Fibrin Facilitates Both Innate and T Cell-Mediated Defense against Yersinia pestis

The gram-negative bacterium Yersinia pestis causes plague, a rapidly progressing and often fatal disease. The formation of fibrin at sites of Y. pestis infection supports innate host defense against plague, perhaps by providing a non-diffusible spatial cue that promotes the accumulation of inflammatory cells expressing fibrin-binding integrins. This report demonstrates that fibrin is an essential component of T cell-mediated defense against plague but can be dispensable for antibody-mediated defense. Genetic or pharmacologic depletion of fibrin abrogated innate and T cell-mediated defense in mice challenged intranasally with Y. pestis. The fibrin-deficient mice displayed reduced survival, increased bacterial burden, and exacerbated hemorrhagic pathology. They also showed fewer neutrophils within infected lung tissue and reduced neutrophil viability at sites of liver infection. Depletion of neutrophils from wild type mice weakened T cell-mediated defense against plague. The data suggest that T cells combat plague in conjunction with neutrophils, which require help from fibrin in order to withstand Y. pestis encounters and effectively clear bacteria

Regulation of PERK Signaling and Leukemic Cell Survival by a Novel Cytosolic Isoform of the UPR Regulator GRP78/BiP

The unfolded protein response (UPR) is an evolutionarily conserved mechanism to allow cells to adapt to stress targeting the endoplasmic reticulum (ER). Induction of ER chaperone GRP78/BiP increases protein folding capacity; as such it represents a major survival arm of UPR. Considering the central importance of the UPR in regulating cell survival and death, evidence is emerging that cells evolve feedback regulatory pathways to modulate the key UPR executors, however, the precise mechanisms remain to be elucidated. Here, we report the fortuitous discovery of GRP78va, a novel isoform of GRP78 generated by alternative splicing (retention of intron 1) and alternative translation initiation. Bioinformatic and biochemical analyses revealed that expression of GRP78va is enhanced by ER stress and is notably elevated in human leukemic cells and leukemia patients. In contrast to the canonical GRP78 which is primarily an ER lumenal protein, GRP78va is devoid of the ER signaling peptide and is cytosolic. Through specific knockdown of endogenous GRP78va by siRNA without affecting canonical GRP78, we showed that GRP78va promotes cell survival under ER stress. We further demonstrated that GRP78va has the ability to regulate PERK signaling and that GRP78va is able to interact with and antagonize PERK inhibitor P58IPK. Our study describes the discovery of GRP78va, a novel cytosolic isoform of GRP78/BiP, and the first characterization of the modulation of UPR signaling via alternative splicing of nuclear pre-mRNA. Our study further reveals a novel survival mechanism in leukemic cells and other cell types where GRP78va is expressed

Functions of CD8 T Cells in Protective Immune Response to Histoplasmosis

CD4 T 細胞在對抗巨噬細胞內之非病毒性病原菌扮演重要角色。在人類免疫不全病毒感染中，CD4 T 細胞會逐漸喪失。受感染個體發展成後天免疫缺乏症候群並死於伺機性感染。故了解 CD8 T 細胞在 CD4 T 細胞缺少時之功能是重要的研究課題。 首先，我計算在組織胞漿菌感染後脾臟中對於宿主抵禦感染非常重要之特異性 T 細胞數目，並發現此種巨噬細胞胞內病源菌之感染可以活化 CD4 及CD8 T 細胞。CD8 T 細胞反應強度雖較CD4 T 細胞的反應強度弱，二種細胞反應之擴張與收縮的動力學則是相同的模式。產生干擾素γ與高量 CD44 表現之間的高度相關性不只在反應高峰期可觀察到，在整個感染過程均是如此。此外，廣大系列 Vβ族群均會對全身性以及肺部感染產生反應，代表在抵抗組織胞漿菌之初次免疫反應中並無使用特定T 細胞受體傾向。有趣的是，在高劑量感染或二次感染後功能性 T 細胞擁有較初次感染窄小範圍之T 細胞受體 Vβ 使用。 CD8 T 細胞對於宿主抵抗組織胞漿菌感染的貢獻，在 CD4 T 細胞完整之小鼠是較其次的，因若去除CD8 T 細胞只會延遲但並不影響病菌的清除。然而，CD8 T 細胞在缺乏功能性CD4 T 細胞之宿主是否具有保護能力仍然需要更進一步的探討。在我的研究中發現，MHC class II 基因缺陷小鼠在感染組織胞漿菌後能將病菌數量維持如同第一週之數目長達十六週，顯示CD8 T 細胞能夠限制病菌之複製，但無法清除。研究結果顯示，由組織胞漿菌感染之野生型小鼠體內取出之CD8 T 細胞胞內會表現干擾素γ。此外，CD8 T 細胞也具有細胞毒殺的功能。然而 MHC class II 基因缺陷小鼠的 CD8 T 細胞的功能卻有限制，以致於無法完全清除全身性及肺部感染。 在我的研究中亦發現，巨噬細胞除了擔任組織胞漿菌之宿主細胞及功能細胞外，吞噬組織胞漿菌而凋亡之巨噬細胞亦可作為樹突狀細胞的抗原提供者。呈獻外源性組織胞漿菌抗原之樹突狀細胞可透過直接吞噬酵母菌，或透過取得凋亡巨噬細胞內之真菌抗原，即所謂”交叉抗原呈獻” 可刺激組織胞漿菌特異性 CD8 T 細胞產生反應。根據這些研究，我們提出一個詳述在組織胞漿菌感染宿主中，細胞性免疫反應與真菌清除之前因後果的模式。It is widely accepted that cellular immune response via the activation of CD4+ T cells plays an important role in the protection against endosomal intracellular non-viral pathogens of the macrophage. In HIV infection, the major complication of the disease is the progressive loss of CD4+ T cells. The infected individuals develop AIDS and succumb to opportunistic infections. It is, therefore, important to understand how CD8+ T cells function in the absence of CD4+ T cells. First of all, I enumerated antigen-specific functional T cells, which are critical to host defense against infection, in the infection of Histoplasma capsulatum, an intracellular pathogen of the macrophages. I found that not only CD4 but also CD8 T cells were activated. The magnitude of CD8 T cell response was lower than CD4 T cell, but the expansion and contraction of both cell types followed the same kinetics. Strong correlation between IFNγ production and CD44hi expression was observed not only at the peak of response but also throughout the course of infection. Moreover, a broad spectrum of Vβ populations responded to systemic as well as pulmonary infections, suggesting no obvious T cell receptor bias in primary immune response to histoplasmosis. Interestingly, after high dose challenge or secondary infection the functional T cells had a narrower TCR Vβ repertoire than do primary effector cells. The contribution of CD8 T cells in host defense against histoplasmosis is minor in the CD4 T cell-intact mouse, as it has been shown that depleting CD8 T cells delays but does not affect fungal clearance. However, it remains to be determined whether the CD8 T cells are protective in a host lacking functional CD4 T cells. I found that MHC class II-deficient mice infected with Histoplasma kept the fungus in check for up to 16 weeks, indicating CD8 T cells are able to limit fungal replication but unable to clear the fungus. Ex vivo studies showed that CD8 T cells from Histoplasma-infected wild type mice exhibit cytotoxic activity as well as IFNγ production. However, the CD8 T cells in IIKO mice had functional limitation to clear systemic as well as pulmonary histoplasmosis. It is also demonstrated in this study that the macrophage, being the primary host cell as well as the effector cell of Histoplasma, can also serve as antigen donor to dendritic cells. Histoplasma-specific CD8 T cells are stimulated by dendritic cells that present exogenous Histoplasma antigens, either through direct ingestion of yeasts or through uptake of apoptotic macrophage-associated fungal antigens, a process known as ‘cross-presentation’. Based on these results, I present a model detailing the possible sequence of events leading to a cell-mediated immune response and fungal clearance in Histoplasma-infected hosts.Abstract.............................................. i Abstract (Chinese)....................................iii Abbreviation...........................................v Table of Contents......................................vi Chapter I. Introduction...................................1 Part I. Histoplasma capsulatum infection and host defense.2 Part II. Antigen-specific T cells and their TCR usage in infections................................................4 Part III. CD8 T cell effector mechanisms in resistance to intracellular pathogen infection..........................5 Part IV. Cross-presentation in intracellular pathogen infection.................................................8 Chapter II. Aims of The Study............................11 (I)To characterize specific T cell response to Hisoplasma infection................................................12 (II)To study the mechanisms of CD8 T cells mediating protective immunity against Hisoplasma infection.........12 (III)To study cross-presentation of Histoplasma antigens and activation of CD8 T cells............................13 Chapter III. Materials and Methods.......................14 Part I. Materials........................................15 1.Mice...................................................15 2.Antibodies.............................................15 3.Solutions..............................................19 4.Chemicals and reagents.................................22 5.Equipments.............................................26 Part II. Methods.........................................26 1.Fungus and infection...................................26 2.Quantitation of fungal load............................27 3.Mouse lymphocytes isolation............................27 3.1 Preparation of single cell suspension from mouse spleen...................................................27 3.2 Nylon wool enrichment of splenic T cells.............28 3.3 MACS purification of splenic CD4 and CD8 T cells.....28 3.4 Preparation of single cell suspension from lung tissues and mediastinal lymph............................29 3.5 T cell isolation by panning..........................30 4.In vivo cell depletion.................................30 5.Hybridoma cell supernatant collection and monoclonal antibody purification....................................31 6.Cell surface and intracytoplasmic staining for flow cytometric analysis......................................31 6.1 Cell surface marker staining.........................32 6.2 Intracytoplasmic cytokine staining...................32 6.3 TCR Vβ repertoire staining...........................32 6.4 Intracytoplasmic granzyme B staining.................33 6.5 Flow cytometric analysis.............................33 7.RNA extraction and reverse transcription...............34 8.Competitive PCR........................................35 9.Isolation of resident peritoneal and alveolar macrophages..............................................36 10.Culture of bone marrow-derived dendritic cells........36 11.T cell functional assay...............................37 11.1 [3H] Thymidine incorporation assay..................37 11.2 Cytokine ELISA assay................................37 11.3 Cytotoxicity assay..................................38 12.Preparation of Histoplasma for ingestion by macrophages and dendritic cells.....................................,39 13.Assay for cell apoptosis..............................40 13.1 Induction of macrophage apoptosis...................40 13.2 Cell death detection ELISA..........................40 13.3 TUNEL staining......................................41 14.Statistics............................................41 Chapter IV. Results......................................43 Part I. Functional T cells in Immune Response to Histoplasmosis...........................................44 1.CD8 T cells as well as CD4 T cells are active IFNγ producers................................................44 2.The magnitude and kinetics of specific T cell response.................................................44 3.Most IFNγ-producing cells express CD44hi phenotype.....45 4.Expansion of a broad spectrum of TCR Vβ repertoire and IFNγ-producing populations in mice systemically infected with Histoplasma.........................................46 5.Comparing IFNγ-producing Vβ populations in systemic and pulmonary infections.....................................47 6.The TCR Vβ usage of functional IFNγ-producing T cells in high dose and secondary Histoplasma infection............48 Part II. The Protective Role of CD8 T Cells in Histoplasma Infection................................................48 1.CD8 T cells are critical in host defense against histoplasmosis in the absence of CD4 T cells.............49 2.Histoplasma clearance is delayed in mice lacking CD8 T cells....................................................50 3.CD8 T cell-mediated protection is perforin-dependent as well as perforin–independent............................51 4.CD8 T cells from Histoplasma-infected mice have cytotoxic function and produce IFNγ......................52 Part III. CD8 T Cells in Chronic Histoplasmosis in IIKO Mice.....................................................53 1.CD8 T cells in IIKO mice do not clear low dose Histoplasma inoculum.....................................53 2.CD8 T cells in IIKO mice are activated after Histoplasma infection................................................54 3.IFNγ production in IIKO mice...........................55 4.IIKO mice do not survive high dose infection...........57 Part IV. Dendritic Cells Cross-Present Exogenous Fungal Antigens to Stimulate a Protective CD8 T Cell Response in Infection by Histoplasma.................................58 1.Macrophages are not efficient in presenting Histoplasma antigens to stimulate sensitized T cells.................58 2.Dendritic cells are more potent than macrophages to stimulate CD8 T cell IFNγ production and granzyme B expression...............................................59 3.Engulfing live Histoplasma yeasts induces macrophage apoptosis................................................59 4.Dendritic cells take up apoptotic macrophages..........60 5.Dendritic cells acquiring Histoplasma antigens from apoptotic macrophages efficiently induce MHC class I-dependent CD8 T cell response............................61 6.Alveolar macrophages undergo apoptosis after ingestion of Histoplasma and serve as antigen-donor for dendritic cells to cross-present antigens to CD8 T cells...........62 Chapter V. Discussion....................................63 1.Antigen-specific IFNγ-producing cells and their role in host defense to infection................................64 2.The kinetics of the expansion and contraction of IFNγ-producing cells..........................................65 3.T cell expression of CD44hi phenotype and IFNγ production...............................................66 4.IFNγ production in infection...........................67 5.T cell Vβ repertoires in Histoplasma infection.........69 6.Evolution of the TCR Vβ repertoire in bacterial and viral infections.........................................71 7.The effector functions of CD8 T cells in host defense against histoplasmosis...................................73 8.Functional limitation of CD8 T cells in the absence of CD4 T cells..............................................74 9.CD8 T cells in MHC class II-/- and CD4-/- mouse........76 10.The role of macrophages in Histoplasma infection......77 11.The role of dendritic cells as antigen-presenting cells in Histoplasma infection.................................79 12.The mechanisms of cross-presentation..................81 13.Conclusion............................................82 Reference................................................85 Figures.................................................100 Tables..................................................155 Appendix................................................162 Publication List........................................16

Yersinia pestis Proteomics and Vaccine Development

Pneumonic plague, caused by the gram-negative bacteria Yersinia pestis, has led to millions of human causalities during several major pandemics and has the potential to be employed as a deadly biological warfare agent. For this reason, it is vital that an effective vaccine against Y. pestis be created. Currently, a subunit vaccine exists which works by stimulating B-cells (humoral immunity), but it has failed to protect primates in aerosolized bacterial challenges. In an effort to complement the existing vaccine, an alternative vaccine strategy can use both B-cell stimulation and the targeting of T cells (cell-mediated immunity) with an antigenic peptide isolated from the bacteria. To determine what proteins may stimulate T cells, bacteria from three different strains of Y. pestis, KIM 5, KIM 6, and KIM 8, were cultured on TBA plates for four days and then in BCS liquid medium at either 26 or 37C for four hours. Subproteomic fractionation of different regions of Y. pestis cultures were obtained and examined using 2-D DIGE, size-spin filters, and mixed-mode chromatography to isolate protein fractions that may stimulate T cells. Once differential proteins are selected, future work includes determining structure by mass spectrometry, and testing for antigenic peptides on T4 and T8 T cell stimulation assays

Pre-Clinical Pregnancy Models for Evaluating Zika Vaccines

Zika virus (ZIKV) infection during pregnancy can result in a variety of developmental abnormalities in the fetus, referred to as Congenital Zika Syndrome (CZS). The effects of CZS can range from the loss of the viable fetus to a variety of neurological defects in full-term infants, including microcephaly. The clinical importance of ZIKV-induced CZS has driven an intense effort to develop effective vaccines. Consequently, there are approximately 45 different ZIKV vaccine candidates at various stages of development with several undergoing phase I and II clinical trials. These vaccine candidates have been shown to effectively prevent infection in adult animal models, however, there has been less extensive testing for their ability to block vertical transmission to the fetus during pregnancy or prevent the development of CZS. In addition, it is becoming increasingly difficult to test vaccines in the field as the intensity of the ZIKV epidemic has declined precipitously, making clinical endpoint studies difficult. These ethical and practical challenges in determining efficacy of ZIKV vaccine candidates in preventing CZS have led to increased emphasis on pre-clinical testing in animal pregnancy models. Here we review the current status of pre-clinical pregnancy models for testing the ability of ZIKV vaccines to prevent CZS