-CSF, including CXCL12 (4.2 ± 1.5-fold). In addition to CAR cells, other stromal cells in the bone marrow express CXCL12, including osteoblasts and endothelial cells. To assess the role of CXCL12 production by each of these cell types to B lymphopoiesis, we generated Cxcl12flox mice and crossed them with mice expressing the following tissue-specific Cre-recombinase transgenes: Osteocalcin-Cre (Oc-Cre) targeting mature mineralizing osteoblasts; Osterix-Cre (Osx-Cre) targeting CAR cells and all osteolineage cells; or Prx1-Cre targeting mesenchymal progenitors and their progeny. Deletion of Cxcl12 using Oc-Cre or Osx-Cre had a similar effect on B cell development, with an isolated loss of mature naïve B cells in the bone marrow (2.7 ± 0.5 and 4.1 ± 1.7-fold, respectively). In contrast, deletion of Cxcl12 using Prx1-Cre resulted in severe suppression of B lymphopoiesis that included a loss of CLP (3.3 ± 2.0-fold), BLP (5.6 ± 4.3-fold), and pre-pro-B cells (12.4 ± 5.1-fold). Interestingly, treatment of Prx1-Cre Cxcl12flox/- mice with G-CSF resulted in additional B cell loss, indicating that deletion of Cxcl12 in mesenchymal stromal cells is not sufficient to fully recapitulate G-CSF-induced B cell suppression. In summary, G-CSF treatment results in marked changes in the bone marrow microenvironment that lead to a suppression of B lymphopoiesis. While G-CSF-induced inhibition of CXCL12 expression from stromal cells contributes to B cell suppression, additional alterations in the microenvironment also contribute to this phenotype. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Patients infected with HIV have an 8 to 150-fold risk of lymphoma compared to HIV uninfected patients, with certain aggressive non-Hodgkin lymphoma (NHL) histologies being AIDS-defining. Despite the decrease in incidence of both NHL and Hodgkin lymphoma (HL) in the current anti-retroviral treatment (ART) era, HIV-infected patients remain at significantly increased risk of lymphoma compared to the general population. Over the last decade, advances have been made in the treatment of both NHL and HL with 18 agents approved since 2009. Unfortunately, safety and efficacy data in the HIV-infected population have been lacking in part due to exclusion of such patients from clinical trials. As such, the optimal treatment of HIV-associated lymphomas is unknown. In 2017, the National Comprehensive Care Network (NCCN) and the American Society of Clinical Oncology (ASCO) advocated expanding clinical trial opportunities for HIV-infected patients. However, exclusion criteria precluding HIV-infected patient participation likely persist. We sought to document and describe the current status of clinical trial exclusion criteria for NHL and HL as they relate to HIV infection. Methods We extracted data from the National Institute of Health's (NIH) registry of clinical trials (ClinicalTrials.gov) using the search term 'lymphoma' and reviewed all clinical trials (independent of funding source) in the United States that were recruiting or not yet recruiting adult patients for therapeutic trials (phase 1, 2, or 3) as of June 2019. A total of 734 trials met the above criteria; of these 206 were excluded as they were not primarily adult lymphoma trials (allogeneic transplant [n=87], leukemia [n=79], pediatric [n=87], lung cancer [n=12], and other [n=12]). As such, 526 clinical trials were included in the final sample. The primary outcome measure was whether HIV infection was an exclusion criterion for participation in the clinical trial. With the exception of the sample size, which was measured as a continuous variable, explanatory variables were dichotomized and included funding source (NIH vs. non-NIH), phase (phase 1 vs. phase 2 or 3), start date (start date prior to January 2018 vs. start date on or after January 2018), and recruitment status (recruiting vs. not yet recruiting). If the lymphoma under study was an aggressive subtype it was dichotomized as an AIDS-defining malignancy (aggressive NHL vs. other). We assessed whether these explanatory variables were associated with the primary outcome measure. We utilized bivariate analyses to identify measures with p 〈 0.20 for inclusion in a multivariate logistic regression model. Results: Of the 526 lymphoma-related trials, 352 (66.9%) excluded HIV-infected patients. Among all studies, the target sample sizes of these studies ranged from 6 to 6,542 (median 52) and were primarily phase 1 (219; 41.6%) or phase 2 (277; 52.7%) studies. Only 30 of the clinical trials were phase 3 (5.7%). The majority of studies (493; 93.7%) were actively recruiting and were initiated prior to January 1, 2018 (302; 57.4%). The majority of the 526 trials (320; 60.8%) included patients with aggressive NHL. The remaining trials included patients with indolent lymphoma, HL, or NHL with an unspecified subtype. Only 39 (7.4%) of all studies were funded strictly by NIH; the remainder were funded strictly by industry (192; 36.5%), 'other' (107; 20.3%), or a combination (188; 35.7%).[Table 1] Of the explanatory variables, funding source, sample size, and a start date on or after January 1, 2018 were associated with the outcome measure in the bivariate analysis and were included in a multivariate model. In the multivariate analyses, NIH-funded studies (OR 0.34 [0.17, 0.67], studies which began recruitment after January 1, 2018 (OR 0.66 [0.45, 0.97]) and larger studies (OR 0.998 [0.996, 0.999] were less likely to exclude HIV-infected patients. [Table 2] Conclusions: Despite support from ASCO and specific NCCN guidance which advocates that the participation in cancer clinical trials of patients living with HIV/AIDS "should be encouraged whenever feasible," the majority of interventional clinical trials for the treatment of lymphoma currently exclude participants based on infection with HIV. This goal of inclusion could be accomplished by adding an additional HIV-specific safety and efficacy cohort to most studies which could potentially be mandated by the FDA for all registrational trials. Disclosures Lynch: Rhizen Pharmaceuticals S.A: Research Funding; Takeda Pharmaceuticals: Research Funding; T.G. Therapeutics: Research Funding; Incyte Corporation: Research Funding; Johnson Graffe Keay Moniz & Wick LLP: Consultancy; Juno Therapeutics: Research Funding. Shadman:Sound Biologics: Consultancy; Acerta: Research Funding; Pharmacyclics: Consultancy, Research Funding; Celgene: Research Funding; Gilead: Research Funding; TG Therapeutics: Research Funding; Atara: Consultancy; Verastem: Consultancy; Mustang Biopharma: Research Funding; Bigene: Research Funding; ADC Therapeutics: Consultancy; Merck: Research Funding; AbbVIe: Consultancy, Research Funding; Sunesis: Research Funding; Genentech, Inc.: Consultancy, Research Funding; Emergent: Research Funding; AstraZeneca: Consultancy. Shustov:Spectrum Pharmaceuticals: Consultancy, Research Funding. Smith:AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma BV: Research Funding; Merck Sharp & Dohme Corp: Consultancy, Research Funding; Denovo Biopharma: Research Funding; Genentech: Research Funding; Ignyta (spouse): Research Funding; Bristol-Myers Squibb (spouse): Research Funding; Ayala (spouse): Research Funding; Pharmacyclics: Research Funding; Portola Pharmaceuticals: Research Funding; Seattle Genetics: Research Funding; Incyte Corporation: Research Funding. Till:Mustang Bio: Patents & Royalties, Research Funding. Ujjani:PCYC: Research Funding; Pharmacyclics: Honoraria; Pharmacyclics: Honoraria; Genentech: Honoraria; PCYC: Research Funding; Gilead: Consultancy; Gilead: Consultancy; Astrazeneca: Consultancy; Astrazeneca: Consultancy; Atara: Consultancy; Genentech: Honoraria; Atara: Consultancy; AbbVie: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding. Uldrick:Roche: Other: commercial research support through a CTA with Fred Hutchinson Cancer Research Center; Merck: Other: drug for a clinical trial from Merck through a CRADA with the NCI; Celgene: Other: research support from Celgene through a CRADA at the NCI; Patent: Patents & Royalties: co-inventor on US Patent 10,001,483 entitled . Gopal:Seattle Genetics, Pfizer, Janssen, Gilead, Sanofi, Spectrum, Amgen, Aptevo, BRIM bio, Acerta, I-Mab-pharma, Takeda, Compliment, Asana Bio, and Incyte.: Consultancy; Seattle Genetics, Pfizer, Janssen, Gilead, Sanofi, Spectrum, Amgen, Aptevo, BRIM bio, Acerta, I-Mab-pharma, Takeda, Compliment, Asana Bio, and Incyte: Honoraria; Teva, Bristol-Myers Squibb, Merck, Takeda, Seattle Genetics, Pfizer, Janssen, Takeda, and Effector: Research Funding.

Description: Background: ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) forms the backbone of frontline management of classical Hodgkin lymphoma (CHL) in North America regardless of stage. Expected cure rates with upfront therapy approach 75% in advanced stage, and 85-90% in early stage. A novel regimen incorporating brentuximab vedotin sought to improve upon ABVD in untreated advanced stage CHL patients (brentuximab vedotin + AVD). While it demonstrated a modest modified PFS benefit, it was associated with notable toxicities including higher rates of neuropathy and infection. PD-1 inhibition is highly effective in relapsed/refractory CHL, leading to the FDA approval of nivolumab and pembrolizumab in this setting. The first-line setting may represent the ideal time for a PD-1 inhibitor, with relatively intact host immunity and coexistence of malignant cells and T-cells in the microenvironment. Using a proven chemotherapy backbone, we designed a trial adding pembrolizumab to AVD chemotherapy (APVD) without a PD-1 inhibitor lead-in for untreated CHL (NCT03331341). Methods: This is a single arm pilot study combining pembrolizumab with AVD in untreated CHL of any stage. Eligibility requires ECOG 0-1, adequate organ function, and measurable disease. The trial intends to enroll 30 patients. AVD is given at standard doses on days 1 and 15 of a 28-day cycle. Pembrolizumab (200 mg IV) is given starting cycle 1 day 1 and every 21 days thereafter (cycle 1 day 22, cycle 2 day 15 etc.). The primary objective is to estimate the safety of delivering 2 cycles of APVD. The study will be determined a success if 〉 85% of subjects are able to complete 2 cycles of therapy without a dose delay 〉 3 weeks. Operationally, the stopping rule will be activated if the lower limit of the 95% confidence interval of toxicity crosses 15%. Thus, the trial would stop if 4/10, 7/20, 8/25, or 9/30 had a dose delay of 〉3 weeks due to toxicity. The secondary objective is to estimate the FDG-PET2 negative (Deauville score 1-3) after 2 cycles of APVD. Exploratory objectives include overall and progression free survival, predictive capacity of PET2 after APVD, peripheral blood flow cytometry of T-cell subsets, and analysis of ctDNA. After PET2 response assessment, subjects may continue APVD for up to 6 total cycles, or pursue treatment deemed appropriate for their stage/risk factors (including alternate systemic therapy or radiotherapy) at investigator discretion. Results: Six subjects have enrolled and received 2 cycles of therapy. Median age of these subjects was 28 years (range 18-69). Most subjects have advanced stage (stage II n=1 (17%), stage III n=3 (50%), stage IV n=2, (33%)). 3/6 (50%) of subjects had B symptoms at diagnosis, while 1/6 (17%) had bulky disease. Among the 6 subjects enrolled thus far, all have completed the first 2 cycles of therapy without any treatment delays. 3/6 subjects achieved a complete metabolic response (Deauville 1-3) on PET2, and 3/6 had a partial response (PR) with Deauville 4. The only subject who has completed all 6 cycle of therapy had a PET2 with Deauville 4 which converted to Deauville 2 upon completion of all therapy. There were no grade 2+ AEs attributable to pembrolizumab. No serious AEs have been reported. Non-hematologic grade 1 AEs of note include fatigue (50%), AST/ALT increase (33%), nausea (33%), arthralgia (17%), diarrhea (17%), maculopapular rash (17%), fever (17%), and alkaline phosphatase increased (17%). Conclusion: The concurrent combination of pembrolizumab with AVD chemotherapy for untreated CHL has been safe to date without any dose delays, serious adverse events, or immune-related adverse events of grade 2 or higher. All patients treated thus far achieved an objective response by PET2, with 3/6 achieving a complete metabolic response by interim scan. One subject has completed all therapy with a complete metabolic response (Deauville 2) after PET2 showed Deauville 4. Trial enrollment is ongoing. Disclosures Lynch: Incyte Corporation: Research Funding; T.G. Therapeutics: Research Funding; Johnson Graffe Keay Moniz & Wick LLP: Consultancy; Juno Therapeutics: Research Funding; Takeda Pharmaceuticals: Research Funding; Rhizen Pharmaceuticals S.A: Research Funding. Ujjani:Atara: Consultancy; Astrazeneca: Consultancy; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; Gilead: Consultancy; PCYC: Research Funding; Pharmacyclics: Honoraria. Kurtz:Roche: Consultancy. Gopal:Seattle Genetics, Pfizer, Janssen, Gilead, Sanofi, Spectrum, Amgen, Aptevo, BRIM bio, Acerta, I-Mab-pharma, Takeda, Compliment, Asana Bio, and Incyte.: Consultancy; Teva, Bristol-Myers Squibb, Merck, Takeda, Seattle Genetics, Pfizer, Janssen, Takeda, and Effector: Research Funding; Seattle Genetics, Pfizer, Janssen, Gilead, Sanofi, Spectrum, Amgen, Aptevo, BRIM bio, Acerta, I-Mab-pharma, Takeda, Compliment, Asana Bio, and Incyte: Honoraria.

Description: Abstract 510 Hematopoietic stem cells (HSCs) reside in a specialized microenvironment in the bone marrow that provides key signals required for HSC maintenance. Neither the stromal cell population(s) that comprise this stem cell niche nor their HSC-regulatory signals are well defined. In particular, there is controversy about the relative importance of the endosteal niche, which is comprised of osteoblasts and their progenitors, and the perivascular niche, which is comprised of endothelial cells, CXCL12-abundant reticular (CAR) cells, and mesenchymal stem and progenitor cells (including Nestin-GFP+ cells). CXCL12 is a key component of the stem cell niche that regulates HSC trafficking and function. CXCL12 is constitutively expressed by several stromal cell populations, including endothelial cells, osteoblasts, and perivascular stromal cells. Here we generated a floxed allele of Cxcl12 to conditionally delete Cxcl12 from candidate niche cells in the bone marrow and assess the effect on HSCs. Deletion of Cxcl12 in endothelial cells and mature osteoblasts was mediated by the Tie2-Cre recombinase (Cre) and osteocalcin (Oc)-Cre transgenes, respectively. To target Cxcl12 deletion in CAR cells and osteoprogenitors, we used the osterix (Osx)-Cre transgene. Finally, to target multipotent mesenchymal progenitors, we used the Prx1-Cre transgene. Prx1 is a transcription factor expressed early during limb bud mesoderm development, and Prx1-Cre targets all cells derived from limb bud mesoderm. We first performed lineage mapping studies using transgenic mice carrying a knock-in of the green fluorescent protein (GFP) gene into the Cxcl12 locus. As reported previously, the highest CXCL12 expression was observed in CAR cells, which is a heterogeneous perivascular stromal cell population that contains osteoprogenitors. Lineage mapping with Osx- and Prx1-Cre show that both transgenes mediate efficient and equivalent recombination in nearly all mature osteoblasts, osteoblast progenitors, and CAR cells. Prx1-Cre, but not Osx-Cre, also targets a novel subset of PDGFRα+ Sca+ mesenchymal progenitors. We show that deletion of Cxcl12 from mature osteoblasts has no effect on HSC number or function. Deletion of Cxcl12 from Osx-Cre-targeted stromal cells, which includes osteoprogenitors and CAR cells, results in constitutive mobilization of hematopoietic progenitors; however, HSC number and function are normal. Cxcl12 deletion in endothelial cells results in a modest loss of long-term repopulating activity. Strikingly, deletion of Cxcl12 in mesenchymal progenitors is associated with a marked loss of HSCs and HSC quiescence. In addition to endothelial cells, we thus identify a novel Prx1-Cre targeted subset of mesenchymal progenitors that appears to be necessary to support HSCs. These data show that expression of CXCL12 from stromal cells in the perivascular region is required for HSC maintenance. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 235 Truncation mutations of CXCR4 that cause increased receptor signaling are responsible for most cases of WHIM (warts, hypogammaglobulinemia, infections, myelokathexis) syndrome, which is characterized by the retention of mature neutrophils in the bone marrow despite peripheral neutropenia. This observation and others have established CXCR4 as a key regulator of neutrophil release from the bone marrow. However, it is unclear how modulation of neutrophil CXCR4 signaling is linked to their migration toward the vascular endothelium and subsequent entry into the circulation. Therefore, the question of whether neutrophil egress from the bone marrow is a passive, random process or actively directed and what (if any) signals regulate it remains unanswered. We recently analyzed a myelokathexis pedigree and discovered homozygous, loss-of-function mutations in CXCR2. Based on these observations, we developed a “tug-of-war” model in which opposing chemokine gradients, specifically release-inducing CXCR2 signals and retention-promoting CXCR4 signals, act antagonistically to regulate neutrophil release from the bone marrow. To test this model, we analyzed neutrophil trafficking in CXCR2−/− mice. These mice have a well-characterized defect in neutrophil emigration from the blood to sites of inflammation, leading to chronic subclinical infection and the systemic release of cytokines that stimulate granulopoiesis. To circumvent these potentially confounding effects, we generated mixed bone marrow chimeras reconstituted with a 1:1 ratio of wild-type and CXCR2−/− bone marrow. As expected, approximately 50% (46 ± 4%) of circulating B cells were derived from CXCR2−/− cells. In contrast, a significant decrease in the proportion of CXCR2−/− neutrophils in the blood was observed (23 ± 4%; P 〈 0.001). Consistent with a myelokathexis phenotype, there was a relative accumulation of mature CXCR2−/− neutrophils in the bone marrow (47 ± 9% of Gr-1hi SSChi cells in the bone marrow were derived from CXCR2−/− cells; P 〈 0.01). Neutrophil trafficking from the bone marrow was estimated by calculating the percentage of neutrophils in the blood out of the total amount in the blood and bone marrow (neutrophil distribution index or NDI). We estimated that 1.3 ± 0.2% of wild-type neutrophils were in the blood, but the percentage of CXCR2−/− neutrophils in the blood was reduced to 0.4 ± 0.1% (P 〈 0.01). These data provide genetic evidence that CXCR2 signals promote neutrophil release from the bone marrow in a cell-autonomous manner. To explore the epistatic relationship of CXCR2 and CXCR4 signals to neutrophil trafficking, the neutrophil response to AMD3100, a small molecule CXCR4 antagonist, was examined in the CXCR2−/− mixed chimeras. Consistent with previous reports, treatment with AMD3100 resulted in a 5.2 ± 0.7-fold increase in wild-type neutrophils in the blood one hour after administration. In contrast, AMD3100 induced release of CXCR2−/− neutrophils was impaired, with only a 2.8 ± 0.3-fold increase observed (P 〈 0.05). G-CSF treatment is thought to induce neutrophil release through disruption of CXCR4 signaling. Thus, we next characterized the neutrophil response to 5 days of G-CSF treatment. Wild-type neutrophils displayed a shift from the bone marrow to the blood, with an NDI of 5.0 ± 0.4%. The number of CXCR2−/−neutrophils in the blood increased after treatment, but the percentage in the blood (2.5 ± 0.7%) was less than wild-type (P 〈 0.05). These data show that maximal neutrophil release requires the coordinated regulation of CXCR2 and CXCR4 signals. Studies are underway to assess neutrophil trafficking of CXCR4−/− × CXCR2−/− neutrophils. The tug-of-war model of neutrophil trafficking in the bone marrow predicts that CXCR2 ligands will be highly expressed in bone marrow endothelial cells or other cells closely associated with the endothelium. To test this prediction, endothelial cells (CD45− Ter119− CD31+) were sorted from the bone marrow of wild-type mice at baseline or after 5 days of G-CSF treatment. RNA expression profiling showed constitutive high level expression of the CXCR2 ligands CXCL1 and CXCL2. Moreover, expression of CXCL2 was significantly induced after G-CSF treatment. Chemokine expression was confirmed by real time RT-PCR and ELISA. Taken together, our data suggest that CXCR2 signaling is a second chemokine pathway that, in coordination with CXCR4, controls neutrophil release from the bone marrow. Disclosures: No relevant conflicts of interest to declare.

Description: There is evidence suggesting that N-cadherin expression on osteoblast lineage cells regulates hematopoietic stem cell (HSC) function and quiescence. To test this hypothesis, we conditionally deleted N-cadherin (Cdh2) in osteoblasts using Cdh2flox/flox Osx-Cre mice. N-cadherin expression was efficiently ablated in osteoblast lineage cells as assessed by mRNA expression and immunostaining of bone sections. Basal hematopoiesis is normal in these mice. In particular, HSC number, cell cycle status, long-term repopulating activity, and self-renewal capacity were normal. Moreover, engraftment of wild-type cells into N-cadherin–deleted recipients was normal. Finally, these mice responded normally to G-CSF, a stimulus that mobilizes HSCs by inducing alterations to the stromal micro-environment. In conclusion, N-cadherin expression in osteoblast lineage cells is dispensable for HSC maintenance in mice.

Description: The bone marrow microenvironment plays a key role in regulating hematopoietic stem cell (HSC) function. In particular, bone marrow stromal signals contribute to the maintenance of HSC quiescence, a property that is thought to be associated with long-term repopulating activity. We previously reported that G-CSF treatment disrupts the osteoblast niche by inducing osteoblast apoptosis and inhibiting osteoblast differentiation. In this altered bone marrow microenvironment, we also showed that the number of HSCs in the bone marrow after G-CSF treatment (as defined by CD34− Kit+ Sca+ lineage-cells or CD150+ CD48− CD41− lineage-[SLAM] cells) was unchanged and that the HSCs were more quiescent than HSCs from untreated mice. However, despite the quiescent phenotype, there was a marked loss of HSC long-term repopulating activity. To define mechanisms for this phenotype, we first asked whether G-CSF acts directly on HSCs to inhibit their long-term repopulating activity. Bone marrow chimeras containing wild type and G-CSFR−/− cells were established and treated with G-CSF. The contribution of G-CSFR−/− cells to hematopoiesis remained stable for at least 3 months after G-CSF treatment, demonstrating that the effects of G-CSF on HSC function are not direct. We next performed RNA expression profiling on sorted SLAM cells, a cell population highly enriched for HSCs. These data showed that expression of Cdkn1a (p21cip1/waf1) was increased in HSCs harvested from G-CSF treated mice. To define the contribution of Cdkn1a to HSC quiescence and loss of repopulating activity following treatment with G-CSF, Cdkn1a−/− mice (inbred on a C57BL/6 background) were studied. Wild-type or Cdkn1a−/− mice were treated with G-CSF for 7 days and pulse labeled with bromo-deoxyuridine (BrdU), and the percentage of SLAM cells that labeled with BrdU was determined. Consistent with our previous observations, treatment of wild-type mice with G-CSF resulted in a significant decrease in the percentage of BrdU+ SLAM cells in the bone marrow. In contrast, in Cdkn1a−/− mice, no change in the percentage of BrdU+ SLAM cells after G-CSF treatment was observed [10.08 ± 2.26% (untreated); 10.96 ± 2.80% (G-CSF treated); p = NS]. To assess HSC function, competitive repopulation assays were performed using untreated or G-CSF treated bone marrow from wild type or Cdkn1a−/− mice. Surprisingly, G-CSF had a similar deleterious effect on HSC repopulating activity in both wild type and Cdkn1a−/− mice. Collectively, these data show G-CSF treatment, possibly through disruption of the osteoblast niche, induces HSC quiescence and loss of long-term repopulating activity. HSC quiescence, but not loss of repopulating activity, is dependent upon Cdkn1a−/−. The mechanisms by which G-CSF treatment results in a loss of HSC function are under investigation.