Piezo1 is a mechanosensitive, nonselective Ca2+ channel that is broadly expressed in CD4+ T cells. Using lineage-specific Piezo1 knockout mice (Piezo1cKO), we show that loss of Piezo1 in CD4+ T cells significantly increased IFNγ and IL-17 production in vitro under TH1 and TH17 polarizing conditions, respectively. Despite their intrinsic proinflammatory phenotype, Piezo1cKO T cells are incapable of establishing disease in vivo in 3 separate adoptive transfer T-cell-mediated inflammatory mouse models, including experimental autoimmune encephalomyelitis, inflammatory bowel disease, and graft-vs-host disease. These phenomena coincided with a decreased effector memory (CD44hiCD62Llo) CD4+ T-cell pool derived from donor Piezo1cKO T cells, an observation related to intrinsic T-cell fitness, as a cotransfer inflammatory bowel disease mouse model revealed a deficiency in the CD4+ effector memory population derived only from the naive Piezo1cKO but a not coinfused Piezo1WT CD4+ T-cell source. Taken together, our results support Piezo1 as restraining proinflammatory T-cell differentiation while contributing to the generation and persistence of the effector memory pool during CD4+ T-cell-mediated immunopathology.

Osteosarcoma (OS) is an aggressive malignant bone cancer, with refractory and metastatic disease remaining a significant challenge. Transforming growth factor-β1 (TGF-β) is a potent immune suppressive cytokine in OS and the TGF-β is increased in the sera of OS patients and this increase is associated with high-grade OS and lung metastases. Therefore, blocking TGF-β1 signaling may be a novel therapy for OS treatment. Here we show that blocking TGF-β1 signaling using TGF-βR1 inhibitor, Vactosertib, significantly inhibited OS proliferation in vitro and in vivo. Notably, Vactosertib inhibits c-Myc expression in the OS cells. Vactosertib increased immune effectors (IFNγ+CD8+ cells and NK cells) and inhibited immune suppressors (M2-like TAM, MDSC) in the OS tumor microenvironment. Our results suggest that inhibition of TGF-β1 signaling is an effective therapeutic strategy against OS through a multi-pronged approach that targets tumor intrinsic and extrinsic factors to achieve optimal immune-effector functions and maximal clinical response.

Abstract Osteosarcoma (OS) is an aggressive malignant bone cancer, with the lung as the most frequent site of metastasis. Unresectable pulmonary metastasis remains a significant challenge with a survival rate of less than 20%. Identification of novel therapeutic strategies are desperately needed. Transforming growth factor-β1 (TGF-β) is a potent immune suppressive cytokine in OS tumor microenvironment (TME). TGF-β1 expression is increased in the sera and tumor tissues of OS patients and this increase is associated with high-grade OS and lung metastases. Therefore, blocking TGF-β1 signaling may be a novel therapy for OS treatment. In this study, we show that blocking TGF-β1 signaling using the orally bioavailable small molecule TGF-βR1 inhibitor, Vactosertib, significantly inhibited OS proliferation in vitro and in vivo. Notably, Vactosertib inhibits c-Myc expression in the OS cells and oral administration of Vactosertib significantly reduces OS growth in vivo. Vactosertib increased immune effectors (e.g., IFNγ+CD8+ cells and NK cells) and inhibited immune suppressors (e.g., M2-like TAM, MDSC) in the OS TME. Our results suggest that inhibition of TGF-β1 signaling is an effective therapeutic strategy against OS through a multi-pronged approach that targets tumor intrinsic and extrinsic factors to achieve optimal immune-effector functions and maximal clinical response. Citation Format: Sung Hee Choi, Jay Myers, Suzanne Tomchuck, Melissa Bonner, Saada Eid, Daniel Kingsley, Kristen VanHeyst, Seong-Jin Kim, Byung-Gyu Kim, Alex Y. Huang. Oral TGF-beta receptor1 inhibitor vactosertib promotes osteosarcoma regression by targeting tumor proliferation and enhancing anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 728.

BackgroundDespite its potential utility in delivering direct tumor killing and in situ whole-cell tumor vaccination, tumor cryoablation produces highly variable and unpredictable clinical response, limiting its clinical utility. The mechanism(s) driving cryoablation-induced local antitumor immunity and the associated abscopal effect is not well understood. MethodsThe aim of this study was to identify and explore a mechanism of action by which cryoablation enhances the therapeutic efficacy in metastatic tumor models. We used the subcutaneous mouse model of the rhabdomyosarcoma (RMS) cell lines RMS 76-9(STINGwt) or RMS 76-9(STING-/-), along with other murine tumor models, in C57BL/6 or STING(-/-) (TMEM173(-/-)) mice to evaluate local tumor changes, lung metastasis, abscopal effect on distant tumors, and immune cell dynamics in the tumor microenvironment (TME). ResultsThe results show that cryoablation efficacy is dependent on both adaptive immunity and the STING signaling pathway. Contrary to current literature dictating an essential role of host-derived STING activation as a driver of antitumor immunity in vivo, we show that local tumor control, lung metastasis, and the abscopal effect on distant tumor are all critically dependent on a functioning tumor cell-intrinsic STING signaling pathway, which induces inflammatory chemokine and cytokine responses in the cryoablated TME. This reliance extends beyond cryoablation to include intratumoral STING agonist therapy. Additionally, surveys of gene expression databases and tissue microarrays of clinical tumor samples revealed a wide spectrum of expressions among STING-related signaling components. ConclusionsTumor cell-intrinsic STING pathway is a critical component underlying the effectiveness of cryoablation and suggests that expression of STING-related signaling components may serve as a potential therapy response biomarker. Our data also highlight an urgent need to further characterize tumor cell-intrinsic STING pathways and the associated downstream inflammatory response evoked by cryoablation and other STING-dependent therapy approaches.

Insights regarding the biodistribution and homing of mesenchymal stromal cells (MSCs), as well as their interaction with alloreactive T-cells are critical for understanding how MSCs can regulate graft-versus-host disease (GVHD) following allogeneic (allo) bone marrow transplantation (BMT). We developed novel assays based on 3D, microscopic, cryo-imaging of whole-mouse-sized volumes to assess the therapeutic potential of human MSCs using an established mouse GVHD model. Following infusion, we quantitatively tracked fluorescently labeled, donor-derived, T-cells and third party MSCs in BMT recipients using multispectral cryo-imaging. Specific MSC homing sites were identified in the marginal zones in the spleen and the lymph nodes, where we believe MSC immunomodulation takes place. The number of MSCs found in spleen of the allo BMT recipients was about 200% more than that observed in the syngeneic group. To more carefully define the effects MSCs had on T cell activation and expansion, we developed novel T-cell proliferation assays including secondary lymphoid organ (SLO) enlargement and Carboxyfluoescein succinimidyl ester (CFSE) dilution. As anticipated, significant SLO volume enlargement and CFSE dilution was observed in allo but not syn BMT recipients due to rapid proliferation and expansion of labeled T-cells. MSC treatment markedly attenuated CFSE dilution and volume enlargement of SLO. These assays confirm evidence of potent, in vivo, immunomodulatory properties of MSC following allo BMT. Our innovative platform includes novel methods for tracking cells of interest as well as assessing therapeutic function of MSCs during GVHD induction. Our results support the use of MSCs treatment or prevention of GVHD and illuminate the wider adoption of MSCs as a standard medicinal cell therapy.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the gastrointestinal tract. CD4+ T cells are especially known to be the main drivers of IBD when they show an elevated level of activation. Elevation of intracellular Ca2+ is one of the key triggering signals for T cell activation. Piezo1 is a mechanosensitive nonselective Ca2+-permeable cation channel, which is broadly expressed in mammalian cells. However, the role of Piezo1 in the pathogenesis of T cell-mediated colitis remains unknown. We have generated T cell-specific Piezo1 knockout (Piezo1fl/flxCD4-cre) mice and observed that loss of Piezo1 in CD4+ T cell increased Th1 and Th17 cell polarization. RNA-sequence analysis of Piezo1fl/flxCD4-cre T cells identified elevated pathogenic Th17 cell pathway, IFN-γ signaling pathways and inflammatory response gene signature compared to that of wild type. These results suggest that Piezo1 controls the inflammatory response of pathogenic T cells. Next, we examined the function of Piezo1 on intestinal inflammation in vivo using acute and chronic colitis mouse model. For the acute colitis mouse model, we used a chemically induced mouse model of colitis using DSS in drinking water. Piezo1fl/flxCD4-cre mice with DSS developed severe colitis compared to Piezo1fl/fl mice with DSS. However, in chronic colitis mouse model, which is the adaptive transfer of naïve CD4+ T cells (CD4+CD45RBhigh) from Piezo1fl/fl or Piezo1fl/flxCD4cre into Rag1−/− mice, T cells from Piezo1fl/flxCD4cre mice failed to induce colon inflammation, while mice that received T cells from Piezo1fl/fl mice developed severe intestinal inflammation. Thus, our data demonstrate a critical role of Piezo1 in CD4+ T cell-mediated intestinal inflammation. Supported by R03 CA230840, P30 CA043703, St. Baldrick’s Foundation, Hyundai Hope-on-Wheels Scholar Hope Grant, Andrew McDonough B+ Foundation, Curing Kids Cancer, Center for Pediatric Immunotherapy at Rainbow

Graft-vs-host disease (GvHD) limits successful outcomes following allogeneic blood and marrow transplantation (allo-BMT). We examined whether the administration of human, bone marrow-derived, multipotent adult progenitor cells (MAPCs™) could regulate experimental GvHD. The immunoregulatory capacity of MAPC cells was evaluated in vivo using established murine GvHD models. Injection of MAPC cells on day +1 (D1) and +4 (D4) significantly reduced T-cell expansion and the numbers of donor-derived, Tumor Necrosis Factor Alpha (TNFα) and Interferon Gamma (IFNγ)-producing, CD4+ and CD8+ cells by D10 compared with untreated controls. These findings were associated with reductions in serum levels of TNFα and IFNγ, intestinal and hepatic inflammation and systemic GvHD as measured by survival and clinical score. Biodistribution studies showed that MAPC cells tracked from the lung and to the liver, spleen, and mesenteric nodes within 24 hours after injection. MAPC cells inhibited mouse T-cell proliferation in vitro and this effect was associated with reduced T-cell activation and inflammatory cytokine secretion and robust increases in the concentrations of Prostaglandin E2 (PGE2) and Transforming Growth Factor Beta (TGFβ). Indomethacin and E-prostanoid 2 (EP2) receptor antagonism both reversed while EP2 agonism restored MAPC cell-mediated in vitro T-cell suppression, confirming the role for PGE2. Furthermore, cyclo-oxygenase inhibition following allo-BMT abrogated the protective effects of MAPC cells. Importantly, MAPC cells had no effect on the generation cytotoxic T lymphocyte activity in vitro, and the administration of MAPC cells in the setting of leukemic challenge resulted in superior leukemia-free survival. Collectively, these data provide valuable information regarding the biodistribution and regulatory capacity of MAPC cells, which may inform future clinical trial design.

We demonstrated the use of multispectral cryo-imaging and software to analyze human mesenchymal stromal cells (hMSCs) biodistribution in mouse models of graft-versus-host-disease (GVHD) following allogeneic bone marrow transplantation (BMT). We injected quantum dot labeled MSCs via tail vein to mice receiving BMT and analyzed hMSC biodistribution in major organs (e.g. lung, liver, spleen, kidneys and bone marrow). We compared the biodistribution of hMSCs in mice following allogeneic BMT recipients (with GVHD) to the biodistribution following syngeneic BMT (without GVHD). Cryo-imaging system revealed cellular biodistribution and redistribution patterns in the animal model. We initially found clusters of cells in the lung that eventually dissociated to single cells and redistributed to other organs within 72 h. The in vivo half-life of the exogenous MSCs was about 21 h. We found that the biodistribution of stromal cells was not related to blood flow, rather cells preferentially homed to specific organs. In conclusion, cryo-imaging was suitable for analyzing the cellular biodistribution. It could provide capabilities of visualizing cells anywhere in the mouse model with single cell sensitivity. By characterizing the biodistribution and anatomical specificity of a therapeutic cellular product, we believe that cryo-imaging can play an important role in the advancement of stem and stromal cell therapies and regenerative medicine.

Late-term complications of hematopoietic cell transplantation (HCT) are numerous and include incomplete engraftment. One possible mechanism of incomplete engraftment after HCT is cytokine-mediated suppression or dysfunction of the bone marrow microenvironment. Mesenchymal stromal cells (MSCs) elaborate cytokines that nurture or stimulate the marrow microenvironment by several mechanisms. We hypothesize that the administration of exogenous MSCs may modulate the bone marrow milieu and improve peripheral blood count recovery in the setting of incomplete engraftment. In the current study, we demonstrated that posttransplant intramuscular administration of human placental derived mesenchymal-like adherent stromal cells [PLacental eXpanded (PLX)-R18] harvested from a three-dimensional in vitro culture system improved posttransplant engraftment of human immune compartment in an immune-deficient murine transplantation model. As measured by the percentage of CD45(+) cell recovery, we observed improvement in the peripheral blood counts at weeks 6 (8.4 vs. 24.1%, p < 0.001) and 8 (7.3 vs. 13.1%, p < 0.05) and in the bone marrow at week 8 (28 vs. 40.0%, p < 0.01) in the PLX-R18 cohort. As measured by percentage of CD19(+) cell recovery, there was improvement at weeks 6 (12.6 vs. 3.8%) and 8 (10.1 vs. 4.1%). These results suggest that PLX-R18 may have a therapeutic role in improving incomplete engraftment after HCT.

Lymph nodes (LNs) represent an area of interest for targeting metastatic tumors because they play an essential role in tumor survival in non-native tissues. The chemokine CCL3 is important in recruiting first-responder immune cells to areas of epithelial insult and orchestrating cellular contacts in the LNs of vaccinated mice that subsequently lead toward the enhancement of memory CD8+ T cell generation. We hypothesize that by introducing a continuous supply of CCL3 into the microenvironment of a metastatic tumor, we can redirect a LN destined for tumor-tolerance toward the production of greater antitumor cellular responses. To interrogate our hypothesis, we subcutaneously inoculated naïve murine recipients with a Balb/c colon metastatic tumor (CT26) that is either the wild-type (WTTUs) or WTTUs transfected to secrete CCL3 (L3TUs). Immunocompetent mice injected with L3TUs resulted in a suppression of tumor growth compared to the WTTU. In vivo analysis across 7-days post-tumor injections with WTTUs or L3TUs, revealed an enhanced accumulation of endogenous DCs, NKs, and lymphocytes in the TDLNs. In vitro analysis showed that OVA-peptide or whole-protein-pulsed bone marrow derived dendritic cells (BMDCs) cultured with CCL3, showed an enhanced capacity to induce proliferation of antigen-specific T cells. Examination of the day-5 TDLNs for direct signs of adaptive antitumor responses also revealed an enhanced production of the antitumor cytokine, IFNγ compared to WTTUs and the specific recruitment of antitumor-associated DCs to the TDLN.

Lymph node (LN) plays a critical role in tumor cell survival outside of the primary tumor sites and dictates overall clinical response in many tumor types (1, 2). Previously, we and others have demonstrated that CCL3 plays an essential role in orchestrating T cell-antigen-presenting cell (APC) encounters in the draining LN following vaccination, and such interactions enhance the magnitude of the memory T cell pool (3-5). In the current study, we investigate the cellular responses in the tumor-draining lymph nodes (TDLNs) of a CCL3-secreting CT26 colon tumor (L3TU) as compared to wild-type tumor (WTTU) during the priming phase of an antitumor response (<= 10 days). In comparison to WTTU, inoculation of L3TU resulted in suppressed tumor growth, a phenomenon that is accompanied by altered in vivo inflammatory responses on several fronts. Autologous tumor-derived CCL3 (aCCL3) secretion by L3TU bolstered the recruitment of T-and B-lymphocytes, tissue-migratory CD103(+) dendritic cells (DCs), and CD49b(+) natural killer (NK) cells, resulting in significant increases in the differentiation and activation of multiple Interferon-gamma (IFN gamma)-producing leukocytes in the TDLN. During this early phase of immune priming, NK cells constitute the major producers of IFN gamma in the TDLN. CCL3 also enhances CD8+ T cell proliferation and differentiation by augmenting DC capacity to drive T cell activation in the TDLN. Our results revealed that CCL3-dependent IFN. production and CCL3-induced DC maturation drive the priming of effective antitumor immunity in the TDLN.

The survival of patients with metastatic or relapsed Ewing sarcoma (ES) remains dismal despite intensification of combination chemotherapy and radiotherapy, precipitating the need for novel alternative therapies with minimal side effects. Natural killer (NK) cells are promising additions to the field of cellular immunotherapy. Adoptive NK cell therapy has shown encouraging results in hematological malignancies. Despite these initial promising successes, however, NK cell therapy for solid tumors remains to be investigated using in vivo tumor models. The purpose of this study is to evaluate the efficacy of ex vivo expanded human NK cells in controlling primary and metastatic ES tumor growth in vitro and in vivo. Using membrane-bound IL-21 containing K562 (K562-mbIL-21) expansion platform, we were able to obtain sufficient numbers of expanded NK (eNK) cells that display favorable activation phenotypes and inflammatory cytokine secretion, along with a strong in vitro cytotoxic effect against ES. Furthermore, eNK therapy significantly decreased lung metastasis without any significant therapeutic effect in limiting primary tumor growth in an in vivo xenograft model. Our data demonstrate that eNK may be effective against pulmonary metastatic ES, but challenges remain to direct proper trafficking and augmenting the cytotoxic function of eNK to target primary tumor sites.

Abstract Any therapeutic approach toward the eradication of metastatic tumor cells must involve targeting both the tumor's primary site of origin and those seeding in secondary tissues where metastasis has occurred. Lymph nodes represent an area of interest for targeting metastatic tumors because they play an essential role in tumor survival in non-native tissues. Interrogation of early cellular events in the draining lymph nodes (DLNs) during the initial priming of the adaptive T cell response can reveal new insights into how LN cells respond to a metastasizing tumor. The inflammatory chemokine CCL3 is important in orchestrating cellular contacts in vaccinated lymph nodes (LNs) and enhancing memory T cell generation. CCL3 has also been implicated in the modification and recruitment of natural killer (NK) cells and dendritic cells (DCs) to sites of epithelial insult and are important in establishing whether tumors will be tolerated or rejected. We hypothesize that by introducing a continuous supply of CCL3 into the microenvironment of a metastatic tumor, we can redirect a lymph node destined for tumor-tolerance toward the production of greater antitumor cellular responses. To interrogate our hypothesis, we subcutaneously inoculated naïve murine recipients with a Balb/c colon metastatic tumor (CT26) that is either the wild-type (WTTUs) or WTTUs transfected to secrete CCL3 (L3TUs). Immunocompetent mice injected with L3TUs resulted in a suppression of tumor growth compared to the WTTU group in a CD8+ T cell dependent manner. 1, 3, and 5-days after injection with WTTUs or L3TUs, there was an enhanced accumulation of endogenous DCs, NKs, and T cells in the DLNs and non-draining LNs (NDLNs). In vivo analysis of DCs in the DLN of L3TUs showed increased numbers of CD11c+ cells that upregulated the T cell costimulatory molecule, CD86+, while in vitro, SIINFEKL-pulsed BMDCs cultured with CCL3, showed an enhance capacity to induce proliferation of OT-I (CD8+) T cells. Examination of the day-5 DLN for direct signs of adaptive antitumor responses revealed an enhanced production of the antitumor cytokine, IFNγ, in the L3TU group, while the WTTU group showed a greater accumulation of CD4+ T-regulatory cells (Treg) over convention CD4+ T cells. Together these data suggest that CCL3 may enhance the overall immune response in the DLN in three ways. First, CCL3 attracts key cell-types such as NK cells to the DLN in larger quantities that can directly interact with DCs or T cells to enhance the development of IFNγ producing immune cells. Second, CCL3 directly influences DC maturation and indirectly enhances T cell proliferation. Third, CCL3 directly enhances the overall systemic accumulation of lymphocyte and myeloid cells in the DLN and NDLNs. Citation Format: Frederick Allen, Joseph M. Nthale, Saada K. Eid, Peter Rauhe, David Askew, Jay Myers, Alexander Tong, Alex Y. Huang. CCL3 in the tumor microenvironment augments antitumor immune priming in the lymph node [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A119.

Molecular intermediates in T-cell activation pathways are crucial targets for the therapy and prevention of graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (allo-HCT). We recently identified an essential role for cyclin-dependent kinase 5 (Cdk5) in T-cell activation and effector function, but the contribution of Cdk5 activity to the development of GVHD has not been explored. Using an established, preclinical, murine, GVHD model, we reveal that Cdk5 activity is increased in key target organs early after allo-HCT. We then generated chimeric mice (Cdk5 (+/+C) or Cdk5 (-/-C)) using hematopoietic progenitors from either embryonic day 16.5 Cdk5 (+/+) or Cdk5 (-/-) embryos to enable analyses of the role of Cdk5 in GVHD, as germ line Cdk5 gene deletion is embryonically lethal. The immunophenotype of adult Cdk5(-/-C) mice is identical to control Cdk5 (+/+C) mice. However, transplantation of donor Cdk5 (-/-C) bone marrow and T cells dramatically reduced the severity of systemic and target organ GVHD. This phenotype is attributed to decreased T-cell migration to secondary lymphoid organs (SLOs), reduced in vivo proliferation within these organs, and fewer cytokine-producingdonorT cells during GVHD development. Moreover, these defects inCdk5 (-/-) T-cell function are associated with altered CCR7 signaling following ligation by CCL19, a receptor: ligand interaction critical for T-cell migration into SLOs. Although Cdk5 activity in donor T cells contributed to graft-versus-tumor effects, pharmacologic inhibition of Cdk5 preserved leukemia-free survival. Collectively, our data implicate Cdk5 in allogeneic T-cell responses after HCT and as an important new target for therapeutic intervention.

Late term complications of hematopoietic cell transplantation (HCT) are numerous and include incomplete engraftment. One possible mechanism of incomplete engraftment after HCT is cytokine-mediated suppression or dysfunction of the bone marrow microenvironment. Mesenchymal stromal cells (MSC) elaborate cytokines that nurture or stimulate the marrow microenvironment by several mechanisms. Administration of exogenous MSCs, in the setting of incomplete engraftment, may modulate the bone marrow milieu and improve blood count recovery.

Haplo-HCT has emerged as an alternative for allogeneic transplantation for patients who do not have a matched donor. Delayed immune reconstitution, GVHD, and relapse remain obstacles to widespread use of haplo-HCT. FTY720 is a novel immunosuppressant that is FDA approved for multiple sclerosis (MS). It had been shown previously that FTY720 ameliorates GVHD in murine model without impairing graft vs leukemia (GVL) effect (Kim YM et al. J. Clin. Invest. 2003; 111:659-69); however, the FTY720 doses given in these studies were 20 times higher than the comparative human doses that are approved for treatment of MS. It had been shown that FTY720 has an activity against lymphoid malignancies. FTY720, administered in clinically-comparabledoses, ameliorates GVHD in a murine haplo-HCT model. To model GVHD in the haplo-HCT setting, we transplanted 2 x 106 T cells + 5 x 106 total bone marrow cells from B6D2F1 into 5 B6D2F1 mice (as control group) and from C57BL/6 into 20 B6D2F1 mice (as experimental group). Among the 20 B6D2F1 recipient mice, 10 mice received 5 μcg/kg/day of FTY720 starting on day 2 throughout the transplant, while 10 mice received placebo. Mice were monitored daily for survival and scored weekly for GVHD; by weight, posture, mobility, skin, and fur changes (Cooke et al. 1996 Blood88:3230). Mice were bled weekly to monitor engraftment of granulocytes and mononuclear cells and for reconstitution of T and B-cells. All B6D2F1 mice that received haplo-HCT, but none of the B6D2F1 mice that received syngeneic HCT, developed signs of GVHD. B6D2F1 mice that were treated with FTY720 had significantly lower GVHD scores compared to untreated mice (p=0.0071; Figure A). All haplo-HCT recipient mice died from GVHD but there was a trend toward improved survival in mice that received FTY720 (37.5 vs. 30 days), although the difference was not statistically significant (p=0.3). All recipient mice reconstituted mononuclear cells and granulocytes in a median of 7 days with no significant difference between the groups. B-cells were not reconstituted in recipient B6D2F1 mice during the observed period. Of interest, FTY720 treated mice showed enhanced recovery of T cell compartment (biased towards CD4+ T cells with na•ve, CD44loCD62L+phenotype) when compared to untreated B6D2F1 mice (Figure B). Fingolimod ameliorates GVHD in a murine model of haplo-HCT and may lead to improved T-cell reconstitution. Given its demonstrated safety profile in MS, its activity against GVHD in clinically-achievable doses, and its ability to stimulate recovery of T-cells, further clinical development of fingolimod in haplo-HCT setting may be warranted; particularly for lymphoid malignancies.Figure B: Recovery of T-cell compartment by Day +49 after transplant.2View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Mesenchymal stromal cells (MSCs) have shown promise as treatment for graft-versus-host disease (GvHD) following allogeneic bone marrow transplantation (alloBMT). Mechanisms mediating in vivo effects of MSCs remain largely unknown, including their biodistribution following infusion. To this end, human bone-marrow derived MSCs (hMSCs) were injected via carotid artery (IA) or tail vein (TV) into allogeneic and syngeneic BMT recipient mice. Following xenogeneic transplantation, MSC biodistribution was measured by bioluminescence imaging (BLI) using hMSCs transduced with a reporter gene system containing luciferase and by scintigraphic imaging using hMSCs labeled with [(99m)Tc]-HMPAO. Although hMSCs initially accumulated in the lungs in both transplant groups, more cells migrated to organs in alloBMT recipient as measured by in vivo BLI and scintigraphy and confirmed by ex vivo BLI imaging, immunohistochemistry and quantitative RT-PCR. IA injection resulted in persistent whole-body hMSC distribution in alloBMT recipients, while hMSCs were rapidly cleared in the syngeneic animals within one week. In contrast, TV-injected hMSCs were mainly seen in the lungs with fewer cells traveling to other organs. Summarily, these results demonstrate the potential use of IA injection to alter hMSC biodistribution in order to more effectively deliver hMSCs to targeted tissues and microenvironments.

Idiopathic pneumonia syndrome (IPS) is an acute, noninfectious lung disorder associated with high morbidity and mortality after hematopoietic cell transplantation. Previous studies have suggested a role for TNFα in the pathogenesis of IPS. We report a multicenter phase II trial investigating a soluble TNF-binding protein, etanercept (Enbrel, Amgen, Thousand Oaks, CA), for the treatment of pediatric patients with IPS. Eligible patients were < 18 years old, within 120 days after transplantation, and with radiographic evidence of a diffuse pneumonitis. All patients underwent a pretherapy broncho-alveolor lavage (BAL) to establish the diagnosis of IPS. Systemic corticosteroids (2.0 mg/kg/day) plus etanercept (.4 mg/kg twice weekly × 8 doses) were administered. Response was defined as survival and discontinuation of supplemental oxygen support by day 28 of study. Thirty-nine patients (median age, 11 years; range, 1 to 17) were enrolled, with 11 of 39 patients nonevaluable because of identification of pathogens from their pretherapy BAL. In the remaining 28 patients, the median fraction of inspired oxygen at study entry was 45%, with 17 of 28 requiring mechanical ventilation. Complete responses were seen in 20 (71%) patients, with a median time to response of 10 days (range, 1 to 24). Response rates were higher for patients not requiring mechanical ventilation at study entry (100% versus 53%, P = .01). Overall survival at 28 days and 1 year after therapy were 89% (95% confidence interval [CI], 70% to 96%) and 63% (95% CI, 42% to 79%), respectively. Plasma levels of proinflammatory cytokines were significantly increased at onset of therapy, subsequently decreasing in responding patients. The addition of etanercept to high-dose corticosteroids was associated with high response rates and survival in children with IPS.