The activation of cytotoxic T-cell (CTL) responses requires antigen presentation by professional antigen presenting cells. Macrophages (MØ) can regulate CTL responses but little is known about the role played by splenic macrophages (Sp-MØ) in antigen cross-presentation. Here, we established, and characterized, an efficient culture method for generating Sp-MØ. By monitoring MØ markers, we found that 7-days Sp-MØ resembles the red pulp macrophages (RPMØ) phenotypic characteristics. The phagocytic capacity of Sp-MØ was increased as the cells become more differentiated. Thus, increased differentiation of Sp-MØ in vitro can be macrophage-colony stimulating factor (M-CSF) driven, which allows for an optimal condition to increase the yield of the spleen-derived MØ. As a result of examining the antigen presentation of Sp-MØ during differentiation, we reported that Sp-MØ down-regulated their ability to cross-present the cell-associated lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) but not the soluble OVA proteins without altering their capacity to directly present LCMV antigens after infection. Mechanistically, we defined the cytosolic pathway as the dominant cross-presentation pathway used by Sp-MØ. Further analysis revealed a direct relationship between Sp-MØ differentiation, phagosomal acidification, and antigen cross-presentation. As Sp-MØ become more mature, their vesicular phagosomal system acquired high acidic characteristics, which adversely affected antigen cross-presentation due to potent and enhanced antigen degradation. We also addressed the capacity of diverse LCMV antigens, generated during virus infection, to induce LCMV-specific CTL responses via cross-presentation by employing antigen donor cells (ADCs) that provide sufficient LCMV antigens after virus inactivation with no possible direct antigen presentation. Our results demonstrated that the ADCs induced LCMV-specific CTL responses in vitro and in vivo. Out of the four CTL epitopes tested (NP396, NP205, GP33, and GP276), in vitro cross-presentation were dominated by LCMV-NP396 epitope; while the in vivo cross-priming has shifted towards LCMV-GP33 and NP396 epitopes. Collectively, the data presented in this thesis have defined for the first time important factors that influence Sp-MØ culturing in vitro and highlighted a potential role for the Sp-MØ in regulating CTL iii responses via cross-presentation, and characterized how different epitopes from LCMV are cross presented in vitro and in vivo. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2010-04-12 15:07:38.492

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

No description available.

No description available.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: pp. 124-137

Indiana University-Purdue University Indianapolis (IUPUI) / Triple-negative breast cancer (TNBC), which accounts for 15-20% of all breast cancers, is highly aggressive and metastatic with the poorest overall rates. While surgery, radiation, and chemotherapy remain the main treatment options, TNBC represents an unmet medical need for better treatment strategies. Tremendous efforts have been made to develop effective therapies over the past years. However, TNBC treatment options are still very limited due to the lack of good drug targets and the low response rate of current therapies. In this study, we developed two different strategies to treat TNBC based on its cancer genomic features: 1) heterozygous loss of chromosome 17p (17p loss) and 2) high mutation load. 17p loss is one of the most frequent genomic events in breast cancer including TNBC, rendering cancer cells vulnerable to the inhibition of POLR2A via α-amanitin (POLR2A-specific inhibitor). Here, we developed a new drug T-Ama (α-amanitin-conjugated trastuzumab) targeting HER2-low TNBC with 17p loss by combining the effects of α-amanitin and trastuzumab (HER2+ breast cancer therapy). Our results showed that T-Ama exhibited superior efficacy in treating HER2-low TNBC with 17p loss in vitro and in vivo, and surprisingly induced immunogenic cell death (ICD) which further enhanced T cell infiltration and cytotoxicity levels and delivered greater efficacy in combination with immune checkpoint blockade therapy. Collectively, the therapeutic window created by 17p loss and HER2 expression will make HER2-low TNBC clinically feasible targets of T-Ama. As another genetic feature of TNBC, the higher genomic instability and mutational burden results in more neoantigens presented on MHC-I, along with the higher level of tumor-infiltrating T cells, making TNBC a perfect model for immunotherapy compared to the other breast cancer subtypes. Here, we designed a deconvolution-algorithm-derived library screening to find new therapeutic targets and identified PIK3C2α as a key player that determines MHC-I turnover and reduces the MHC-I-restricted antigen presentation on tumor cells. In preclinical models, inhibition of PIK3C2α profoundly suppressed breast tumor growth, increased tumor-infiltrating CD8+ T cells, and showed high potential enhancing the efficacy of anti-PD-1 therapy, suggesting that PIK3C2α is a potential therapeutic target for TNBC immunotherapy. / 2025-05-22

No description available.

<p>In this Ph.D. dissertation, the capacity of cultured bovine polymorphonuclear neutrophils (PMNs) to passively acquire functional membrane proteins from apoptotic or necrotic cells was examined. The rapid transfer of membrane proteins from a variety of syngeneic, allogeneic and xenogeneic donor cells to PMNs was observed. In contrast to PMNs from other species, bovine PMNs did not express endogenous major histocompatability class II (MHC II) protein, either constitutively or inducibly. The entire bovine PMN population was, however, able to acquire detectable levels of surface MHC II or cluster of differentiation (CD) 3 protein following PMN co-culture with cells in conditions which permitted close contact with dieing cells. Therefore, it was hypothesized that membrane lipids and proteins were acquired by bovine PMN following fusion with microparticles (MPs) shed from either apoptotic or necrotic cells. </p> <p>It was then determined whether the lifespan of bovine PMNs could be sufficient to provide an opportunity for PMNs to interact with T cells. Lymphocyte recruitment to sites of inflammation often occurs 3-5 days after the initial PMN recruitment. PMN survival would need to span this interval to provide an opportunity for an interaction between PMNs and lymphocytes. Pro-inflammatory cytokines, such as interferon (IFN)-ã and granulocyte macrophage colony stimulating factor (GM-CSF), and bacterial lipopolysaccharide (LPS) were observed to prolong the lifespan of cultured PMNs beyond 96 hours. These observations supported the conclusion that it was biologically possible for PMNs and T cells to interact at sites of inflammation.</p> <p>Using confocal microscopy, direct evidence was provided for the formation and release of MPs from peripheral blood mononuclear cells (PBMCs) and the attachment of these MPs to bovine PMNs. A time-dependent integration of both MP membranes and integral membrane proteins into the PMN plasma membrane was also observed. The passively acquired membrane lipids and proteins then diffused throughout the PMN plasma membrane. Another observation was the formation of MPs which contained donor cell cytoplasmic proteins and subsequent transfer this cytoplasmic protein to recipient PMNs. These observations raised the possibility that MPs could also transfer genetic material. Thus, confocal microscopy provided direct evidence that MPs were one mechanism by which bovine PMNs could passively acquire membrane lipids and integral membrane proteins.</p> <p>Finally, the functional consequences of passive acquisition of membrane proteins were examined using two different approaches. A significant increase in green fluorescent protein (GFP) transgene expression was observed following PMN infection using the GFP expressing bovine adenovirus vector (BAV304). These PMNs had passively acquired membranes from an adenovirus permissive cell line. This observation provided indirect evidence for the passive acquisition of a functional viral receptor protein. Direct evidence that PMNs passively acquired functional membrane proteins was provided by the observation that the passive transfer of ovine MHC II molecules to bovine PMNs enabled these cells to induce antigen-specific proliferation and cytokine expression by xenoreactive T cell lines. Despite a reduction in amplitude and duration, T cell responses induced by PMNs were qualitatively similar to those observed following activation by the stimulator B cell line. These observations supported the conclusion that PMNs could function as antigen presenting cells (APCs) following the passive acquisition of MHC II protein.</p> <p>In conclusion, this research project provided evidence that bovine PMNs have an impressive ability to acquire membranes and functional integral membrane proteins from dead or dying cells. The implications of this transfer of immunological information are discussed within the context of the role which PMNs might play in both innate and adaptive immune responses. </p>

<p>In this Ph.D. dissertation, the capacity of cultured bovine polymorphonuclear neutrophils (PMNs) to passively acquire functional membrane proteins from apoptotic or necrotic cells was examined. The rapid transfer of membrane proteins from a variety of syngeneic, allogeneic and xenogeneic donor cells to PMNs was observed. In contrast to PMNs from other species, bovine PMNs did not express endogenous major histocompatability class II (MHC II) protein, either constitutively or inducibly. The entire bovine PMN population was, however, able to acquire detectable levels of surface MHC II or cluster of differentiation (CD) 3 protein following PMN co-culture with cells in conditions which permitted close contact with dieing cells. Therefore, it was hypothesized that membrane lipids and proteins were acquired by bovine PMN following fusion with microparticles (MPs) shed from either apoptotic or necrotic cells. </p> <p>It was then determined whether the lifespan of bovine PMNs could be sufficient to provide an opportunity for PMNs to interact with T cells. Lymphocyte recruitment to sites of inflammation often occurs 3-5 days after the initial PMN recruitment. PMN survival would need to span this interval to provide an opportunity for an interaction between PMNs and lymphocytes. Pro-inflammatory cytokines, such as interferon (IFN)-ã and granulocyte macrophage colony stimulating factor (GM-CSF), and bacterial lipopolysaccharide (LPS) were observed to prolong the lifespan of cultured PMNs beyond 96 hours. These observations supported the conclusion that it was biologically possible for PMNs and T cells to interact at sites of inflammation.</p> <p>Using confocal microscopy, direct evidence was provided for the formation and release of MPs from peripheral blood mononuclear cells (PBMCs) and the attachment of these MPs to bovine PMNs. A time-dependent integration of both MP membranes and integral membrane proteins into the PMN plasma membrane was also observed. The passively acquired membrane lipids and proteins then diffused throughout the PMN plasma membrane. Another observation was the formation of MPs which contained donor cell cytoplasmic proteins and subsequent transfer this cytoplasmic protein to recipient PMNs. These observations raised the possibility that MPs could also transfer genetic material. Thus, confocal microscopy provided direct evidence that MPs were one mechanism by which bovine PMNs could passively acquire membrane lipids and integral membrane proteins.</p> <p>Finally, the functional consequences of passive acquisition of membrane proteins were examined using two different approaches. A significant increase in green fluorescent protein (GFP) transgene expression was observed following PMN infection using the GFP expressing bovine adenovirus vector (BAV304). These PMNs had passively acquired membranes from an adenovirus permissive cell line. This observation provided indirect evidence for the passive acquisition of a functional viral receptor protein. Direct evidence that PMNs passively acquired functional membrane proteins was provided by the observation that the passive transfer of ovine MHC II molecules to bovine PMNs enabled these cells to induce antigen-specific proliferation and cytokine expression by xenoreactive T cell lines. Despite a reduction in amplitude and duration, T cell responses induced by PMNs were qualitatively similar to those observed following activation by the stimulator B cell line. These observations supported the conclusion that PMNs could function as antigen presenting cells (APCs) following the passive acquisition of MHC II protein.</p> <p>In conclusion, this research project provided evidence that bovine PMNs have an impressive ability to acquire membranes and functional integral membrane proteins from dead or dying cells. The implications of this transfer of immunological information are discussed within the context of the role which PMNs might play in both innate and adaptive immune responses. </p>

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 96-108).