Graft-versus-host disease (GVHD) represents a major hurdle impeding the efficacy of allogeneic bone marrow transplantation (BMT). Bortezomib is a proteasome inhibitor that was recently approved for treatment of myeloma. We found that bortezomib potently inhibited in vitro mixed lymphocyte responses and promoted the apoptosis of alloreactive T cells. Bortezomib given at the time of allogeneic BMT in mice resulted in significant protection from acute GVHD. Reductions in GVHD-associated parameters and biological evidence of proteasome inhibition were observed with this regimen but with no adverse effects on long-term donor reconstitution. Assessment of graft-versus-tumor responses in advanced leukemia-bearing mice demonstrated that only the combination of allogeneic BMT and T cells with bortezomib promoted significant increases in survival. Increased cytotoxic T cell killing of the tumor was also observed. Thus, the combination of proteasome inhibition with selective immune attack can markedly increase the efficacy of BMT in cancer

We previously reported that interleukin-10 (IL-10) and transforming growth factor (TGF)-␤ treatment of primary mixed lymphocyte reaction (MLR) cultures resulted in secondary alloantigen-specific hyporesponsiveness and protection from graft-versus-host disease (GVHD) lethality. Here, we report that CD4

IntroductionThe wider application of bone marrow transplantation (BMT) has been limited, in part, by graft-versus-host disease (GVHD) complications. Human and mouse mesenchymal stem cells (MSCs) have been shown to suppress allogeneic-induced and nonspecific mitogen-induced T-cell proliferation in vitro (reviewed in detail 1,2 ). Implicated suppressive mechanisms have included interleukin (IL)-10, 3 transforming growth factor (TGF)-␤, hepatocyte growth factor, 4 indoleamine 2,3 dioxygenase (IDO), 5 nitric oxide, 6 prostaglandin (PG) E 2 , 7 increased T-regulatory cells (Tregs), 8 and activation of the PD-1-negative costimulatory pathway. 9 In vivo, there have been conflicting data regarding the potential of MSCs to suppress GVHD. [10][11][12] Multipotent adult progenitor cells (MAPCs) are nonhematopoietic stem cells that can be copurified with MSCs from BM cells. MAPCs express the pluripotent state-specific transcription factors Oct-3/4 and Rex-1, and can differentiate into cell types representative of all 3 germ layers 13,14 ; thus, MAPCs are generally believed to be a more primitive cell type than MSCs. MSCs kept for prolonged periods in culture tend to lose their differentiation capabilities and undergo senescence at approximately 20 to 40 population doublings. 15,16 In contrast to MSCs, MAPCs have an average telomere length that remained constant for up to 100 population doublings in vitro. 13 Based upon their differential potential and reduced senescence, MAPCs have been considered as a potentially desirable nonhematopoietic stem cell source for use in allogeneic BMT. In fact, a multicenter phase 1 open label clinical trial of MultiStem, based upon MAPC technology, was initiated in 2008.In this study, we sought to determine whether MAPCs might be useful for GVHD prevention. We demonstrate that murine MAPCs are potently immune suppressive in vitro and can reduce GVHD lethality in vivo when present in the spleen, a site of initial allopriming, early post-BMT. Furthermore, we identify the mechanism of action MAPCs use to elicit T-cell inhibition and reduce GVHD-induced tissue injury in vivo. Methods MiceBALB/c (H2 d ), C57BL/6 (H2 b ; termed B6), and B10.BR (H2 k ) mice were purchased from The Jackson Laboratory or the National Institutes of Health (NIH). All mice were housed in a specific pathogen-free facility in microisolator cages and used at 8 to 12 weeks of age in protocols approved by the Institutional Animal Care and Use Committee of the University of Minnesota. MAPC isolation and cultureMAPCs were isolated from B6 and BALB/c BM, as described. 13 Briefly, BM was plated in Dulbecco modified Eagle medium/MCDB containing 10 ng/mL epidermal growth factor (Sigma-Aldrich), platelet-derived growth factor-BB (R&D Systems), leukemia inhibitory factor (Chemicon International), 2% fetal calf serum (HyClone), 1ϫ selenium-insulin-transferrinethanolamine, 0.2 mg/mL linoleic acid-bovine serum albumin (BSA), 0.8 mg/mL BSA, 1ϫ chemically defined lipid concentrate, and 1ϫ ␣-mercaptoethanol (all from Sigma-Aldrich). Cells...

Multipotent adult progenitor cells (MAPCs) are marrow-derived pluripotent stem cells with a broad differentiation potential. We sought to identify factors that affect adoptively transferred MAPCs. In vitro, MAPCs expressed low levels of major histocompatibility complex (MHC) antigens, failed to stimulate CD4 ؉ and CD8 ؉ T-cell alloresponses, and were targets of NK cytolysis. To study in vivo biodistribution, we labeled MAPCs with luciferase for sequential quantification of bioluminescence and DsRed2 for immunohistochemical analysis. C57BL /6 MAPCs were infused intravenously into C57BL /6, Rag-2 ؊/؊ (T-and B-cell-deficient), and Rag-2 ؊/؊ /IL-2R␥ c ؊/؊ (T-, B-, and NK-cell-deficient) mice. In C57BL /6 mice, MAPCs were transiently detected only in the chest compared with long-term persistence in T-and B-celldeficient mice. NK depletion reduced MAPC elimination. Because the lungs were the major uptake site after intravenous injection, intra-arterial injections were tested and found to result in more widespread biodistribution. Widespread IntroductionMultipotent adult progenitor cells (MAPCs) are a novel class of stem cells derived from adult tissues, including bone marrow, muscle, and brain. [1][2][3][4][5] MAPCs are pluripotent, and a single MAPC injected into a blastocyst contributes to all tissues, including skeletal muscle, cardiac muscle, liver, lung, intestine, central nervous system, skin, spleen, blood, and marrow. 2 Clonal MAPCs are able to differentiate in vitro into various lineages of mesodermal, ectodermal, and endodermal origin and contribute to terminally differentiated tissues when postnatally grafted. [1][2][3][4][5] This capacity is enhanced during injury, 2 suggesting a possible role for MAPCs in the regeneration of tissues injured by chemotherapy or radiation during conditioning for hematopoietic stem cell transplantation.For any clinical application of MAPCs, it is critical to understand the mechanisms that can lead to immune rejection of the infused cells. Because undifferentiated MAPCs express low levels of major histocompatibility complex (MHC) class I, MAPCs may be ideal targets for NK cell-mediated killing. If so, NK-cell resistance may serve as a barrier to the infusion of undifferentiated MAPCs in vivo. As a part of innate immune defense, NK cells can kill infected or transformed tissues before sensitization and without restriction by MHC antigens. [6][7][8] Because NK function can influence the outcome of hematopoietic cell transplantation, 9,10 we reasoned that NK activity may have a role in MAPC homing and rejection.Other important considerations in the use of MAPCs include understanding the host factors that might affect in vivo MAPC biodistribution and persistence. Therefore, we analyzed the role of MAPCs as targets of immune response as a function of engraftment and persistence of infused MAPCs using a 2-reporter gene system with red fluorescence protein derived from Discosoma coral (DsRed2) 11 and firefly luciferase. 12 Donor MAPC-derived optical signatures of fluorescent (DsRed2...

PD-1/PD-L1 pathway inhibition is effective against advanced renal cell carcinoma, although results are variable and may depend on host factors, including the tumor microenvironment. Vascular-targeted photodynamic (VTP) therapy with the photosensitizer WST11 induces a defined local immune response, and we sought to determine whether this could potentiate the local and systemic antitumor response to PD-1 pathway inhibition. Using an orthotopic Renca murine model of renal cell carcinoma that develops lung metastases, we treated primary renal tumors with either VTP alone, PD-1/PD-L1 antagonistic antibodies alone, or a combination of VTP and antibodies and then examined treatment responses, including immune infiltration in primary and metastatic sites. Modulation of PD-L1 expression by VTP in human xenograft tumors was also assessed. Treatment of renal tumors with VTP in combination with systemic PD-1/PD-L1 pathway inhibition, but neither treatment alone, resulted in regression of primary tumors, prevented growth of lung metastases, and prolonged survival in a preclinical mouse model. Analysis of tumor-infiltrating lymphocytes revealed that treatment effect was associated with increased CD8:regulatory T cell (Treg) and CD4FoxP3-:Treg ratios in primary renal tumors and increased T-cell infiltration in sites of lung metastasis. Furthermore, PD-L1 expression is induced following VTP treatment of human renal cell carcinoma xenografts. Our results demonstrate a role for local immune modulation with VTP in combination with PD-1/PD-L1 pathway inhibition for generation of potent local and systemic antitumor responses. This combined modality strategy may be an effective therapy in cancers resistant to PD-1/PD-L1 pathway inhibition alone. .

OBJECTIVE To evaluate a multimodal strategy aimed at treating all sites of disease that provides a rapid readout of success or failure in men presenting with non-castrate metastatic prostate cancers that are incurable with single modality therapy. MATERIALS AND METHODS Twenty selected men with oligometastatic M1a (extrapelvic nodal disease) or M1b (bone disease) at diagnosis were treated using a multimodal approach that included androgen deprivation, radical prostatectomy plus pelvic lymphadenectomy (retroperitoneal lymphadenectomy in the presence of clinically positive retroperitoneal nodes), and stereotactic body radiotherapy to osseous disease or the primary site. Outcomes of each treatment were assessed sequentially. Androgen deprivation was discontinued in responding patients. The primary end point was an undetectable prostate-specific antigen (PSA) after testosterone recovery. The goal was to eliminate all detectable disease. RESULTS Each treatment modality contributed to the outcome: 95% of the cohort achieved an undetectable PSA with multimodal treatment, including 25% of patients after androgen deprivation alone and an additional 50% and 20% after surgery and radiotherapy, respectively. Overall, 20% of patients (95% confidence interval: 3%–38%) achieved the primary end point, which persisted for 5, 6, 27+, and 46+ months. All patients meeting the primary end point had been classified with M1b disease at presentation. CONCLUSION A sequentially applied multimodal treatment strategy can eliminate detectable disease in selected patients with metastatic spread at diagnosis. The end point of undetectable PSA after testosterone recovery should be considered when evaluating new approaches to rapidly set priorities for large-scale testing in early metastatic disease states and to shift the paradigm from palliation to cure.

For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40–80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 103-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs.

Consumption of bifidobacteria as a dietary adjunct has received considerable attention for its possible role in the maintenance of gastrointestinal health. However, speculation exists about these presumed health benefits because of an inability to assess the fate and mechanism of action of ingested bifidobacteria. Thus, our objective was to examine the fate of ingested bifidobacteria through the gastrointestinal tract. Variations in the highly conserved 16S ribosomal DNA (rDNA) of bifidobacteria from six male subjects (18 to 35 y old) were assessed by restriction fragment length polymorphism (RFLP) analysis. During the 16-d study, 10(10) colony-forming units (CFU) of a commercially available bifidobacteria were delivered to subjects in fluid milk for each of 8 d. During the remaining 8 d, subjects consumed milk without bifidobacteria. Feces were collected at 4-d intervals and plated on selective media. For each subject, 10-15 colonies were randomly selected and used as template for PCR-amplification of 16S rDNA. 16S rDNA was restriction digested and resolved by electrophoresis. The 16S rDNA-RFLP of the ingested bifidobacteria was unique compared with bifidobacteria found in subjects prior to the feeding study. When subjects consumed bifidobacteria, a 16S rDNA-RFLP identical to that of the ingested bifidobacteria was observed in feces. The concentration of the ingested bifidobacteria in feces increased to 67.2 +/- 8.5% (mean +/- SEM) of total bifidobacteria. After feeding stopped, the ingested bifidobacteria diminished and became undetectable. Using this molecular approach to monitor ingested bifidobacteria, we demonstrate the kinetics of passage of this organism through the gastrointestinal tract of healthy humans.