HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells

OBJECTIVE: Mesenchymal stem cells (MSC) do not elicit alloreactive lymphocyte responses due to immune modulations. We investigated the immunologic properties of MSC after differentiation along three lineages: bone, cartilage, and adipose. METHODS AND RESULTS: Flow cytometry showed that undifferentiated MSC express HLA class I but not class II, although HLA class II was present intracellularly as detected by Western blot. Addition of interferon gamma (IFN-gamma) for 48 hours induced greater than 90% of cells to express HLA class II. No lymphocyte response was induced by allogeneic irradiated MSC as stimulators. Results were similar using MSC pretreated with IFN-gamma. After growth of cells in medium to induce differentiation to bone, cartilage, or adipose for 6 or 12 days, the expression of HLA class I increased but no class II was detected on the cell surface. The ability to upregulate HLA class II on the cell surface after exposure to IFN-gamma for 48 hours was clearly diminished after the cells had been cultured in differentiation medium for 6 or 12 days, with only 10% of cells expressing HLA class II. Using MSC grown in osteogenic, chondrogenic, or adipogenic medium as stimulator cells, no lymphocyte alloreactivity was seen, even if differentiated MSC had been pretreated with IFN-gamma. MSC inhibit mixed lymphocyte cultures, particularly after osteogenic differentiation. This suppression was further enhanced by IFN-gamma. CONCLUSIONS: Undifferentiated and differentiated MSC do not elicit alloreactive lymphocyte proliferative responses and modulate immune responses. The findings support that MSC can be transplantable between HLA-incompatible individuals.     

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8- and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T = 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions.     

Microtransplantation in older patients with AML: A pilot study of safety,efficacy and immunologic effects

Older AML patients have low remission rates and poor survival outcomes with standard chemotherapy. Microtransplantation (MST) refers to infusion of allogeneic hematopoietic stem cells without substantial engraftment. MST has been shown to improve clinical outcomes compared with chemotherapy alone. This is the first trial reporting on broad correlative studies to define immunologic mechanisms of action of MST in older AML patients. Older patients with newly diagnosed AML were eligible for enrollment, receiving induction chemotherapy with cytarabine (100 mg/m2) on days 1-7 and idarubicin (12 mg/m2) on days 1-3 (7 + 3). MST was administered 24 hours later. Patients with complete response (CR) were eligible for consolidation with high dose cytarabine (HiDAC) and a second cycle of MST. Responses were evaluated according to standard criteria per NCCN. Immune correlative studies were performed. Sixteen patients were enrolled and received 7 + 3 and MST (median age 73 years). Nine (56%) had high-risk and seven (44%) had standard-risk cytogenetics. Ten episodes of CRS were observed. No cases of GVHD or treatment-related mortality were reported. Event-free survival (EFS) was 50% at 6 months and 19% at 1 year. Overall survival (OS) was 63% at 6 months and 44% at 1 year. Donor microchimerism was not detected. Longitudinal changes were noted in NGS, TCR sequencing, and cytokine assays. Addition of MST to induction and consolidation chemotherapy was well tolerated in older AML patients. Inferior survival outcomes in our study may be attributed to a higher proportion of very elderly patients with high-risk features. Potential immunologic mechanisms of activity of MST include attenuation of inflammatory cytokines and emergence of tumor-specific T cell clones.     

免疫因子对慢性免疫性血小板减少性紫癜骨髓巨核祖细胞体外生长的影响

目的 :探讨慢性免疫性血小板减少性紫癜 (CITP)骨髓巨核祖细胞生长成熟与免疫因素的关系。方法 :通过巨核祖细胞体外培养 ,观察CITP骨髓巨核祖细胞生长和成熟情况 ,并观察抗CD80抗体及干扰素α 2b(IFN α 2b)对CITP和对照组巨核祖细胞生长和成熟的影响 ,并与对照组比较。结果 :①CITP组的各种集落数(CFU MK、BFU MK、mCFU MK)均低于对照组 ,两组间集落数的均值比较差异有统计学意义 (P <0 .0 5 )。CITP组的直径和面积均低于对照组 ,两组间直径和面积均值比较差异有统计学意义 (P <0 .0 5 )。②加入抗CD80抗体的CITP的巨核祖细胞集落生成数明显增多 (P <0 .0 5 )。③IFN α 2b对两组的总集落均有抑制 ,CITP比对照组的巨核祖细胞集落生成受抑制程度低 (P <0 .0 5 )。结论 :CITP巨核祖细胞的集落生成和成熟度较低。抗CD80抗体和IFN α 2b对CITP巨核祖细胞的集落形成均有影响     

Naive B cells generate regulatory T cells in the presence of a mature immunologic synapse

Naive B cells are ineffective antigen-presenting cells and are considered unable to activate naive T cells. However, antigen-specific contact of these cells leads to stable cell pairs that remain associated over hours in vivo. The physiologic role of such pairs has not been evaluated. We show here that antigen-specific conjugates between naive B cells and naive T cells display a mature immunologic synapse in the contact zone that is absent in T-cell-dendritic-cell (DC) pairs. B cells induce substantial proliferation but, contrary to DCs, no loss of L-selectin in T cells. Surprisingly, while DC-triggered T cells develop into normal effector cells, B-cell stimulation over 72 hours induces regulatory T cells inhibiting priming of fresh T cells in a contact-dependent manner in vitro. In vivo, the regulatory T cells home to lymph nodes where they potently suppress immune responses such as in cutaneous hypersensitivity and ectopic allogeneic heart transplant rejection. Our finding might help to explain old observations on tolerance induction by B cells, identify the mature immunologic synapse as a central functional module of this process, and suggest the use of naive B-cell-primed regulatory T cells, "bTregs," as a useful approach for therapeutic intervention in adverse adaptive immune responses.     

Effects of lipid A on the immunologic properties of liposomal model membranes

Incorporation of lipid A influences the immunogenic properties of liposomal model membranes that carry N-(hapten)-substituted derivatives of phosphatidylethanolamine as antigenic determinants. These effects of lipid A include a marked enhancement of the hapten-specific response; abolition of the requirement of critical threshold epitope density for liposomal immunogenicity; and conversion of liposomes from a thymus-independent type 2 to a thymus-independent type 1 immunogen. Recent experiments demonstrate that an optimal in vitro response to liposomes--although originally classified as thymus-independent on the basis of conventional criteria--requires two T cell factors, interleukin-2 and interleukin-X, which are also involved in the response to a classic thymus-dependent immunogen. Incorporation of lipid A has no qualitative effect on the dependence of liposomal immunogenicity on these lymphokines.     

Effect of silencing a disintegrin and metalloprotease 12 expression on self-renewal capacity of CD133 positive giloma cells

<正>Objective To investigate the inhibitory effect of a disintegrin and metalloprotease 12 silenced by shR-NA on self-renewal capacity of CD133 positive giloma cells.Methods The shRNA recombinant lentivirus aimed at silencing ADAM12 was prepared.Human glioma cells U87were employed in this study and assigned into three     

Leukemic cells and aberrant phenotypes in acute leukemia patients: a flow cytometry analysis

The aim of this study was to evaluate the heterogeneity of immunophenotype features in acute leukemia patients and to detect the presence of leukemia-associated immunophenotypes. We prospectively investigated the phenotype of blast cells from 44 adult acute leukemia patients using a large panel of monoclonal antibodies by multiparametric flow cytometry. Thirty-three patients were classified as AML according to the FAB classification. Eleven patients were diagnosed as ALL (10 cases B-ALL, 1 case T-ALL) according to both FAB and immunnophenotyping. We found leukemia-associated phenotypes in 28 of 33 AML patients (84.8%) and in 8 of 11 ALL patients (72.7%). In 61.1% of patients more than one aberrant phenotype was observed. Linear infidelity was the most frequent aberrancy in both AML (64.3%) and ALL (37.5%) subgroups. The present study shows that MFC is a helpful method for sufficient identification of leukemic cells and for determination of blast cells immunophenotype heterogeneity. The double stain flow cytometry in our study revealed aberrant phenotypes in up to 81.8% patients.     

Receptor specificity in the self-renewal and differentiation of primary multipotential hemopoietic cells.

To determine whether cytokine-induced signals generate unique responses in multipotential hemopoietic progenitor cells, the signaling domains of 3 different growth factor receptors (Mpl, granulocyte-colony-stimulating factor [G-CSF] receptor, and Flt-3) were inserted into mouse primary bone marrow cells. To circumvent the activation of endogenous receptors, each signaling domain was incorporated into an FK506 binding protein (FKBP) fusion to allow for its specific activation using synthetic FKBP ligands. Each signaling domain supported the growth of Ba/F3 cells; however, only Mpl supported the sustained growth of transduced marrow cells, with a dramatic expansion of multipotential progenitors and megakaryocytes. These findings demonstrate that the self-renewal and differentiation of multipotential progenitor cells can be influenced through distinct, receptor-initiated signaling pathways.     

Immunomodulation with thiabendazole: a review of immunologic properties and efficacy in combined modality cancer therapy.

Thiabendazole (TBZ) appears to be an immunorestorative agent, demonstrating maximum immunopotentiation in the immunosuppressed host. Initial in vivo and in vitro immune studies indicate that the drug is most effective when given 24 hours prior to or at the time of administration of antigen. Single doses are more effective than multiple daily doses. One cell population potentiated by TBZ is the macrophage, either by direct activation or secondary to increased lymphokine production. As an adjuvant to conventional cancer treatment modalities, TBZ needs the same host setting as seen with many immunopotentiators. Tumor bulk must be reduced by the primary modality. A dose schedule must be developed in conjunction with each primary modality employed, ie, timing is critical. As an adjuvant in cancer therapy, the drug is most effective when given every other day. Depending upon the primary modality employed, adjuvant TBZ may require dose adjustment.     

Maintaining the self-renewal and differentiation potential of human CD34+ hematopoietic cells using a single genetic element

Hematopoiesis is a complex process involving hematopoietic stem cell (HSC) self-renewal and lineage commitment decisions that must continue throughout life. Establishing a reproducible technique that allows for the long-term ex vivo expansion of human HSCs and maintains self-renewal and multipotential differentiation will allow us to better understand these processes, and we report the ability of the leukemia-associated AML1-ETO fusion protein to establish such a system. AML1-ETO-transduced human CD34+ hematopoietic cells routinely proliferate in liquid culture for more than 7 months, remain cytokine dependent for survival and proliferation, and demonstrate self-renewal of immature cells that retain both lymphoid and myeloid potential in vitro. These cells continue to express the CD34 cell surface marker and have ongoing telomerase activity with maintenance of telomere ends, however they do not cause leukemia in nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. Identification of the signaling pathways that are modulated by AML1-ETO and lead to the self-renewal of immature human progenitor cells may assist in identifying compounds that can efficiently expand human stem and progenitor cells ex vivo.     

Asymmetric segregation and self-renewal of hematopoietic stem and progenitor cells with endocytic Ap2a2

The stem cell-intrinsic model of self-renewal via asymmetric cell division (ACD) posits that fate determinants be partitioned unequally between daughter cells to either activate or suppress the stemness state. ACD is a purported mechanism by which hematopoietic stem cells (HSCs) self-renew, but definitive evidence for this cellular process remains open to conjecture. To address this issue, we chose 73 candidate genes that function within the cell polarity network to identify potential determinants that may concomitantly alter HSC fate while also exhibiting asymmetric segregation at cell division. Initial gene-expression profiles of polarity candidates showed high and differential expression in both HSCs and leukemia stem cells. Altered HSC fate was assessed by our established in vitro to in vivo screen on a subcohort of candidate polarity genes, which revealed 6 novel positive regulators of HSC function: Ap2a2, Gpsm2, Tmod1, Kif3a, Racgap1, and Ccnb1. Interestingly, live-cell videomicroscopy of the endocytic protein AP2A2 shows instances of asymmetric segregation during HSC/progenitor cell cytokinesis. These results contribute further evidence that ACD is functional in HSC self-renewal, suggest a role for Ap2a2 in HSC activity, and provide a unique opportunity to prospectively analyze progeny from HSC asymmetric divisions.     

In vivo proliferative and differential properties of murine bone marrow cells separated on the basis of rhodamine-123 retention

We have studied the in vivo proliferation and differential properties of murine bone marrow cells (BMC) differing in mitochondrial activity. Following centrifugal elutriation, the cells were sorted on the basis of rhodamine-123 (Rh) fluorescence intensity within a predetermined light scatter window. Unfractionated BMC colonized the spleen with a characteristic preponderance of erythrocytic nodules upon infusion into heavily irradiated mice. In contrast, Rh-dull BMC formed few macroscopic spleen colonies up to day 12, but relatively more microscopic colonies when spleens were sectioned, and the majority of the colonies showed megakaryocytic (Meg) and/or granulocytic (G) differentiation. On day 16 many megakaryocytes were found in these spleens, and very large erythroid colonies had developed since day 12. In contrast, Rh-bright BMC were highly enriched for cells forming erythrocytic spleen colonies that disintegrated after day 12, but the percentage of G and Meg colonies was low. Spleens obtained from recipients of Rh-bright cells contained a negligible number of megakaryocytes on day 16. Apparently, the mitochondrial content of cells that form hemopoietic colonies in the spleen related to the onset and duration of their lineage expression, and to the relative sizes of the lineage compartments. Thus, the colony founders of the majority of spleen surface nodules observed on days 8 and 12 are cells with high mitochondrial activity forming transient erythrocytic nodules. Such spleen colonies represent the bulk of the countable nodules that form the basis of the widely applied murine "stem cell" assay.     

Hemagglutinin polymorphism as the basis for low- and high-yield phenotypes of swine influenza virus.

Single amino acid substitutions at the rim of the receptor binding site of the hemagglutinin molecule of swine influenza virus markedly influence the replicative capacity of the virus in chicken embryos, Madin-Darby canine kidney cells (MDCK), and swine as well as its antigenic phenotype. Mutants of low-yield (L) phenotype replicate poorly in chicken embryos and produce small plaques in MDCK cells but are highly infective for swine. Such mutants have lysine at position 153 and glycine at position 155 of the hemagglutinin (residues 156 and 158 in the H3 model). High-yield (H) mutants have the converse replicative characteristics and can be antigenically distinguished from L mutants (and from each other) based on their differential reactivity with two monoclonal antibodies, 9C8 and Sa-13. H mutants differ from L mutants in that the H mutants express glutamic acid at either position 153 or 155. L and H mutants act in an allelic fashion in effecting predictable one-step adaptation to different hosts. Selection for replication (e.g., high-yielding) phenotype results in concordant pleiotropic change in antigenic phenotype and in genotype. Conversely, immunoselection leads to change in replicative phenotype. Although the mechanism by which these mutations affect viral replication has not yet been defined, they may reflect differences in the affinity of each mutant for different host receptors.