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We have previously shown that a granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-15 (IL-15) 'fusokine' (GIFT15) exerts immune suppression via aberrant signaling through the IL-15 receptor on lymphomyeloid cells. We show here that ex vivo GIFT15 treatment of mouse splenocytes generates suppressive regulatory cells of B cell ontogeny (hereafter called GIFT15 B(reg) cells). Arising from CD19+ B cells, GIFT15 B(reg) cells express major histocompatibility complex class I (MHCI) and MHCII, surface IgM and IgD, and secrete IL-10, akin to previously described B10 and T2-MZP B(reg) cells, but lose expression of the transcription factor PAX5, coupled to upregulation of CD138 and reciprocal suppression of CD19. Mice with experimental autoimmune encephalomyelitis went into complete remission after intravenous infusion of GIFT15 B(reg) cells paralleled by suppressed neuroinflammation. The clinical effect was abolished when GIFT15 B(reg) cells were derived from mmicroMT (lacking B cells), MHCII-knockout, signal transducer and activator of transcription-6 (STAT-6)-knockout, IL-10-knockout or allogeneic splenocytes, consistent with a pivotal role for MHCII and IL-10 by sygeneic B cells for the observed therapeutic effect. We propose that autologous GIFT15 B(reg) cells may serve as a new treatment for autoimmune ailments.

Recent studies involving bone marrow mesenchymal stromal cells (MSCs) demonstrated that interferon (IFN)-gamma stimulation induces major histocompatibility complex (MHC) class II-mediated antigen presentation in MSCs both in vitro and in vivo. Concordantly, we investigated the ability of MSCs to present extracellular antigen through their MHC class I molecules, a process known as cross-presentation. Using an in vitro antigen presentation assay, we demonstrated that murine MSCs can cross-present soluble ovalbumin (OVA) to naive CD8(+) T cells from OT-I mice. Cross-presentation by MSC was proteasome dependent and partly dependent on transporter associated with antigen-processing molecules. Pretreatment of MSC with IFN-gamma increased cross-presentation by up-regulating antigen processing and presentation. However, although the transcription of the transporter associated with antigen processing-1 molecules and the immunoproteasome subunit LMP2 induced by IFN-gamma was inhibited by transforming growth factor-beta, the overall cross-presentation capacity of MSCs remained unchanged after transforming growth factor-beta treatment. These observations were validated in vivo by performing an immune reconstitution assay in beta(2)-microglobulin(-/-) mice and show that OVA cross-presentation by MSCs induces the proliferation of naive OVA-specific CD8(+) T cells. In conclusion, we demonstrate that MSCs can cross-present exogenous antigen and induce an effective CD8(+) T-cell immune response, a property that could be exploited as a therapeutic cell-based immune biopharmaceutic for the treatment of cancer or infectious diseases.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and remains a major cause of mortality in children with recurrent disease and in adults. Despite observed graft-versus-leukemia effects after stem cell transplantation, successful immune therapies for ALL have proven elusive. We previously reported immunostimulatory oligodeoxynucleotides containing CpG motifs (CpG ODN) enhance allogeneic T(h)1 responses and reduce leukemic burden of primary human ALL xenografts. To further the development of CpG ODN as a novel ALL therapy, we investigated the antileukemia activity induced by CpG ODN in a transplantable syngeneic pre-B ALL model. CpG ODN induced early killing of leukemia by innate immune effectors both in vitro and in vivo. Mice were treated with CpG ODN starting 7 days after injection with leukemia to mimic a minimal residual disease state and achieved T cell-dependent remissions of more than 6 months. In addition, mice in remission after CpG ODN treatment were protected from leukemia rechallenge, and adoptive transfer of T cells from mice in remission conferred protection against leukemia growth. To our knowledge, this is the first demonstration that CpG ODN induce a durable remission and ongoing immune-mediated protection in ALL, suggesting this treatment may have clinical utility in patients with minimal residual disease.

BACKGROUND: Despite the promising therapeutic potential of regulatory T cells (Treg) in animal studies of graft-versus-host disease (GVHD), little is known about their effect on human GVHD. Whether Treg are capable of ameliorating GVHD tissue damage has never been demonstrated in humans. It is also unknown whether Treg modulation of GVH histopathologic damage relies on their presence during effector T-cell priming, or whether allogeneic Treg are safe to use clinically. METHODS: To address these questions, we used an in vitro human skin explant GVHD model, which mimics the physiopathology of GVHD. First, donor"-derived CD8 T cells were stimulated with human leukocyte antigen-unmatched "recipient" dendritic cells (priming phase)�

Natural killer T (NKT) cells are innate-like lymphocytes that recognize lipid antigens and have been shown to enhance B-cell activation and antibody production. B cells typically recruit T-cell help by presenting internalized antigens recognized by their surface antigen receptor. Here, we demonstrate a highly efficient means whereby human B cells present lipid antigens to NKT cells, capturing the antigen using apolipoprotein E (apoE) and the low-density lipoprotein receptor (LDL-R). ApoE dramatically enhances B-cell presentation of alpha-galactosylceramide (alphaGalCer), an exogenous CD1d presented antigen, inducing activation of NKT cells and the subsequent activation of B cells. B cells express the LDL-R on activation, and the activation of NKT cells by B cells is completely LDL-R dependent, as shown by blocking experiments and the complete lack of presentation when using apoE2, an isoform of apoE incapable of LDL-R binding. The dependence on apoE and the LDL-R is much more pronounced in B cells than we had previously seen in dendritic cells, which can apparently use alternate pathways of lipid antigen uptake. Thus, B cells use an apolipoprotein-mediated pathway of lipid antigen presentation, which constitutes a form of innate help for B cells by NKT cells.

The ability of NK cells to directly recognize pathogens and be activated via Toll-like receptors (TLR) is increasingly recognized. Nevertheless, controversial results on the NK cell ability to be directly activated by lipopolysaccharide (LPS), the ligand of TLR4, have been recently reported. To start elucidating the reasons explaining the contrasting observations of the literature, we focused on the potential role of currently used NK cell purification procedures to condition putative NK cell responsiveness to LPS. To do so, human NK cells were isolated by negative selection, using three different commercial kits, to be comparatively evaluated for the production of IFNgamma in response to ultra-pure LPS and/or IL-2. Despite the lack of surface TLR4, we found that two out of the three NK cell-enriched populations released IFNgamma (and one of the two, IL-12p70 as well) in response to the LPS plus IL-2 combination, whereas the last one did not. However, the two LPS plus IL-2-responsive NK cell populations were found variably contaminated with 6-sulfo LacNAc(+) dendritic cells (slanDC), demonstrated responsible for triggering, via the production of IL-12p70 in response to LPS, the release of IFNgamma by IL-2-stimulated NK cells. Accordingly, slanDC depletion completely abrogated the capacity to produce both IL-12p70 and IFNgamma in response to LPS plus IL-2 by slanDC-containing NK cells. Taken together, our data uncover that two commercially available kits, specifically designed to isolate NK cells by negative selection, also co-purify variable amounts of slanDC. The latter cells may dramatically affect the outcome of experiments carried on to evaluate NK cell responsiveness to TLR agonists such as LPS.

Here, we describe a procedure and first-in-man clinical effects of adoptive transfer of ex vivo expanded CD4+CD25+CD127- T regulatory cells (Tregs) in the treatment of graft versus host disease (GvHD). The cells were sorted from buffy coats taken from two family donors, expanded ex vivo and transferred to respective recipients who suffered from either acute or chronic GvHD. The therapy allowed for significant alleviation of the symptoms and reduction of pharmacologic immunosuppression in the case of chronic GvHD, while in the case of grade IV acute GvHD it only transiently improved the condition, for the longest time within all immunosuppressants used nonetheless.

The Msh2 mismatch repair (MMR) protein is critical for class switch recombination (CSR) events that occur in mice that lack the Smu tandem repeat (SmuTR) region (SmuTR(-/-) mice). The pattern of microhomology among switch junction sites in Msh2-deficient mice is also dependent on the presence or absence of SmuTR sequences. It is not known whether these CSR effects reflect an individual function of Msh2 or the function of Msh2 within the MMR machinery. In the absence of the SmuTR sequences, Msh2 deficiency nearly ablates CSR. We now show that Mlh1 or Exo1 deficiencies also eliminate CSR in the absence of the SmuTR. Furthermore, in SmuTR(-/-) mice, deficiencies of Mlh1 or Exo1 result in increased switch junction microhomology as has also been seen with Msh2 deficiency. These results are consistent with a CSR model in which the MMR machinery is important in processing DNA nicks to produce double-stranded breaks, particularly in sequences where nicks are infrequent. We propose that double-stranded break paucity in MMR-deficient mice leads to increased use of an alternative joining pathway where microhomologies are important for CSR break ligation. Interestingly, when the SmuTR region is present, deficiency of Msh2 does not lead to the increased microhomology seen with Mlh1 or Exo1 deficiencies, suggesting that Msh2 might have an additional function in CSR. It is also possible that the inability to initiate MMR in the absence of Msh2 results in CSR junctions with less microhomology than joinings that occur when MMR is initiated but then proceeds abnormally due to Mlh1 or Exo1 deficiencies.

The presence of interleukin-2 (IL-2)-producing human immunodeficiency virus type 1 (HIV-1)-specific CD4(+) T-cell responses has been associated with the immunological control of HIV-1 replication; however, the causal relationship between these factors remains unclear. Here we show that IL-2-producing HIV-1-specific CD4(+) T cells can be cloned from acutely HIV-1-infected individuals. Despite the early presence of these cells, each of the individuals in the present study exhibited progressive disease, with one individual showing rapid progression. In this rapid progressor, three IL-2-producing HIV-1 Gag-specific CD4(+) T-cell responses were identified and mapped to the following optimal epitopes: HIVWASRELER, REPRGSDIAGT, and FRDYVDRFYKT. Responses to these epitopes in peripheral blood mononuclear cells were monitored longitudinally to textgreater1 year postinfection, and contemporaneous circulating plasma viruses were sequenced. A variant of the FRDYVDRFYKT epitope sequence, FRDYVDQFYKT, was observed in 1/21 plasma viruses sequenced at 5 months postinfection and 1/10 viruses at 7 months postinfection. This variant failed to stimulate the corresponding CD4(+) T-cell clone and thus constitutes an escape mutant. Responses to each of the three Gag epitopes were rapidly lost, and this loss was accompanied by a loss of antigen-specific cells in the periphery as measured by using an FRDYVDRFYKT-presenting major histocompatibility complex class II tetramer. Highly active antiretroviral therapy was associated with the reemergence of FRDYVDRFYKT-specific cells by tetramer. Thus, our data support that IL-2-producing HIV-1-specific CD4(+) T-cell responses can exert immune pressure during early HIV-1 infection but that the inability of these responses to enforce enduring control of viral replication is related to the deletion and/or dysfunction of HIV-1-specific CD4(+) T cells rather than to the fixation of escape mutations at high frequencies.

Persistent viral infections are a major health concern worldwide. During persistent infection, overwhelming viral replication and the rapid loss of antiviral T-cell function can prevent immune-mediated clearance of the infection, and therapies to reanimate the immune response and purge persistent viruses have been largely unsuccessful. Adoptive immunotherapy using memory T cells is a highly successful therapeutic approach to eradicate a persistent viral infection. Understanding precisely how therapeutically administered memory T cells achieve clearance should improve our ability to terminate states of viral persistence in humans. Mice persistently infected from birth with lymphocytic choriomeningitis virus are tolerant to the pathogen at the T-cell level and thus provide an excellent model to evaluate immunotherapeutic regimens. Previously, we demonstrated that adoptively transferred memory T cells require recipient dendritic cells to effectively purge an established persistent viral infection. However, the mechanisms that reactivate and sustain memory T-cell responses during clearance of such an infection remain unclear. Here we establish that therapeutic memory T cells require CD80 and CD86 costimulatory signals to efficiently clear an established persistent viral infection in vivo. Early blockade of costimulatory pathways with CTLA-4-Fc decreased the secondary expansion of virus-specific CD8(+) and CD4(+) memory T cells as well as their ability to produce antiviral cytokines and purge the persistent infection. Late costimulation blockade also reduced virus-specific T-cell numbers, illustrating that sustained interactions with costimulatory molecules is required for efficient T-cell expansion. These findings indicate that antiviral memory T cells require costimulation to efficiently clear a persistent viral infection and that costimulatory pathways can be targeted to modulate the magnitude of an adoptive immunotherapeutic regimen.

In recent years, the differentiation of bone marrow cells (BMCs) into myocytes has been extensively investigated, but the findings remain inconclusive. The purpose of this study was to determine the conditions necessary to induce myogenic differentiation in short-term cultures of adult BMCs, and to identify the BMC subpopulation responsible for this phenomenon. We report that high-density cultures of murine hematopoietic BMCs gave rise to spontaneous beating cell clusters in the presence of vascular endothelial and fibroblast growth factors. These clusters originated from c-kit(pos) cells. The formation of the clusters could be completely blocked by adding a c-kit/tyrosine kinase inhibitor, Gleevec (imatinib mesylate; Novartis International, Basel, Switzerland, http://www.novartis.com), to the culture. Cluster formation was also blunted in BMCs from c-kit-deficient (Kit(W)/Kit(W-v)) mice. Clustered cells expressed cardiomyocyte-specific transcription factor genes Gata-4 and Nkx2.5, sarcomeric proteins beta-MHC and MLC-2v, and ANF and connexin-43. Immunostaining revealed alpha-sarcomeric actinin expression in more than 90% of clustered cells. Under electron microscopy, the clustered cells exhibited a sarcomeric myofiber arrangement and z-bands. This study defines the microenvironment required to achieve a reproducible in vitro model of beating, myogenic cell clusters. This model could be used to examine the mechanisms responsible for the postnatal myogenic differentiation of BMCs. Our results identify c-kit(pos) bone marrow hematopoietic cells as the source of the myogenic clusters.

The rising incidence of autoimmune diseases such as multiple sclerosis (MS) in developed countries might be due to a more hygienic environment, particularly during early life. To investigate this concept, we developed a model of neonatal exposure to a common pathogen-associated molecular pattern, LPS, and determined its impact on experimental autoimmune encephalomyelitis (EAE). Mice exposed to LPS at 2 wk of age showed a delayed onset and diminished severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE, induced at 12 wk, compared with vehicle-exposed animals. Spinal cord transcript levels of CD3epsilon and F4/80 were lower in LPS- compared with PBS-exposed EAE animals with increased IL-10 levels in the LPS-exposed group. Splenic CD11c(+) cells from LPS-exposed animals exhibited reduced MHC class II and CD83 expression but increased levels of CD80 and CD86 both before and during EAE. MOG-treated APC from LPS-exposed animals stimulated less T lymphocyte proliferation but increased expansion of CD4(+)FoxP3(+) T cells compared with APC from PBS-exposed animals. Neuropathological studies disclosed reduced myelin and axonal loss in spinal cords from LPS-exposed compared with PBS-exposed animals with EAE, and this neuroprotective effect was associated with an increased number of CD3(+)FoxP3(+) immunoreactive cells. Analyses of human brain tissue revealed that FoxP3 expression was detected in lymphocytes, albeit reduced in MS compared with non-MS patients' brains. These findings support the concept of early-life microbial exposure influencing the generation of neuroprotective regulatory T cells and may provide insights into new immunotherapeutic strategies for MS.