Nanobodies as potential tools for microbiological testing of live biotherapeutic products.

Nanobodies are highly specific binding domains derived from naturally occurring single chain camelid antibodies. Live biotherapeutic products (LBPs) are biological products containing preparations of live organisms, such as Lactobacillus, that are intended for use as drugs, i.e. to address a specific disease or condition. Demonstrating potency of multi-strain LBPs can be challenging. The approach investigated here is to use strain-specific nanobody reagents in LBP potency assays. Llamas were immunized with radiation-killed Lactobacillus jensenii or L. crispatus whole cell preparations. A nanobody phage-display library was constructed and panned against bacterial preparations to identify nanobodies specific for each species. Nanobody-encoding DNA sequences were subcloned and the nanobodies were expressed, purified, and characterized. Colony immunoblots and flow cytometry showed that binding by Lj75 and Lj94 nanobodies were limited to a subset of L. jensenii strains while binding by Lc38 and Lc58 nanobodies were limited to L. crispatus strains. Mass spectrometry was used to demonstrate that Lj75 specifically bound a peptidase of L. jensenii, and that Lc58 bound an S-layer protein of L. crispatus. The utility of fluorescent nanobodies in evaluating multi-strain LBP potency assays was assessed by evaluating a L. crispatus and L. jensenii mixture by fluorescence microscopy, flow cytometry, and colony immunoblots. Our results showed that the fluorescent nanobody labelling enabled differentiation and quantitation of the strains in mixture by these methods. Development of these nanobody reagents represents a potential advance in LBP testing, informing the advancement of future LBP potency assays and, thereby, facilitation of clinical investigation of LBPs.

GLI1+ perivascular, renal, progenitor cells: The likely source of spontaneous neoplasia that created the AGMK1-9T7 cell line.

The AGMK1-9T7 cell line has been used to study neoplasia in tissue culture. By passage in cell culture, these cells evolved to become tumorigenic and metastatic in immunodeficient mice at passage 40. Of the 20 x 106 kidney cells originally plated, less than 2% formed the colonies that evolved to create this cell line. These cells could be the progeny of some type of kidney progenitor cells. To characterize these cells, we documented their renal lineage by their expression of PAX-2 and MIOX, detected by indirect immunofluorescence. These cells assessed by flow-cytometry expressed high levels of CD44, CD73, CD105, Sca-1, and GLI1 across all passages tested; these markers have been reported to be expressed by renal progenitor cells. The expression of GLI1 was confirmed by immunofluorescence and western blot analysis. Cells from passages 13 to 23 possessed the ability to differentiate into adipocytes, osteoblasts, and chondrocytes; after passage 23, their ability to form these cell types was lost. These data indicate that the cells that formed the AGMK1-9T7 cell line were GLI1+ perivascular, kidney, progenitor cells.

Protective efficacy and correlates of immunity of immunodominant recombinant Babesia microti antigens.

Babesia microti, an intraerythrocytic apicomplexan parasite, is the primary causative agent of human babesiosis and an emerging threat to public health in the United States and elsewhere. An effective vaccine against B. microti would reduce disease severity in acute babesiosis patients and shorten the parasitemic period in asymptomatic individuals, thereby minimizing the risk of transfusion-transmitted babesiosis. Here we report on immunogenicity, protective efficacy, and correlates of immunity following immunization with four immunodominant recombinantly produced B. microti antigens-Serine Reactive Antigen 1 (SERA1), Maltese Cross Form Related Protein 1 (MCFRP1), Piroplasm ß-Strand Domain 1 (PißS1), and Babesia microti Alpha Helical Cell Surface Protein 1 (BAHCS1)-delivered subcutaneously in Montanide ISA 51/CpG adjuvant in three doses to BALB/c mice. Following B. microti parasite challenge, BAHCS1 led to the highest reduction in peak parasitemia (67.8%), followed by SERA1 (44.8%) and MCFRP1 (41.9%); PißS1 (27.6%) had minimal protective effect. All four B. microti antigens induced high ELISA total IgG and each isotype; however, antibody levels did not directly correlate with anti-parasitic activity in mice. Increased prechallenge levels of some cell populations including follicular helper T cells (TFH) and memory B cells, along with a set of six cytokines [IL-1α, IL-2, IL-3, IL-6, IL-12(p40), and G-CSF] that belong to both innate and adaptive immune responses, were generally associated with protective immunity. Our results indicate that mechanisms driving recombinant B. microti antigen-induced immunity are complex and multifactorial. We think that BAHCS1 warrants further evaluation in preclinical studies.

Evaluation of association of anti-PEG antibodies with anaphylaxis after mRNA COVID-19 vaccination.

BACKGROUND: The mechanism for anaphylaxis following mRNA COVID-19 vaccination has been widely debated; understanding this serious adverse event is important for future vaccines of similar design. A mechanism proposed is type I hypersensitivity (i.e., IgE-mediated mast cell degranulation) to polyethylene glycol (PEG). Using an assay that, uniquely, had been previously assessed in patients with anaphylaxis to PEG, our objective was to compare anti-PEG IgE in serum from mRNA COVID-19 vaccine anaphylaxis case-patients and persons vaccinated without allergic reactions. Secondarily, we compared anti-PEG IgG and IgM to assess alternative mechanisms. METHODS: Selected anaphylaxis case-patients reported to U.S. Vaccine Adverse Event Reporting System December 14, 2020-March 25, 2021 were invited to provide a serum sample. mRNA COVID-19 vaccine study participants with residual serum and no allergic reaction post-vaccination ("controls") were frequency matched to cases 3:1 on vaccine and dose number, sex and 10-year age category. Anti-PEG IgE was measured using a dual cytometric bead assay (DCBA). Anti-PEG IgG and IgM were measured using two different assays: DCBA and a PEGylated-polystyrene bead assay. Laboratorians were blinded to case/control status. RESULTS: All 20 case-patients were women; 17 had anaphylaxis after dose 1, 3 after dose 2. Thirteen (65 %) were hospitalized and 7 (35 %) were intubated. Time from vaccination to serum collection was longer for case-patients vs controls (post-dose 1: median 105 vs 21 days). Among Moderna recipients, anti-PEG IgE was detected in 1 of 10 (10 %) case-patients vs 8 of 30 (27 %) controls (p = 0.40); among Pfizer-BioNTech recipients, it was detected in 0 of 10 case-patients (0 %) vs 1 of 30 (3 %) controls (p >n 0.99). Anti-PEG IgE quantitative signals followed this same pattern. Neither anti-PEG IgG nor IgM was associated with case status with both assay formats. CONCLUSION: Our results support that anti-PEG IgE is not a predominant mechanism for anaphylaxis post-mRNA COVID-19 vaccination.

Toll-like Receptor-9 (TLR-9) Signaling Is Crucial for Inducing Protective Immunity following Immunization with Genetically Modified Live Attenuated Leishmania Parasites.

No human vaccine is available for visceral leishmaniasis (VL). Live attenuated centrin gene-deleted L. donovani (LdCen-/-) parasite vaccine has been shown to induce robust innate immunity and provide protection in animal models. Toll-like receptors (TLRs) are expressed in innate immune cells and are essential for the early stages of Leishmania infection. Among TLRs, TLR-9 signaling has been reported to induce host protection during Leishmania infection. Importantly, TLR-9 ligands have been used as immune enhancers for non-live vaccination strategies against leishmaniasis. However, the function of TLR-9 in the generation of a protective immune response in live attenuated Leishmania vaccines remains unknown. In this study, we investigated the function of TLR-9 during LdCen-/- infection and found that it increased the expression of TLR-9 on DCs and macrophages from ear-draining lymph nodes and spleen. The increase in TLR-9 expression resulted in changes in downstream signaling in DCs mediated through signaling protein myeloid differentiation primary response 88 (MyD88), resulting in activation and nuclear translocation of nuclear factor-κB (NF-κB). This process resulted in an increase in the DC's proinflammatory response, activation, and DC-mediated CD4+T cell proliferation. Further, LdCen-/- immunization in TLR-9-/- mice resulted in a significant loss of protective immunity. Thus, LdCen-/- vaccine naturally activates the TLR-9 signaling pathway to elicit protective immunity against virulent L. donovani challenge.

IL-10 Signaling Elicited by Nivolumab-Induced Activation of the MAP Kinase Pathway Does Not Fully Contribute to Nivolumab-Modulated Heterogeneous T Cell Responses.

Immune checkpoint inhibitor (ICI) therapy has revolutionized anti-cancer treatment for many late-stage cancer patients. However, ICI therapy has thus far demonstrated limited efficacy for most patients, and it remains unclear why this is so. Interleukin 10 (IL-10) is a cytokine that has been recognized as a central player in cancer biology with its ability to inhibit anti-tumor T cell responses. Recent studies suggest that IL-10 might also exert some intrinsic anti-tumor T cell responses, and clinical studies using recombinant IL-10 alone or in combination with ICI are underway. This paradoxical effect of IL-10 and its underlying mechanisms impacting ICI-modulated T cell responses remain poorly understood. In this study, using an in vitro mixed lymphocyte reaction assay, we found that treatment with ICIs such as the anti-programmed cell death receptor-1 (PD-1) mAb nivolumab elicits a strong expression of IL-10. While neutralization of IL-10 signaling with an anti-IL-10 specific mAb significantly decreases the production of IFN-γ by T cells in a cohort of donor cells, the opposite effect was observed in other donor cells. Similarly, neutralization of IL-10 signaling significantly decreases the expression of T cell activation markers Ki67 and CD25, as well as the production of Granzyme B in a cohort of donor cells, whereas the opposite effect was observed in others. Furthermore, we found that nivolumab and IL-10 differentially modulate the signal transducer and activator of transcription 3 (STAT3) and AKT serine-threonine kinase pathways. Finally, we found that nivolumab activates the mitogen-activated protein kinase (MAPK) pathway, which in turn is responsible for the observed induction of IL-10 production by nivolumab. These findings provide new insights into the mechanisms underlying anti-PD-1-modulated T cell responses by IL-10, which could lead to the discovery of novel combination treatments that target IL-10 and immune checkpoint molecules.

Functional heterogeneity of IFN-γ-licensed mesenchymal stromal cell immunosuppressive capacity on biomaterials.

Mesenchymal stromal cells (MSCs) are increasingly combined with biomaterials to enhance their therapeutic properties, including their immunosuppressive function. However, clinical trials utilizing MSCs with or without biomaterials have shown limited success, potentially due to their functional heterogeneity across different donors and among different subpopulations of cells. Here, we evaluated the immunosuppressive capacity, as measured by the ability to reduce T-cell proliferation and activation, of interferon-gamma (IFN-γ)-licensed MSCs from multiple donors on fibrin and collagen hydrogels, the two most commonly utilized biomaterials in combination with MSCs in clinical trials worldwide according to ClinicalTrials.gov Variations in the immunosuppressive capacity between IFN-γ-licensed MSC donors on the biomaterials correlated with the magnitude of indoleamine-2,3-dioxygenase activity. Immunosuppressive capacity of the IFN-γ-licensed MSCs depended on the αV/α5 integrins when cultured on fibrin and on the α2/ß1 integrins when cultured on collagen. While all tested MSCs were nearly 100% positive for these integrins, sorted MSCs that expressed higher levels of αV/α5 integrins demonstrated greater immunosuppressive capacity with IFN-γ licensing than MSCs that expressed lower levels of these integrins on fibrin. These findings were equivalent for MSCs sorted based on the α2/ß1 integrins on collagen. These results demonstrate the importance of integrin engagement to IFN-γ licensed MSC immunosuppressive capacity and that IFN-γ-licensed MSC subpopulations of varying immunosuppressive capacity can be identified by the magnitude of integrin expression specific to each biomaterial.

Exploring the Potential Use of a PBMC-Based Functional Assay to Identify Predictive Biomarkers for Anti-PD-1 Immunotherapy.

The absence of reliable, robust, and non-invasive biomarkers for anti- Programmed cell death protein 1 (PD-1) immunotherapy is an urgent unmet medical need for the treatment of cancer patients. No predictive biomarkers have been established based on the direct assessment of T cell functions, the primary mechanism of action of anti-PD-1 therapy. In this study, we established a model system to test T cell functions modulated by Nivolumab using anti-CD3 monoclonal antibody (mAb)-stimulated peripheral blood mononuclear cells (PBMCs), and characterized T cell functions primarily based on the knowledge gained from retrospective observations of patients treated with anti-PD-1 immunotherapy. During a comprehensive cytokine profile assessment to identify potential biomarkers, we found that Nivolumab increases expression of T helper type 1 (Th1) associated cytokines such as interferon-γ (IFN-γ) and interleukin-2 (IL-2) in a subset of donors. Furthermore, Nivolumab increases production of Th2, Th9, and Th17 associated cytokines, as well as many proinflammatory cytokines such as IL-6 in a subset of donors. Conversely, Nivolumab treatment has no impact on T cell proliferation, expression of CD25, CD69, or Granzyme B, and only modestly increases in the expansion of regulatory T cells. Our results suggest that assessment of cytokine production using a simple PBMC-based T cell functional assay could be used as a potential predictive marker for anti-PD-1 immunotherapy.

BRAF and MEK inhibitors differentially affect nivolumab-induced T cell activation by modulating the TCR and AKT signaling pathways.

Immune checkpoint inhibitors (ICIs) such as the anti-PD-1 antibody Nivolumab, achieve remarkable clinical efficacy in patients with late stage cancers. However, only a small subset of patients benefit from this therapy. Numerous clinical trials are underway testing whether combining ICIs with other anti-cancer therapies can increase this response rate. For example, anti-PD-1/PD-L1 therapy combined with MAP kinase inhibition using BRAF inhibitors (BRAFi) and/or MEK inhibitors (MEKi) are in development for treatment of late stage melanomas. However, the benefits and underlying mechanisms of these combinatorial therapies remain unclear. In the current study, we assess the effects of MAPK inhibition on Nivolumab-induced T cell responses. Using an in vitro mixed lymphocyte reaction assay, we demonstrate that Nivolumab-induced T cell activation is highly heterogeneous. While BRAFi inhibits Nivolumab-induced cytokine production, T cell proliferation, activation markers (CD69, CD25), and Granzyme B in a substantial proportion of donor pairs, a small subset of donor pairs shows an additive effect. MEKi alone significantly inhibits Nivolumab-induced T cell activation; the addition of BRAFi significantly enhances this inhibitory effect. Mechanistically, the effects of BRAFi and/or MEKi on Nivolumab-induced T cell activation may be due to alteration of the activation of the AKT and T cell receptor (TCR) signaling pathways. Our results suggest that MAPK inhibition may not provide a clinical benefit for most melanoma patients being treated with anti-PD-1 therapy.

TCRß-expressing macrophages induced by a pathogenic murine malaria correlate with parasite burden and enhanced phagocytic activity.

Macrophages express a wide array of invariant receptors that facilitate host defense and mediate pathogenesis during pathogen invasion. We report on a novel population of CD11bhighCD14+F4/80+ macrophages that express TCRß. This population expands dramatically during a Plasmodium berghei ANKA infection and sequesters in the brain during experimental cerebral malaria. Importantly, measurement of TCRß transcript and protein levels in macrophages in wildtype versus nude and Rag1 knockout mice establishes that the observed expression is not a consequence of passive receptor expression due to phagocytosis or trogocytosis of peripheral T cells or nonspecific antibody staining to an Fc receptor or cross reactive epitope. We also demonstrate that TCRß on brain sequestered macrophages undergoes productive gene rearrangements and shows preferential Vß usage. Remarkably, there is a significant correlation in the proportion of macrophages that express TCRß and peripheral parasitemia. In addition, presence of TCRß on the macrophage also correlates with a significant increase (1.9 fold) in the phagocytosis of parasitized erythrocytes. By transcriptional profiling, we identify a novel set of genes and pathways that associate with TCRß expression by the macrophage. Expansion of TCRß-expressing macrophages points towards a convergence of the innate and adaptive immune responses where both arms of the immune system cooperate to modulate the host response to malaria and possibly other infections.

TCRß Combinatorial Immunoreceptor Expression by Neutrophils Correlates with Parasite Burden and Enhanced Phagocytosis during a Plasmodium berghei ANKA Malaria Infection.

Recent studies have demonstrated that a subpopulation of neutrophils express the TCRαß combinatorial immunoreceptor in humans and mice. Here, we report that a Plasmodium berghei ANKA murine malaria infection induces expansion of TCRß expressing CD11b+ Ly6G+ neutrophils in the spleen during the early phase of infection. Measurement of TCRß transcript and protein levels of neutrophils in wild-type versus nude and Rag1 knockout mice establishes that the observed expression is not a consequence of nonspecific antibody staining or passive receptor expression due to phagocytosis or trogocytosis of peripheral T cells. Remarkably, on day 3 postinfection, we observed a highly significant correlation between the proportion of neutrophils that express TCRß and peripheral blood parasite burden. In addition, TCRß+ neutrophils phagocytose parasitized erythrocytes with 4-fold greater efficiency than TCRß- neutrophils. Together these results signify that TCR expression by the neutrophil plays an important role in the regulation of parasite burden by enhancing the phagocytic capacity of the neutrophil.

Live Attenuated Leishmania donovani Centrin Gene-Deleted Parasites Induce IL-23-Dependent IL-17-Protective Immune Response against Visceral Leishmaniasis in a Murine Model.

No vaccine exists against visceral leishmaniasis. To develop effective vaccines, we have previously reported protective role of live attenuated centrin gene-deleted Leishmania donovani (LdCen-/- ) parasites through induction of Th1 type immune response in mice, hamsters, and dogs. In this study, we specifically explored the role of Th17 cells in LdCen-/- -induced host protection in mice. Our results showed that compared with wild-type L. donovani infection, LdCen-/- parasites induce significantly higher expression of Th17 differentiation cytokines in splenic dendritic cells. There was also induction of IL-17 and its promoting cytokines in total splenocytes and in both CD4 and CD8 T cells following immunization with LdCen-/- Upon challenge with wild-type parasites, IL-17 and its differentiating cytokines were significantly higher in LdCen-/- -immunized mice compared with nonimmunized mice that resulted in parasite control. Alongside IL-17 induction, we observed induction of IFN-γ-producing Th1 cells as reported earlier. However, Th17 cells are generated before Th1 cells. Neutralization of either IL-17 or IFN-γ abrogated LdCen-/- -induced host protection further confirming the essential role of Th17 along with Th1 cytokines in host protection. Treatment with recombinant IL-23, which is required for stabilization and maintenance of IL-17, heightened Th17, and Tc17 responses in immunized mice splenocytes. In contrast, Th17 response was absent in immunized IL-23R-/- mice that failed to induce protection upon virulent Leishmania challenge suggesting that IL-23 plays an essential role in IL-17-mediated protection by LdCen-/- parasites. This study unveiled the role of IL-23-dependent IL-17 induction in LdCen-/- parasite-induced immunity and subsequent protection against visceral leishmaniasis.

Live Attenuated Leishmania donovani Centrin Knock Out Parasites Generate Non-inferior Protective Immune Response in Aged Mice against Visceral Leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania donovani causes severe disease. Age appears to be critical in determining the clinical outcome of VL and at present there is no effective vaccine available against VL for any age group. Previously, we showed that genetically modified live attenuated L. donovani parasites (LdCen-/-) induced a strong protective innate and adaptive immune response in young mice. In this study we analyzed LdCen-/- parasite mediated modulation of innate and adaptive immune response in aged mice (18 months) and compared to young (2 months) mice. METHODOLOGY: Analysis of innate immune response in bone marrow derived dendritic cells (BMDCs) from both young and aged mice upon infection with LdCen-/- parasites, showed significant enhancement of innate effector responses, which consequently augmented CD4+ Th1 cell effector function compared to LdWT infected BMDCs in vitro. Similarly, parasitized splenic dendritic cells from LdCen-/- infected young and aged mice also revealed induction of proinflammatory cytokines (IL-12, IL-6, IFN-γ and TNF) and subsequent down regulation of anti-inflammatory cytokine (IL-10) genes compared to LdWT infected mice. We also evaluated in vivo protection of the LdCen-/- immunized young and aged mice against virulent L. donovani challenge. Immunization with LdCen-/- induced higher IgG2a antibodies, lymphoproliferative response, pro- and anti-inflammatory cytokine responses and stimulated splenocytes for heightened leishmanicidal activity associated with nitric oxide production in young and aged mice. Furthermore, upon virulent L. donovani challenge, LdCen-/- immunized mice from both age groups displayed multifunctional Th1-type CD4 and cytotoxic CD8 T cells correlating to a significantly reduced parasite burden in the spleen and liver compared to naïve mice. It is interesting to note that even though there was no difference in the LdCen-/- induced innate response in dendritic cells between aged and young mice; the adaptive response specifically in terms of T cell and B cell activation in aged animals was reduced compared to young mice which correlated with less protection in old mice compared to young mice. CONCLUSIONS: Taken together, LdCen-/- immunization induced a significant but diminished host protective response in aged mice after challenge with virulent L. donovani parasites compared to young mice.

A New Signaling Pathway for HCV Inhibition by Estrogen: GPR30 Activation Leads to Cleavage of Occludin by MMP-9.

Poor outcome in response to hepatitis C virus, including higher viral load, hepatocellular carcinoma and cirrhosis, is more associated with men and postmenopausal women than with premenopausal women and women receiving hormone replacement therapy, suggesting that ß-estradiol plays an innate role in preventing viral infection and liver disease. Consequently, most research in the field has concluded that estrogen affects HCV replication through viral interactions with estrogen receptor-α. Previously, estrogen-like antagonists, including Tamoxifen, were shown to reduce HCV RNA production and prevent viral entry, although the authors did not identify host factors involved. Estrogen can act alternatively through the membrane-bound G-protein-coupled estrogen receptor, GPR30. Here, human hepatoma Huh7.5 cells were infected with HCV J6/JFH-1 and treated with estrogen or Tamoxifen, resulting in a marked decrease in detectable virus. The effect was mimicked by G1, a GPR30-specific agonist, and was reversed by the GPR30-specific antagonist, G15. While previous studies have demonstrated that estrogen down-regulated occludin in cervical cancer cells, its action on liver cells was unknown. Occludin is a tight junction protein and HCV receptor and here we report that activation and cellular export of MMP-9 led to the cleavage of occludin upon estrogen treatment of liver cells. This is the first report of the cleavage of an HCV receptor in response to estrogen. We also identify the occludin cleavage site in extracellular Domain D; the motif required for HCV entry and spread. This pathway gives new insight into a novel innate antiviral pathway and the suboptimal environment that estrogen provides for the proliferation of the virus. It may also explain the disparate host-virus responses to HCV demonstrated by the two sexes. Moreover, these data suggest that hormone replacement therapy may have beneficial antiviral enhancement properties for HCV-infected postmenopausal women and show promise for new antiviral treatments for both men and women.

Genetically Modified Live Attenuated Leishmania donovani Parasites Induce Innate Immunity through Classical Activation of Macrophages That Direct the Th1 Response in Mice.

Visceral leishmaniasis (VL) causes significant mortality and there is no effective vaccine. Previously, we have shown that genetically modified Leishmania donovani parasites, here described as live attenuated parasites, induce a host protective adaptive immune response in various animal models. In this study, we demonstrate an innate immune response upon infection with live attenuated parasites in macrophages from BALB/c mice both in vitro and in vivo. In vitro infection of macrophages with live attenuated parasites (compared to that with wild-type [WT] L. donovani parasites) induced significantly higher production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-12 [IL-12], gamma interferon [IFN-γ], and IL-6), chemokines (monocyte chemoattractant protein 1/CCL-2, macrophage inflammatory protein 1α/CCL-3, and IP-10), reactive oxygen species (ROS), and nitric oxide, while concomitantly reducing anti-inflammatory cytokine IL-10 and arginase-1 activities, suggesting a dominant classically activated/M1 macrophage response. The classically activated response in turn helps in presenting antigen to T cells, as observed with robust CD4(+) T cell activation in vitro. Similarly, parasitized splenic macrophages from live attenuated parasite-infected mice also demonstrated induction of an M1 macrophage phenotype, indicated by upregulation of IL-1ß, TNF-α, IL-12, and inducible nitric oxide synthase 2 and downregulation of genes associated with the M2 phenotype, i.e., the IL-10, YM1, Arg-1, and MRC-1 genes, compared to WT L. donovani-infected mice. Furthermore, an ex vivo antigen presentation assay showed macrophages from live attenuated parasite-infected mice induced higher IFN-γ and IL-2 but significantly less IL-10 production by ovalbumin-specific CD4(+) T cells, resulting in proliferation of Th1 cells. These data suggest that infection with live attenuated parasites promotes a state of classical activation (M1 dominant) in macrophages that leads to the generation of protective Th1 responses in BALB/c mice.

Rescue of embryonic epithelium reveals that the homozygous deletion of the retinoblastoma gene confers growth factor independence and immortality but does not influence epithelial differentiation or tissue morphogenesis.

The ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb-/-) fetal mice has allowed for the isolation and characterization of the first Rb-/- prostate epithelial cell line. This cell line, designated Rb-/-PrE, was utilized for experiments examining the consequences of Rb loss on an epithelial population. These findings demonstrated that Rb deletion has no discernible effect on prostatic histodifferentiation in Rb-/-PrE cultures. When Rb-/-PrE cells were recombined with embryonic rat urogenital mesenchyme and implanted into athymic male, nude mouse hosts, the recombinants developed into fully differentiated and morphologically normal prostate tissue. The Rb-/-PrE phenotype was characterized by serum independence in culture and immortality in vivo, when compared with wild type controls. Cell cycle analysis revealed elevated S phase DNA content accompanied by increased expression of cyclin E1 and proliferating cell nuclear antigen. Rb-/-PrE cultures also exhibited a diminished ability to growth arrest under high density culture conditions. We believe that the development of Rb-/- prostate tissue and cell lines has provided a unique experimental platform with which to investigate the consequences of Rb deletion in epithelial cells under various physiological conditions. Additionally, the development of this technology will allow similar studies in other tissues and cell populations rescued from Rb-/- fetuses.

Flt3 ligand therapy for recipients of allogeneic bone marrow transplants expands host CD8 alpha(+) dendritic cells and reduces experimental acute graft-versus-host disease.

Recent evidence suggests that dendritic cells (DCs) can regulate and amplify immune responses. Flt3 ligand (FL)-derived DC function was tested as a stimulator of allogeneic lymphocytes in vitro and in vivo. Treatment of mice with FL dramatically expanded DC number, but DCs isolated from FL-treated mice (FL DCs) were poor stimulators of allogeneic T-cell responses in vitro. Further activation of FL DCs did not restore their stimulatory ability, and FL DCs did not suppress the stimulation of the allogeneic T cells by normal DCs. FL treatment significantly increased the CD8 alpha(+) DC subset, which appeared to be the reason for their poor stimulatory capacity. These observations were confirmed in vivo using a mouse model of acute graft-versus-host disease (GVHD) wherein host DCs play a critical role. FL treatment of recipients before allogeneic bone marrow transplantation dramatically suppressed donor T-cell responses to host antigens, thereby reducing GVHD mortality (P <.01). These data represent a novel strategy that alters host DCs and reduces acute GVHD.