Kinetics of CD4 T Cell Cytokine Production, Chemokine Production and Activation after Influenza ...

Kinetics of CD4 T Cell Cytokine Production, Chemokine Production and Activation after Influenza Vaccination by Xi Li Submitted in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy

Supervised By Professor Tim R. Mosmann

Department of Microbiology and Immunology School of Medicine and Dentistry University of Rochester Rochester, New York 2012

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Curriculum Vitae The author was born on September 1980 in Kunming, P. R. China. He attended the University of Science and Technology of China, Hefei, P.R. China, during 1998-2003 and obtained primary Bachelor of Science degree in Biology Science and secondary degree in Business Management. During the fall of 2005, he joined the University of Rochester and started his Ph.D. study in immunology investigating expression of cytokines and chemokines in CD4 T cells and T cell responses after influenza vaccination under the guidance of Dr. Tim R. Mosmann. He received Master of Science degree from the University of Rochester in 2008.

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Publications [1] Li X, Miao HY, Henn A, Topham D, Wu HL, Zand M, Mosmann TR. Ki-67 expression reveals strong, transient influenza specific CD4 T cell responses after adult vaccination. (Vaccine in press) [2] Li X, Mosmann T. The delayed expression of chemokine by human CD4 T cells is a sensitive marker for recent influenza vaccination. (Manuscript in progress) [3] Li X, Miao HY, Henn A, Topham D, Wu HL, Zand M, Mosmann TR. Different CD4 kinetics of the proliferative responses to TIV and LAIV vaccination. (Manuscript in progress) [4] Gao FH, Wang Q, Wu YL, Li X, Zhao KW, Chen GQ. c-Jun N-terminal kinase mediates AML1-ETO protein-induced connexin-43 expression. Biochem Biophys Res Commun. 2007; 356(2): 505-11. [5] Li X, Xu YB, Wang Q, Lu Y, Zheng Y, Wang YC, Lubbert M, Zhao KW, Chen GQ. Leukemogenic AML1-ETO fusion protein upregulates expression of connexin 43: the role in AML 1-ETO-induced growth arrest in leukemic cells. J Cell Physiol. 2006; 208(3): 594-601. [6] Zhao KW*, Li X*, Zhao Q, Huang Y, Li D, Peng ZG, Shen WZ, Zhao J, Zhou Q, Chen Z, Sims PJ, Wiedmer T, Chen GQ. Protein kinase Cdelta mediates retinoic acid and phorbol myristate acetate-induced phospholipid scramblase 1 gene expression: its role in leukemic cell differentiation. Blood. 2004; 104(12): 3731-8. (* Equal contribution) [7] Zhao Q, Tao J, Zhu Q, Jia PM, Dou AX, Li X, Cheng F, Waxman S, Chen GQ, Chen SJ, Lanotte M, Chen Z, Tong JH. Rapid induction of cAMP/PKA pathway during retinoic acid-induced acute promyelocytic leukemia cell differentiation. Leukemia. 2004; 18(2): 285-92.

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Acknowledgements I would like to take this opportunity to express my deepest gratitude to the following people who have helped me, encouraged me and supported me during the time of my Ph.D. training. There are no words to express how thankful I am to my advisor, Professor Tim R. Mosmann, for his guidance, mentoring, understanding and encouragement during this wonderful journey. His dedicated and excellent supervision made this thesis possible. His serious attitude toward science and great spirit of scientific adventure have made a great influence on me during the past years and will continue to benefit me on my future career. I am very grateful to the members of my supervisory thesis committee for their guidance and encouragement. I sincerely thank Professor Jim Miller, Professor Burns Blaxall, Professor Sanjay Maggirwar and Professor Andrea Bottaro for their valuable suggestions and questions which helped me improve my research plan, gain a wide and deeper perspective, and focus on my graduate research career. I feel so fortunate to have had such a great committee mentoring and supporting. My appreciation also goes to all my colleagues in the research group, both past and present. It is a wonderful experience to work with them and this thesis would not have been possible without their support. I especially thank Dr. James Kobie, Dr. Jason Weavers, Dr. Bruce Chung, DaeHee Kim and Gen Maupin for their experimental and theoretical assistance. It was an unforgettable memory in my life. I would like to thank Dr. David Roumanes, Yilin Qi, Nan Deng, Cong Shen and Steven Baker for their numerous help and support in many aspects of my Ph.D. study. I’m also grateful to Dr. James Cavenaugh, Jonathan Rebhahn and Courtney Bishop for their consistent

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support in the lab. Finally I must specially thank to Deanna Maffett and Jennifer Scantlin for their great work in sample collection. Beyond lab members, I would like to express deep appreciation to all my collaborators. I must particularly thank Professor Hulin Wu, Professor Martin Zand, Professor David Topham, Dr. Hongyu Miao, Dr. Alica Henn and Yanfang Huang for supporting the collaborative CBIM projects and giving me the opportunity to work with their wonderful team of biostatistician and scientists. I have truly enjoyed working with them on these projects. I would like to thank Professor Tom Gasiewicz and Fanny Casado of Department of Environment Medicine for their providing of the AhR knockout splenocyte. I’m also grateful to Professor Michael Bulger and George Fromm for their technical support in performing the CHIP assay. I also thanks for Professor Alexandra Livingstone, Professor Eun-Hyung Lee, Professor Sally Quataert and Dr. Jyh-Chiang Wang for their support and inspiring discussion. I would like thank Dr. Tim Bushnell, Matthew Cochran and Flow core for their help in flow cytometry experiment. Finally, I want to express my deep appreciation to Nathan Laniewski for his great work and support in cell sorting. It was always enjoyable working with him. I am deeply grateful to my family for their unlimited love, unconditional support and everlasting encouragement. Their love is always the source of my courage, spirit and strength.

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Abstract The amount and timing of effector molecule secretion are tightly regulated in CD4 T cells during the immune response. T cell cytokine profiles have been studied extensively, but how chemokines are expressed during activation is less clear. This study showed that human CD4 T cells, activated with either influenza antigen or polyclonal stimulation, produce chemokines and cytokines with different kinetics. IL-2, IFNγ and TNFα were quickly induced, while chemokines CCL1, CCL3 and CCL4 were secreted later. Further analysis of sorted early cytokine positive cells showed that even though the IFNγ and IL-2 secreting cells have a preference to subsequently produce chemokines, the majority of chemokine producing cells did not secrete cytokines at early times. In addition to analyzing expression kinetics in individual cells, the kinetics of expansion of cytokine/chemokine-secreting cells during the human immune response to influenza vaccination were measured. The numbers of influenza-responsive CD4 T cells able to secrete chemokines increased transiently, 7 days after influenza vaccination, while the cytokine response did not change significantly. The response was then tracked more precisely by daily sampling, and monitoring of the proliferation marker Ki-67. These two improvements revealed that a substantial fraction of influenza-specific CD4 T cells responded to vaccination.

After 4-6 days, there was a sharp rise in the numbers of Ki-67-

expressing cells that produced cytokines or chemokines in vitro in response to influenza vaccine or peptide. Ki-67+ cell numbers then declined rapidly, and ten days after vaccination, both Ki-67+ and overall influenza-specific cell numbers were similar to pre-vaccination levels. The response to Live Attenuated Influenza Vaccine was similar, but had slightly slower kinetics and higher peak responding cell numbers. Overall, these results demonstrate that CD4 T cells secrete cytokines and chemokines with different kinetics. Ki-67 and chemokine expression are sensitive tools for assessing the quality and quantity of responses to different influenza vaccines, and reveal a response to inactivated influenza vaccine that was difficult to

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detect by previous methods. These results also raise the possibility that vaccination may substantially reshape the anti-influenza T cell memory response, even without significant changes in the overall memory cell numbers.

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Table of Contents Curriculum Vitae…………………………………………………..……………....-iiPublications………………………………………………………………………..-iiiAcknowledgements………………………………………………………………...-ivAbstract…………………………………………………………………………….-viTable of contents…………………………………………………………………-viiiList of Tables……………………………………………………………………..- xi List of Figures ……………………………………………………………………-xii Foreword……………………………………………………………………………-1Chapter I: Introduction 1.1 CD4 T cells regulate immune response………………………………….-21.2 Chemokines are another group of secreting molecule…………………..-51.3 CD4 T cell response during influenza challenge………………………..-81.4 Project and questions. ………………………………………………… -11Chapter II: Material and Method 2.1 Different Kinetics of Cytokine and Chemokine production in human CD4 T Cells. ………………………………………………………………. -122.2 Ki-67 expression reveals strong, transient influenza specific CD4 T cell responses after adult vaccination. …………………………………… -162.3 Expression of CCL1 in mouse CD4 T cell. ……………………………-18Chapter III: Different Kinetics of Cytokine and Chemokine production in human CD4 T Cells 3.1 Introduction…………………………………………………………….-223.2 Results

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- Kinetics of cytokines and chemokines expression were different in human CD4 T cells after TCR stimulation. ………………………………...-23 - Different kinetics of cytokines and chemokines production in response to antigen stimulation.…………………………………………………..-25- Most chemokine producing cells do not secret cytokine during early T cell activation. ……………………………………………………….-26- Transient increase of chemokine expression during influenza vaccination. …………………………………………………………………………..-273.3 Discussion …………………………………………………………….- 28Chapter IV: Ki-67 expression reveals strong, transient influenza specific CD4 T cell responses after adult vaccination 4.1 Introduction ……………………………………………………………-404.2 Results -CD4 T cell responds to TIV vaccination ……………………………...- 41-CD4 T cell responds to LAIV vaccination. ………….………………...-424.3 Discussion ……………………………………………………………..-43Chapter V: Expression of CCL1/TCA3 in mouse CD4 T cells. 5.1 Introduction ……………………………………………………………-545.2 Results - CD4 T cell chemokine induction is dependent on TCR stimulation strength and duration, except CCL1 synthesis which is restricted to memory CD4 T cells. ….………………………………………………- 55 - CCL1 gene promoter histone acetylation is similar between naïve and memory CD4 T cells. .………………………………………………......-56- The transcriptional regulation of CCL1 expression. ………………….-57- The expression of CCL1 is not restricted to specific memory cell subsets. ………………………………………………………………………..-59-

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- CCL1 is expressed in T cell with high proliferation and cytokine production.. …………………………………………………………..-605.3 Discussion …………………………………………………………….-61Chapter VI: General discussion and future projects…………………………- 79 References………………………………………………………………………..- 86-

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List of Tables

Table Table 3-1

Title

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The half maximum secretion time of cytokines and

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chemokines.

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List of Figures Figure

Title

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Figure 3-1

Secretion profiles of human CD4 T cell

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Figure 3-2

Secretion of cytokine and chemokine in human CD4 T cell.

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Figure 3-3

Analysis CD4 T cell cytokine and chemokine secretion.

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Figure 3-4

Kinetics of cytokine and chemokine mRNA expression in

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human CD4 T cells after TCR stimulation. Figure 3-5

Induction of cytokines and chemokines by different

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antigens. Figure 3-6

Tracking chemokine secretion in cytokine producing cells

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Figure 3-7

Secretion of chemokine in sorted cytokine producing cell

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Figure 3-8

Kinetics of cytokine and chemokine secretion during

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influenza vaccination Figure 4-1

Identification of influenza-specific CD4 T cells by flow

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cytometry. Figure 4-2

Transient increase in Ki-67+ cytokine-expressing T cells

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after TIV immunization. Figure 4-3

Increased proportion of Ki-67 expression in influenza- but not SEB-responsive cells after vaccination

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Figure Figure 4-4

Title

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The expression of Ki-67 in bulk memory CD4 T cells after

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TIV immunization. Figure 4-5

T cells Cytokine responses after LAIV immunization.

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Figure 4-6

Cytokine producition of T cells after LAIV vaccination and

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in vitro SEB stimulation. Figure 4-7

Increased proportion of Ki-67 expression in influenza

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responsive cells after LAIV vaccination. Figure 5-1

The expression of CCL1 after TCR stimulation.

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Figure 5-2

The acetylation state of CCL1 gene.

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Figure 5-3

Dependence of CCL1 expression on different signal

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pathways. Figure 5-4

Lack of regulation of CCL1 expression by AhR.

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Figure 5-5

The induction of cytokine and chemokine expression in the

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presence of cycloheximide (CHX). Figure 5-6

Non-specific expression of CCL1 in different cell subsets.

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Figure 5-7

Characterization of CCL1 producing T cells.

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Figure Figure 5-8

Title

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High proliferation and cytokine production in CCL1

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producing cells. Figure 5-9

Expression of Ki-67 in CCL1 secreting cells.

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Figure 5-10

The multi-cytokine producing cell has more CCL1.

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Foreword The author performed all experimental procedures in this thesis unless specified below: Chapter 4, PBMC samples in this section were prepared by Martin Zand’s lab, Department of Medicine, Division of Nephrology, University of Rochester Medical Center, Rochester, NY. Chapter 4, Figure 2 and 3: The statistic analysis in these figures was done by Dr. Hulin Wu and Dr. Hongyu Miao, Department of Biostatistics and Computational Biology, University of Rochester Medical Center, Rochester, NY. Chapter 4, Figure 2: H1N1 peptide pool used in this experiment was prepared by Dr. Jason Weaver, David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY.

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Chapter I: Introduction 1.1 CD4 T cells regulate immune response CD4 T cells have critical roles in the generation of immune responses. They promote B cell isotype switching, somatic mutation and the differentiation of plasma cells and memory cells [1, 2]. In additional to B cell regulation, CD4 T cells also enhance naïve CD8 T cell differentiation into cytotoxic effectors and memory cells, and maintain the cytotoxic T cell response [3, 4]. Furthermore, they regulate macrophage function as well as antigen presenting cells and orchestrate other immune components against a wide variety of pathogenic microorganisms. Besides generating new immunity, CD4 T cells also mediate tolerance to self-antigens and prevent inflammatory damage during pathogen clearance [5]. Differentiation of naïve T cells To acquire effector function which is necessary for regulatory activities, naïve T cells must undergo many steps of division and differentiation. Based on function and patterns of characteristic cytokines, Th1, Th2, Th17, Treg and Tfh lineages have been defined. The pathogen and the combination of cytokine milieu determine the direction of differentiation of naïve T cell after antigen encounter [5]. Differentiation of Th1 cells is related to the sequential activity of IFNγ and IL-12. The development of Th2 cells requires IL-4. TGFβ is critical for the in vitro induction of both Treg and Th17 cells, and with the addition of IL-6 the antigen primed naïve T cells can differentiate into the Th17 lineage. Besides the different effector subsets, antigen stimulated naïve cells may also remain as primed, proliferating cells (Thpp) that could subsequently differentiate into effector cells. T cell differentiation states and cytokine functions Different T cell lineages mediate the clearance of pathogen partly by their ability to produce cytokines. Th1 cells mediate the immune response against intracellular

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pathogens. They contribute to the infection resistance mainly through secreting IFNγ, lymphotoxin (LTA) and IL-2. IFNγ produced by Th1 cells enhances the microbicidal activity of macrophages and neutrophils [6]. Lymphotoxin mediates a large variety of inflammatory, immune stimulatory, and antiviral responses. IL-2 is important for cell proliferation and generation of CD8 memory T cell responses [7]. Th2 cells mediate defense against extracellular parasites including helminthes. They are also involved in the induction of asthma and other allergic disease [8]. Th2 cells produce IL-4, IL-5, IL-9, IL-10, IL-13 and in mouse amphiregulin. IL-4 is the major cytokine that drives Th2 cell differentiation and also mediates the IgE switching of B cells [9-11]. IL-5 stimulates B cell growth, increases immunoglobulin secretion, recruits and activates eosinophils [12, 13]. IL-9 can induce mucin production in epithelial cells during allergic reactions [14]. IL-10 has inhibitory effects. It suppresses differentiation toward the Th1 direction and also suppresses antigen presentation by dendritic cells [15]. IL-13 modulates resistance to intracellular organisms and is the central mediator for allergic asthma. It also upregulates class II expression, promotes IgE class switching and inhibits inflammatory cytokine production [16]. Amphiregulin is a member of epidermal growth factor family. It induces epithelial cell proliferation and participates in the expulsion of the nematode Trichuris muris [17]. Th17 cells were initially defined as a T cell subset involved in chronic inflammatory disease and autoimmunity, but they also mediate host defense to various pathogens, especially extracellular bacteria and fungi [18]. They were characterized as preferential producers of interleukin-17A (IL-17A), IL-17F, IL-21, and IL-22 [19]. IL-17A and IL-17F are genetically linked and often coexpress at the single cell level although there are also single producing cells. They bind to the same receptor of IL17RA, although IL-17A has much higher affinity [20]. Both IL-17A and IL-17F recruit and activate neutrophils during immune responses and IL-17A can induce

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many inflammatory cytokines as well as chemokines [21]. IL-21 made by Th17 cells serves as a positive feedback for Th17 cell differentiation. IL-22 is produced by Th17 cells through a Stat-3 mediated pathway. It mediates host defense against pathogens such as Gram-negative bacterial pneumonia and Citrobacter rodentium [22, 23]. Such defensive functions may largely depend on IL-23 stimulation of innate cells to produce IL-22 rather than the action of Th17 cells. Treg cells have an essential role in the control of immune response-mediated inflammation. They maintain tolerance to self and environment antigens and guard against over reaction to pathogens [24]. They inhibit proliferation of other immune cells and function through both cell-cell contact and/or secreted mediators. IL-10 and TGFβ are two major cytokines secreted by Treg cells that mediate regulatory function [25, 26]. Although TGFβ is not absolutely required for the suppression by Treg cells, it’s a very important mediator for Treg cell inhibition. Tfh cells are considered a new lineage of effector T cells with distinct developmental programming and distinguishable effector function. They are characterized by specific expression of the chemokine receptor CXCR5 and location in the follicular regions of secondary lymphoid tissues [27]. They regulate effector and memory B cell responses and modify B cell immunoglobulin class switching by producing IL-4, IFNγ, IL-10 and IL-21. The plasticity of T cell subsets has recently been described [28]. Inducible Treg (iTreg) and Th17 cells show effector differentiation plasticity [29]. Expression of Foxp3 by iTreg cells or IL-17 by Th17 cells may not be stable and there is a great degree of flexibility in their differentiation options. TGFβ induces both Foxp3 and RORγt in naïve T cells, but Foxp3 is dominant and antagonizes RORγt function unless IL-6 is present [30]. The expression of Foxp3 is stable in thymus derived Treg cells, but not in in vitro generated iTreg [31]. In another example, in vitro Th17

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differentiation shows a STAT4- and T-bet-dependent plasticity towards a Th1 profile in mouse and human [32-34]. Beyond this, even stably committed Th2 cells can reexpress IL-12Rβ2 and produce both IL-4 and IFNγ following a viral infection in vivo [35].

1.2 Chemokines are another group of secreting molecule T helper cells mediate the generation of humoral and cellular immunity through secreting cytokines. In addition to cytokines, another group of secreting molecules chemokines - are also produced by T cells [36-39]. Classification of chemokines Chemokines are a group of small heparin-binding proteins that exhibit very specific cysteine motifs in their amino acid sequence. Depending on the motif displayed by the first two of the four cysteine residues in the molecule, the approximately 50 human chemokines are segregated into four families [40, 41]. CC chemokines, named from CCL1 to CCL28, are the largest family with the first two of four cysteine residues in these molecules adjacent to each other. The second family of chemokines is CXC chemokines, named from CXC1 to CXC16, with a single amino acid residue interposed between the first two canonical cysteines. The third family is the CX3C family, of which CX3CL1 (Fractalkine) is the only member [42]. XCL1 (Lymphotactin) is the sole member in mouse and XCL1 and XCL2 in human of the fourth family with a single cysteine residue. Chemokine functions Chemokines were originally defined as host defensive proteins directing the movement of circulating leukocytes to the site of inflammation or injury. They are also involved in a number of other physiological and pathological processes.

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Chemokines attract mononuclear cells to sites of inflammation. CCL2 is a potent agonist for monocytes, dendritic cells, memory T cells, and basophils [43]. The monocyte chemoattractant proteins - CCL2, CCL7, CCL8 and CCL13- recruit monocytes to sites of trauma, bacterial and mycobacterial infection, toxin exposure, and ischemia. They can also attract basophils and eosinophils and induce degranulation of these cells. CCL3 and CCL4 which are major factors produced by macrophages under bacterial endotoxins stimulation, activate human granulocytes and induce the synthesis and release of other pro-inflammatory cytokines such as IL1, IL-6 and TNFα from fibroblasts and macrophages [44]. Besides the CC chemokines, CXCL8 can also attract neutrophils to sites of inflammation and induce granule exocytosis and the respiratory burst [43]. Chemokines and their receptors control T cell homing during cell development [45]. The homing of lymphoid progenitors in bone marrow is chemokine dependent. High amounts of CXCL12 produced by bone-marrow reticular cells in the bone marrow enhance the hematopoietic stem cell homing to the niches [46]. The increase of CXCL12 concentration in the peripheral blood results in the common lymphoid progenitors mobilizing from bone marrow [47]. The trafficking of T cell progenitors to the thymus is also affected by chemokines. CCL21, CCL25 and CXCL12 are produced or increased in fetal thymus or adult thymus with intrathymic T cell depletion [48, 49]. Although no chemokine has yet been directly linked to T-cell thymus homing, deficiency in chemokine and receptor pairs CCL21 and CCR7, CCL25 and CCR9, CXCL12 and CXCR4 results in delayed thymus colonization or decreased number of total fetal thymocytes [48, 50]. CCR7, CCR9 and CXCR4 not only enhance lymphocyte entry into the thymus, but also control their pilgrimage inside the organ and egress [45]. After thymic egress, interaction between CCR7 and its ligands CCL19 and CCL21 is critical for T cell extravasation to secondary lymphoid organs [51]. Furthermore, chemokine receptors determine the localization of T cells inside lymph nodes. One example is that the chemokine receptor CXCR5

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on primed T cells directs the localization to the follicle where CXCL13 is produced [52]. Besides controlling T cell homing during development, chemokines and their receptors also help the tissue-specific homing of T cells in the steady state and inflammation [53, 54]. CCR7 and L-selectin provide a molecular mechanism for T cells to home to the secondary lymphoid tissue. CCR9 and integrin α4β7 enhance the ability of T cells to home to the gut. CCR4 and CCR10 regulate cutaneous T cell recruitment in the steady state and during inflammation. More recently, in a model of atopic dermatitis, interaction between CCL8 and its receptor CCR8 is crucial for the homing of Th2 cells that drive IL-5 mediated chronic allergic inflammation [53, 55, 56]. Chemokine expression The regulation of cytokine production in CD4 T cells has extensively studied, but the regulation of chemokine expression, especially in T cells, is less clear. Some chemokines are produced in very large amounts by many different cell types, whereas other have very high specificity for particular tissues or cell types. Chemokines can be divided into two major groups based on their expression patterns and functions. Such a division is oversimplified but useful. Those expressed by immune cells or related cells (epithelial, endothelial and fibroblast and so on) upon activation in inflammatory conditions are called “inflammatory”, whereas those expressed in discrete locations in the absence of apparent activating stimuli have been classified as homeostatic. Genomic organization analysis further revealed that most inflammatory chemokines are located in two major clusters of CC chemokine genes and CXC genes at particular chromosomal locations in both mouse and human genomes [57]. Besides these two major clusters, numerous non-clustered or mini cluster genes of both types are located separately in unique chromosomal locations. Such a chromosomal arrangement may be developed from a series of tandem gene duplications that

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occurred during evolution [58]. The common origin and cluster distribution of chemokine superfamilies suggest that there may be some common mechanism that regulates the expression of these inflammatory chemokines during the immune response. Several transcription factors have been reported to be involved in chemokine expression. The promoters of the CCL2, CCL5 and CCL8 genes contain binding sites for the redox-responsive transcription factors AP-1 and NF-κB. H2O2 induction of CCL8 gene expression is linked with the selective binding of AP-1 to the CCL8 promoter [59], whereas TNFα and respiratory syncytial virus [59] induction of CCL8 correlates with the activation of NF-κB binding [60]. Differential activation and binding of inducible transcription factors to the promoter regions of chemokine genes provides a critical regulatory mechanism by which the CXC and CC chemokines can be selectively expressed in a cell type-specific and stimulus-specific manner [61]. 1.3 CD4 T cell response during influenza challenge Human influenza virus is the major pathogen threat for all age groups, especially for infants and elderly people, in the respiratory tract. Influenza epidemics lead worldwide to millions of cases of severe illness and up to 500000 deaths annually [62, 63]. The devastating 1918-1919 pandemic led to more than half million deaths in the United States and 40 million deaths worldwide [64]. Effectively preventing both epidemics and pandemics of influenza will have great benefit both economically and for public health [65]. The current influenza vaccine is not perfect Vaccination with partially purified subunit vaccines (TIV) or live attenuated viruses (LAIV) can reduce both the risk of influenza infection and the associated morbidity and mortality [66]. Due to the rapid antigenic change of influenza, annual immunization is recommended, with a vaccine containing the predominant strains of

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A/H3N2, A/H1N1 and B viruses that are expected to circulate during the following season. However, the predictions of predominant strains are sometimes inaccurate, and the antibodies generated against circulating influenza strains often do not effectively neutralize emergent pandemic strains [62, 67]. Weakened immune functions in elderly subjects compromise the effectiveness of influenza vaccination [65, 66, 68, 69]. Understanding how the immune system responds to influenza challenge will be helpful to either the development of new vaccination strategy or to set up age-specific vaccine plans. Immune response to influenza infection in mice Based on studies from mouse models infected with influenza virus, cytotoxic CD8 T cells play a major role in the clearance of influenza virus in a MHC class-I-dependent mechanism. MHC class I deficient mice (β2 microglobulin-/-), which lack CD8 T cells, display delayed viral clearance of HKx31 influenza in the respiratory tract. The infection with the highly pathogenic A/Puerto Rico/8/34 (PR8) strain of influenza leads to eventual morbidity for mouse lacking CD8 T cells [70, 71]. B cells and neutralizing antibody are also important in immunity to influenza virus. B cell deficient µMT mice could clear HKx31 influenza with delayed kinetics, but are compromised for resistance to PR8 influenza with higher morbidity compared with wild type control [72-74]. CD4 T cells themselves may not be required for elimination of virulent influenza strains, but they are required for the generation of both B cell and cytotoxic T cell responses. The µMT mouse treated with anti-CD4 depleting antibody cannot clear PR8 virus and shows a high mortality to this virulent strain [75]. This suggests that CD8 T cells alone cannot clear the virus, and CD4 T cells may be important for sustaining the cytotoxic response. A similar situation is also observed in B cell deficient mice with anti-CD8 depleting antibody. The remained CD4 T cells cannot effectively clear the virus alone [74, 76]. Therefore CD4 T cells may only participate indirectly to provide help for CD8 and B cells. All these studies indicate that for a virulent strain of influenza neither CD8 cells, CD4

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cells or B cells alone can effectively clear virus. However, combination of these cells can clear infection with slightly delayed kinetics and increased survival [77]. The response to influenza vaccine in humans In humans, when predicted strains match epidemic virus, after influenza vaccination, the enhanced neutralizing antibody title is highly correlated with protection although defects in seroconversion and seroprotection were observed in elderly people [66, 68, 78, 79]. Transient expansion of antibody secreting cells (ASC) following immunization is associated with the enhancement of neutralizing antibody and long lasting humoral memory [80-82].

However, CD4 and CD8 T cell responses

contribute to successful resolution of infection in mice, and may play a similar role in humans [83, 84]. CD4 T cells may contribute to local inflammatory responses, and also provide help for B cell antibody responses. Elevation of CD4 T cell responses is not detected after TIV vaccination in adult. Despite the remarkable expansion of ASC after vaccination, a change in the mean level of T cell responses was not observed in TIV vaccinated adults based on the IFNγ secretion assay [85, 86]. Almost all adults have detectable IFNγ memory T cell responses to influenza due to early and repeated exposure to infection and/or vaccination. The reverse correlation between the magnitude of the CD4 T cell response after vaccination and the baseline level of influenza specific IFNγ+ CD4 T cells suggests that CD4 T cells may plateau in response to influenza antigen with repeated exposures due to previous immunization and infection independently from age [86-88]. Thus preexisting memory cells may mask any elevated response from immunization due to the existence of cross-reactive epitopes between different influenza strains [89]. Nevertheless, vaccination or infection may reshape the repertoire or effector functions [90, 91] of the influenza response.

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1.4 Project and questions The timing and magnitude of immune responses are tightly regulated. It raises a question as to whether the kinetics of chemokine expression is different from cytokines in human CD4 T cells. Despite the large volume of studies in cytokine secretion, less research has been performed to address the regulation of chemokine expression in T cells. Revealing the regulatory mechanism of chemokines will contribute to better understanding of T cell function during immune responses. In addition to cytokines, CD4 T cells secrete chemokines. However, using chemokine secretion as a measurement for T cell function is not common. Thus, characterizing chemokine expression in CD4 T cells will facilitate the design of new T cell functional assays. In addition to analyzing expression kinetics in individual cells, the kinetics of expansion of cytokine/chemokine-secreting cells during the human immune response to influenza vaccination is unclear. With daily sampling and incorporating Ki-67 measurement, the influenza specific CD4 T cell response will be analyzed in more detail.

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Chapter II: Material and method 2.1 Different Kinetics of Cytokine and Chemokine production in human CD4 T Cells. Vaccine administration Five healthy subjects, who had not received influenza vaccination for current year were recruited at the University of Rochester Medical Center during winter/spring 2008-2009. Subjects were immunized by intramuscular injection with standard 20082009 seasonal TIV vaccine (FluLaval, GlaxoSmithKline). Blood was obtained prior to immunization and 7 days and 28 days after. All the protocols and procedures were approved by the Research Subjects Review Board at the University of Rochester Medical Center. Peripheral blood mononuclear cell (PBMC) separation Heparinized blood was diluted with PBS at 1:1 ratio. 50ml Falcon polypropylene tube was prepared with 20ml Ficoll solution (Lonza) on the bottom. 20ml of diluted blood were laid carefully on the top surface of the Ficoll solution without disturbing the liquid interface. Tubes were centrifuged for 20 minutes at 800RPM at room temperature with the brake off. There will be a mononuclear cell layer at the interface of serum and Ficoll solution after centrifuge. Mononuclear cells were removed into a new tube and washed once with RPMI medium. The PBMC were ready to use for next step. Sorting of naïve and memory human CD4 T cells PBMCs were washed once with ice cold PBS+1%FBS. Cell surface was stained with fluorochrome conjugated anti-CD4, anti-CD8, anti-CD56, anti-CD45RA, antiCD45RO antibodies and subjected to two-way flow cytometric sorting using a FACSAria (BD Biosciences). CD4 T cells were gated as CD4+CD8-CD56- and

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further sorted into naïve cells as CD45RAhighCD45ROlow and memory cells as CD45RAlowCD45ROhigh. The resultant sorted cell populations had purity > 98%. Cell surface Pacific Blue Succinimidyl Ester (PBSE) label The protocol was modified from reported article [92]. PBSE (Invitrogen Life Technologies) was diluted into 15µg/ml with PBS. Cells were washed twice with PBS and diluted 10 million per ml in PBS. 110µl of PBSE was added into each ml of cells. Cells were stained for 10 minutes at room temperature and washed once with PBS and once with RPMI medium. Cells were ready to use for the next step. Sorting the cytokine secreting cell The PBSE labeled PBMC were diluted to 10 million per ml by medium (RPMI1640 plus 10% FBS) and stimulated with SEB for 10 hours. After stimulation, cytokine secreting cells were labeled by the cytokine secretion assay (Miltenyi Biotec). Briefly, cells were washed twice with cold MACS buffer (0.5% BSA, 2µM EDTA in PBS) and diluted to 1 million in 90µl by MACS buffer. 10µl cytokine capture antibody was added into each million cells and incubated on ice for 5 minutes. After incubation, cells were diluted to 1 million per ml in pre warmed medium and incubated for 45 minutes on slow shaking at 37ºC. Cells were cooled on ice after 45 minutes incubation and washed twice with cold MACS buffer. The cell surface was labeled with CD4-Qdot705, CD8-PE-TexRed, CD14-Qdot800, CD56-PE-Cy5.5, CD69-APC-Cy7, CD45RA-Qdot655 and APC labeled cytokine detection antibody for 15 minutes in ice. Memory/effector CD4 T cells were gated as CD4+CD8-CD14CD56-CD45RAlowPBSE+, further sorting the CD69high cytokine positive and negative cells and CD69low cells. The sorting was performed in an 18-color FACSAria (BD Biosciences). Intracellular cytokine staining

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PBMCs were isolated from the whole blood by Ficoll (Lonza) gradient centrifugation and washed once with RPMI1640 containing 10% FBS. Cells were stimulated with different stimuli for the indicated time. Golgiplug (BD Pharmingen) and 2µM Monensin were added into cells at the last 6-8 hours of culture. Cells were washed twice with ice cold PBS and stained with 10µl Live/dead staining (Invitrogen) for 30 minutes at 4˚C. Cells were washed twice with HBSS containing 1%FBS and surface stained with specific fluorochrome conjugated antibodies. For intracellular cytokine and chemokine staining, surface stained cells were fixed, permeabilized and stained with fluorochrome-conjugated or biotin conjugated cytokine and chemokine antibodies followed by strepavidin staining. The staining process was performed using the intracellular cytokine staining kit (BD Pharmingen) following manufacture’s recommendations. After staining, cells were fixed by 1% formaldehyde and acquired on LSR-II Flow Cytometer (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star). Real-time RT-PCR Sorted naïve or memory CD4 T cells were stimulated with plate bound anti-CD3 and anti-CD28 antibody for the indicated time. The supernatant were collected from the wells and cells were lysed with Trizol (Invitrogen).

The mRNA was extracted

following manufacture’s protocol and reverse transcripted into cDNA using Superscript III first strand DNA synthesis system (Invitrogen). IL-2, IFNγ, TNFα, CCL1, CCL3, CCL4 and CD3ε probes and primers were obtained from Taqman gene expression assays (Applied biosystem). PCR reaction was perform in 10µl reaction volume with 5µl 2X Universal PCR master mixture, 1µl cDNA, 3.5µl H2O, and 0.5µl 20X probe and primers mixture in a 384-well optical PCR plate. PCR begun with 10min at 95ºC followed by 40 cycles with denaturing at 95ºC for 15s and annealing/extending at 60ºC for 1min in an ABI 7900. All data were analyzed using ABI Prism SDS 3.0 software (Perkin-Elmer). Gene expression unit was arbitrary

15

determined by normalizing gene expression to CD3ε expression using the ΔCt method. Luminex assay Concentration of cytokines and chemokines in supernatant was determined by Milliplex Map Kit (Millipore) following manual. Briefly, supernatant was coincubated with premixed human cytokine/chemokine antibody-immobilized beads overnight at 4ºC in filter plate with shaking. Beads were then washed twice with wash buffer. Biotin-labeled detection antibodies were incubated with the beads at room temperature for 1 hour followed by streptavidin-Phycorythrin staining. Beads were run on Luminex 100 and at least 300 beads were acquired for each cytokine/chemokine. The medium fluorescent intensity data was used for calculating cytokine/chemokine concentrations with the 5- parameter logistic method by BioPlexTM Manager 3.0 software. Elispot assay The frequency of cells secreting cytokine and chemokine was measured by Elispot as previously described [38]. Briefly, filter plates (Millititer MAIPN4550, Millipore Corp., Bedford, MA) were pre-coated with capture antibody and anti-CD3, anti-CD28 antibody at room temperature for 2 hours. Then the plates were washed three times with RPMI medium. Serial dilutions of CD4 T cells were added to the wells and incubated (37°C, 8% CO2) for different times. The plates were washed with PBST, then incubated with biotinylated secondary antibody in PBSTB (PBST + 2% BSA), followed by streptavidin-conjugated alkaline phosphatase (Jackson) diluted 1:1,000 in PBSTB, and developed using the AEC substrate kit (Vector Laboratories, Burlingame, CA). Plates were then dried, and the spots were enumerated using C.T.L. ImmunoSpot 5.0.2. Antibody pairs were purchased from R&D system for CCL1 (Cat. No. MAB272, BAF272) and CCL3 (Cat. No. MAB670, BAF270), from Mabtech for CCL4 (Cat. No. 3495-3-250, 3495-6-250) and IFNγ (Cat. No. 3420-3-250, 3420-6-

16

250) from BD Pharmingen for IL-2 (Cat. No. 551884) or from ebioscience for TNFα (Cat. No. 14-7348, 13-7349). 2.2 Ki-67 expression reveals strong, transient influenza specific CD4 T cell responses after adult vaccination Vaccine administration Healthy subjects, who had not received influenza vaccination for the current year, were recruited at the University of Rochester Medical Center during winter/spring of 2010-2011 and 2011-2012. In winter/spring 2010-2011, fourteen subjects were immunized by intramuscular injection with the 2010-2011 seasonal TIV vaccine (FluLaval, GlaxoSmithKline). In winter/spring 2011-2012, eighteen subjects were intranasal immunized with standard 2011-2012 seasonal LAIV vaccine (FluMist, MedImmune). Blood was obtained prior to immunization and daily thereafter for 10 days. All the protocols and procedures were approved by the Research Subjects Review Board at the University of Rochester Medical Center. Antibodies Antibodies used were: CD3-QDot605 (UCHT1), CD4-PE-TexRed (S3.5), CD8QDot705

(3B5),

CD14-QDot800

(TUK4),

CD45RA-QDot655

(MEM-56)

(Invitrogen); CD69-APC-Cy7 (FN50), IL-2-Pacific blue (MQ1-17H12), TNFαPercp-Cy5.5 (MAH1) (Biolegend); IFNγ-PE-Cy7 (B27), Ki-67-Alex700 (B56) (BD Pharmingen). In vitro stimulation PBMC were isolated from heparinized blood by Ficoll (Lonza) gradient centrifugation and incubated with or without antigens for 2 hours in RPMI (Mediatech, Manassas, VA) containing 10% FBS (Sigma-Aldrich, St. Louis, MO), then 2µM Monensin and Golgiplug (BD Pharmingen) were added for an additional 8

17

hours. Overlapping peptides from all proteins of influenza virus A/California/04/2009 (H1N1) (15mers offset by 5), were obtained from Mimotopes (Clayton Victoria, Australia), pooled, and used at 20ng/ml/peptide. TIV influenza vaccine (Fluzone, A/California/7/2009 (H1N1), A/Victoria/210/2009 (H3N2) and B/Brisbane/60/2008) from Sanofi (Swiftwater, PA) was used at 1.25µg/ml HA. LAIV influenza vaccine (FluMist,

A/California/7/2009

(H1N1),

A/Victoria/210/2009

(H3N2)

and

B/Brisbane/60/2008) was used at 2.5µl/sample. Negative control for peptide was 0.1% DMSO, positive contrl for staining was Staphylococcal exotoxin B (SEB) (Sigma-Aldrich) used at 1µg/ml. Intracellular staining PBMC were isolated from heparinized blood by Ficoll gradient centrifugation. Cells were incubated with or without antigen for 2 hours and add 2µM Monensin and Golgiplug (BD Pharmingen) for additional 8 hours. Stimulated cells were washed once with ice cold HBSS containing 1%FBS and surface stained with specific fluorochrome-conjugated antibodies. For intracellular cytokine and chemokine staining, surface stained cells were fixed, permeabilized and stained with fluorochrome-conjugated cytokine, chemokine and Ki-67 antibodies. The staining process was performed using the intracellular cytokine staining kit (BD Pharmingen) following manufacture’s recommendations. After staining, the cells were fixed with 1% formaldehyde and acquired on LSR-II Flow Cytometer (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star). Statistical analysis To deal with repeated measures with missing time points among subjects, the linear mixed-effects (LME) model, together with the AR(1) model were used. Data were normalized and log-transformed before background subtraction, and then used in the LME fitting. Based on the LME fitting results, the Tukey’s all-pair comparison was used to test the difference between the baseline (day 0) and the peak window (pooled

18

data from days 4-6) only, or to test the differences among the baseline, the peak window and the late stage (pooled data from days 9-10) simultaneously. To test the differences in the proportions of Ki-67 expression under different stimulations, the Wilcoxon signed-rank test was applied to data without background subtraction at each time point. 2.3 Expression of CCL1/TCA3 in mouse CD4 T cell. Mice Female C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Aryl hydrocarbon receptor (AhR) deficient mice were kindly provided by Dr. Tom Gasiewicz from the Department of Environment Medicine on University of Rochester Medical Center. Mice were maintained in the pathogen-free animal facility at the University of Rochester, School of Medicine and Dentistry, Rochester, NY. Cell Purification Naïve and memory CD4 T cells were purified from spleen by flow cytometry sorting using FACSVantage cell sorter (BD Bioscience, San Jose, CA). Single-cell suspension was made by passing tissue through a 0.70µm nylon strainer using a syringe plunger. Cells were washed once with cold PBS + 1% FBS and labeled with antibody cocktail prior FACS-based sorting. Following monoclonal antibody were used: anti-CD4-PE, anti-CD8-FITC, anti-CD19-PE-TexRed, anti-CD25-APC, antiCD44-PE-Cy7 anti-CD62L-APC-Cy7. Sorting the naïve CD4 T cell as CD4+CD8CD19-CD25lowCD44lowCD62Lhigh, memory CD4 T cell as CD4+CD8-CD19CD25lowCD44hgihCD62Llow. Resultant population was more than 98% pure. Capture antibody streptavidin conjugation 1µl of LL-Modifier reagent was mixed for each 10µl antibody labeling. Antibody (with added LL-modifier) was directly pipette into the vial of Lightning-LinkTM

19

(Innova Biosciences) and resuspended gently. The vial was re caped and left standing for 3 hours at room temperature. After incubating for 3 hours (or more), 1ml of LLquencher reagent was added for every 10ml of antibody used. The conjugate can be used after 30 minutes. Chemokine secretion assay Cells were washed three times with ice-cold PBS to remove amine-containing culture media and diluted to 10 million per ml in PBS. Sulfo-NHS-LC-Biotin [93] was weighted in the 1.5ml tube and diluted in sterile water to achieve the final concentration of 10mg/ml. Solution should be made freshly. 100µl of biotin were added into 900µl cell suspension to achieve the biotin final concentration of 1µg/ml. Cells were incubated at room temperature for 10 minutes followed by adding on equal volume of FCS to quench the biotin reagents. Cells were washed three times in RPMI medium. Biotin labeled cells were stimulated following the in vitro stimulation protocol. After stimulation, cells were collected and washed twice by cold PBS with 1% FBS. 1µg streptavidin conjugated antibody was added into 107 cells suspending in 90µl of cold buffer. After incubating for 5 minutes on ice, pre warmed (37℃) medium were added to dilute the cells to 1 million per ml. Cells were then incubated for 45mins at 37℃ with slow continuous rotating or turning tube every 5 mins to resuspend settled cells. The chemokine secreting cells were then detected by using fluorescent antibody detection following the previous described protocol. Intracellular staining Cells were stimulated with antigen bearing antigen presentation cell or plate bound anti-CD3/anti-CD28 antibody for 2 hours and added 2µM Monensin and Golgiplug (BD Pharmingen) for additional 6 hours. Stimulated cells were washed once with ice cold PBS containing 1%FBS and stained with fluorochrome-conjugated antibodies. For intracellular cytokine and chemokine staining, surface stained cells were fixed, permeabilized and stained with fluorochrome-conjugated cytokine and biotin

20

conjugated chemokine antibodies followed by strepavidin staining. The staining process was performed using the intracellular cytokine staining kit (BD Pharmingen) following manufacture’s recommendations. After staining, the cells were fixed with 1% formaldehyde and acquired on LSR-II Flow Cytometer (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star). Elispot assay Experiments were performed following protocol of Material and Method section 2-1. Antibody pairs were purchased from R&D system for CCL1 (Cat. No. MAB8451, BAF845), CCL3 (Cat. No. MAB670, BAF270), and CCL4 (Cat. No. 3495-3-250, 3495-6-250), IFNγ (Cat. No. 3420-3-250, 3420-6-250) from BD Pharmingen for IL-2 (Cat. No. 551884) or from ebioscience for TNFα (Cat. No. 14-7348, 13-7349). The spots were counted by using C.T.L. ImmunoSpot 5.0.2. Chromatin Immunoprecipitation Freshly

sorted

cells

were

formaldehyde

crosslinked,

sonicated

and

immunoprecipitated, and DNA isolated following protocol provided by Dr. Michael Bulger of Department of Pediatrics and Biochemistry and Biophysics, University of Rochester Medical Center. Briefly, cells were washed with PBS and cross-linked at room temperature in 10mL volume containing 11% formaldehyde. After adding of 0.5mL of 2.5 M glycine and a phosphate-buffered saline wash, cells were resuspended in 3mL and sonicated for a total of 170 seconds using a Misonix Sonicator 3000 at 36 to 39 W output. An “input” fraction was then removed to serve as a control. Protein A beads were prepared by washing the beads slurry three times with protein wash buffer. 600µl of protein A slurry were aliquoted for each sample with adding 300µl normal rabbit serum for precleaning. Cell lysis was added into beads coated with antibody, and set on the rocker for overnight at 4ºC. Antibodies used for immunoprecipitation were normal rabbit IgG (Upstate-Millipore) as a control, and anti-histone H3 acetylated at K9 and/or K16 (Upstate-Millipore). 100µl

21

of protein A slurry were added into each tube with rotating for additional 2 hours. The beads were washed sequentially in Paro buffer I, II, III on ice and TE at room temperature. The precipitated DNA fragments were eluted 2 times with 100µl elution buffer with 0.15µg/µl proteinase K and purified by Qiagen PCR purification Kit. Analysis was performed using quantitative polymerase chain reaction (PCR) with sybr green. Results are normalized to the percentage of input. Six pairs of primes were designed covering the reported transcriptional regulatory region on the CCL1 genes. The sequences of primers are TCA 150 to -1 F: CTC ACT GCT CTT GCT GTC AAC ATC; TCA 150 to -1 R: GGA GAG AGT TCT TGG CTC CAC C; CCL1 -172 to -329 F: CCT AGT GGC CTT GGG AAC TTC C; CCL1 -172 to -329 R: TGG GTT AGA CAA GCT GTG TAC GG; CCL1 -246 to –395 F: ATA CGT GGG AGC TGT CCT GG; CCL1 -246 to –395 R: CCA TTT GAT GCA GAT TGT ACC TTC; CCL1 -1306 to –1496 F: TTC AGT TGC ACA GGC TAA GGT C; CCL1 1306 to –1496 R: TGG GAT GTA GAT GCA GAT GGG; CCL1 -1343 to –1539 F: AGC CAA CCC ACT GTG TAT AGG C; CCL1 -1343 to –1539 R: AGA GGA ACA CCC TTC ACT CAG C; CCL1 -2112 to –1927 F: GTG GGT GGC AGG AAG CAG TAG; CCL1 -2112 to –1927 R: CAG TAT GGA GGG TGG GAG GG.

22

Chapter III: Different Kinetics of Cytokine and Chemokine production in human CD4 T Cells. 3.1 Introduction CD4 T helper cells mediate inflammation, pathogen clearance, immune memory generation, autoimmunity and tumor regression through secreting cytokines and chemokines. Before acquiring the effector function for their regulatory activities, antigen primed naïve T cells undergo many steps of division and differentiation into effector cells. The combination of cytokine milieu, the quantity and quality of antigen, the duration of antigen presentation, and genetic background determine the direction of the differentiation process [8, 94-97]. Different T cell lineages mediate the clearance of pathogens in part by their ability to produce characteristic cytokines. Type 1 helper T (Th1) cells mediate the clearance of intracellular pathogens mainly through the secretion of IFNγ, IL-2 and lymphotoxin, while type 2 helper T (Th2) cells mainly secrete IL-4, IL-5, IL-9, IL-10 and IL-13 during the immune response against parasites [98, 99]. Th17 cells fight against bacterial and fungal infections with the characteristic secretion of IL-17A, IL-17F, IL-21, and IL-22 [5, 100]. Treg cells control immune response mediated inflammation partially through secreting IL-10 and TGFβ. The secretion of these cytokine is lineage specific and different transcription factors direct expression of distinct soluble mediators that support pathogen clearance [100, 101]. The finding of irreversible commitment of CD4 T cell might be the end result of repeated stimulation with antigen in vitro or chronic disease in vivo. Memory T cell populations are considered flexible with regard to cytokines production rather than irreversibly committed in both mouse and human T cells [102, 103]. Several modes of plasticity of T cell subsets have recently been described. Cytokines and costimulation, network of interactions between transcription factors, cell clonality,

23

and chromatin modifications are the prime factors that impact the differentiation, stability and plasticity [28, 104]. Besides secreting cytokines, effector T cells also secrete chemokines [38, 39, 105]. Chemokines are a group of small heparin-binding proteins that exhibit very specific cysteine motifs in their amino acid sequence. Chemokines are originally defined as host defense proteins directing the migration of circulating leukocytes to sites of inflammation or injure. Besides chemoattraction, they also have broad functions in T cell development, tissue homing, T cell differentiation and generation of adaptive immunity [40, 41, 45]. Both cytokines and chemokines mediate the regulatory function of T cells. The cytokine gene expression profile is extensively studied and chosen to measure T lymphocyte response during the immune reaction, while the expression of chemokines by CD4 T cells is less clear. The amount and timing of effector molecule secretion, which is highly correlated with immune protection, are tightly regulated during the immune response. Considering the functional differences between cytokines and chemokines, it’s reasonable to hypothesize that the kinetics of chemokine produce may differ from cytokines [106, 107]. In this study, we measured the detailed kinetics of cytokine and chemokine production in human CD4 T cells. From that, chemokine assays were developed and tested on measuring T cell response in the influenza vaccination model. 3.2 Results Kinetics of cytokine and chemokine expression were different in human CD4 T cells after TCR stimulation. The immune regulatory functions of CD4 T cells are mediated mainly through their secretion of cytokines and chemokines. Due to the functional difference in mediators, the kinetics of secretion may be different between cytokines and chemokines during T

24

cell activation. To test this, the secretion spectrum was characterized, using a cytokine/chemokine multiplex assay, by measuring supernatant mediators in sorted naïve and effector/memory human CD4 T cells after polyclonal stimulation. 43 cytokines and 22 chemokines were included in the assay. Cytokines and chemokines were produced mainly by effector/memory T cells, except IL-2 and TNFα which were also secreted by naïve T cells but in much lower amounts. The concentration of secretory molecules increased with time. The concentration of 28 cytokines and 12 chemokines secreted by effector/memory CD4 T cells reached detectable levels during the total 96 hours measurement (Figure 3-1). Cytokines IL-2, IL-4, IL-10, IL13, IL-17, IFNγ, TNFα, GMCSF and sCD40L were produced at the highest levels (>20ng/ml) by activated CD4 T cells, while only chemokines CXCL8, CCL3, CCL4, and CCL5 were produced at the same level (>20ng/ml) (Figure 3-1A). The kinetics of cytokine and secretion were different. Quick cytokine induction was observed in activated CD4 T cells, followed by a slight reduction and then gradual accumulation. The early expression phase did not exist for most chemokines, and the concentration of chemokines increased late after T cell activation (Figure 3-1B). Comparing with the highest concentration produced within 96 hours after TCR stimulation, the concentration of most chemokines was less than 1% of that value at 8 hours and greatly increased at 24 hours, whereas the majority of cytokines have already reached a high concentration in 8 hours (Figure 3-2). Thus, the secretion pattern is different between cytokines and chemokines. The detailed expression kinetics of these most abundantly produced mediators were further analyzed by intracellular cytokine staining [59] in anti-CD3/CD28 stimulated human PBMC. Figure 3-3A shows the gating strategy used to identify CD4+CD8CD45RAlow memory/effector cells producing cytokines and chemokines by ICS. In this example, IL-2 was expressed at early times, 6-12 hours, whereas CCL3 was expressed much later, at 24-72 hours. Figure 3-3B shows the summarized results for the ICS experiments, compared with other protein measurements, Elispot, on

25

supernatants. The secretion pattern difference between cytokines and chemokines is contributed by different kinetics. Cytokines IL-2, IFNγ and TNFα were transiently secreted in the first 12 hours even in the persistent TCR stimulation, while cells secreting CCL1, CCL3 and CCL4 were rare in the first 12 hours with a gradual increase later (Figure 3-3). Such kinetics fit the Elispot assays results, with high IL-2, IFNγ and TNFα spots number in the first 12 hours from early secretion and gradually increasing number of CCL1, CCL3 and CCL4 spots from 24 to 48 hours. Moreover, the IL-2 and TNFα spot numbers reached a plateau after 12 hours while IFNγ spots number kept increasing after early secretion. New IFNγ secreting cells appeared at the late stage. The secretion kinetics of cytokines and chemokines were also correlated with the mRNA induction. IL-2, IFNγ and TNFα mRNA were dramatically induced in memory/effector CD4 T cells in the first 12 hours after TCR stimulation. Maximum induction of these cytokine mRNAs was observed between 6 to 10 hours followed by decreasing of IL-2 and TNFα to low levels, while IFNγ expression continued with the secondary induction at 48 hours. The mRNA levels of CCL1, CCL3 and CCL4 were low in the first 12 hours, and gradually reached a peak between 24 to 48 hours (Figure 3-4). Taken together, IL-2, IFNγ and TNFα were quickly induced after T cell activation, whereas CCL1, CCL3 and CCL4 were expressed in late time point after TCR stimulation. Looking only in the first 12 hours will underestimate the number of cells producing some activated products, particularly chemokines. Different kinetics of cytokines and chemokines production in response to antigen stimulation The expression of cytokines is related to the microenvironment, e.g. antigen type, antigen dose and the type of antigen presenting cells. The kinetics of secretion may vary under different antigen stimuli. To directly investigate whether the kinetics difference between cytokines and chemokines still exists under different antigen

26

stimulations, PBMC were stimulated with either Staphylococcus aureus Enterotoxin B (SEB) or influenza vaccine. Cytokines IL-2, TNFα and IFNγ were produced rapidly following the superantigen or antigen stimulation and reached the maximum frequency in the first 24 hours. Chemokines CCL1, CCL3 and CCL4 mostly increased after 12 hours stimulation and reached the maximum frequency after 24 hours under those two antigen stimulations (Figure 3-5). To further compare the relative rates of cytokine and chemokine production, the time to reach half-maximal frequency (t1/2max) for each effector molecule was calculated. There is no significant difference in t1/2max between anti-CD3/28 stimulation and antigens for each effector molecule. The t1/2max of chemokines was significantly larger than cytokines after antigen stimulation (Table 3-1 and Figure 3-5). The difference in cytokine and chemokine secretion was maintained under influenza vaccine or polyclonal stimulation. Most chemokine producing cells do not secret cytokine during early T cell activation. As cytokines and chemokines were mostly produced at different times after T cell activation, this raises a question as to whether cytokines and chemokines were produced sequentially in the same cell or in different CD4 T cell subgroups. To test these two possibilities, cytokine secreting memory/effector CD4 T cells were sorted from eight hours SEB stimulated PBMC, and expression of cytokine and chemokine was measured after prolonged culture (Figure 3-6). Both the cytokine positive and negative SEB responsive CD69+ cells were sorted. T cells with or without cytokine secretion in early stimulation secreted chemokines at later times. The frequency of CCL1, CCL3 and CCL4 positive cells at later times was significantly higher in sorted IFNγ+ or TNFα+ cells than their negative counterparts. Sorted IL-2 expressing cells have significantly higher production of CCL1 than IL-2 negative cells; such difference was not observed for CCL3 and CCL4 (Figure 3-7). Even though the cytokine producing cells had a preference to make chemokines, considering the low

27

percentage of cytokine producing cells in the total SEB responsive CD4 T cell population, most of the chemokine producing cells were negative for cytokine production during early T cell activation (Figure 3-7). Thus, secretion of chemokines does not necessarily follow cytokine secretion during T cell activation. Transient increase of chemokine expression during influenza vaccination. The cytokine profile is commonly used for measuring T cell immune responses and antigen specific T cell expansion after immunization. The test of cytokine expression in short term T cell activation will underestimate the number of cells producing effector molecules, especially chemokines. The measurement of the cytokine profile alone could not fully describe the T cell response after vaccination. Adding the chemokines into the test panel may reveal additional changes after vaccination. The chemokine response was therefore tested in the model of influenza vaccination. The CD4 T cell activation states and expression of cytokines and chemokines may change during the generation of adaptive immune response. Kinetics of chemokine expression in vitro were tested after vaccination. Cytokine and chemokine expression were measured in ex vivo antigen stimulated PBMC from subjects at multiple time points after trivalent inactivated influenza virus (TIV) vaccination. The cytokines were quickly secreted in ex vivo stimulation. The highest expression of IL-2 and TNFα was observed in the first 24 hours while the expression of CCL3 and CCL4 reached highest levels in 48 or 72 hours, in samples taken either before or after vaccination. The expression of IFNγ was relatively constant in all ex vivo testing time points with a minor reduction after 24 hours. The expression pattern of CCL1 was different from other chemokines after vaccination, with highest expression in 24 hours at day 7 after vaccination. The difference in cytokines and chemokines secretion in vitro did not change after vaccination (Figure 3-8, and was present in both ex vivo influenza vaccine and SEB stimulation conditions. The difference in

28

secretion kinetics between cytokines and chemokines in vitro are maintained after influenza vaccination. As previously reported, vaccination of adults with TIV did not induce significant elevation of the memory T cell IFNγ responses [85]. In addition to analyzing expression kinetics in individual cells, the kinetics of in vivo expansion of influenza responsive T cells after vaccination were measured. The expression level of IL-2 and TNFα also did not changed in influenza specific CD4 T cells after vaccination. Interestingly, the frequency of CCL1, CCL3 and CCL4 expressing T cells increased in late stimulation at day 7 after vaccination. This change was influenza antigen specific compared with the constant chemokine expression in SEB stimulation conditions (Figure 3-8). Thus, the elevated chemokine response is induced in influenza responsive T cell after TIV vaccination. Discussion CD4 T cells regulate the direction and strength of immune responses through the secretion of cytokines and chemokines. The kinetics of secretion is not the same for these mediators, with a quick induction of cytokine [107] and relative delay for most chemokines. The kinetics of mRNA induction matched the secretion, while uncoupled mRNA expression and protein secretion were observed from anergic T cell clones [108]. Thus, different posttranscriptional mechanisms regulate the secretion of mediators between T cell activation and anergy. The different expression of cytokines and chemokines is related to their function in the immune response. The monophasic early expression pattern of IL-2 and TNFα may relate to the early inflammatory response. Expression of IFNγ is different from IL-2 and TNFα. It was quickly induced after initial antigen encounter but continued to be produced at later times. The consistent secretion of IFNγ may be required for driving naïve CD4 T cell differentiation and activating cytotoxic cells in pathogen clearance [109]. The secretion of chemokines, which are chemoattractant for

29

monocytes, neutrophils and lymphocytes, may be more correlated to inflammation and the recruitment of other effector cells locally [110, 111]. The delayed expression of chemokines in T cells was more likely to happen in a tissue site after initial antigen encountering in lymph node and migrating through circulation. Besides activating macrophages and monocytes, CCL3 and CCL4 may recruit additional CD4 T cells to the tissue sites through the CC chemokine receptor CCR5 which is widely expressed on the CD4 cell surface [112, 113]. Even though early cytokine positive cells are more likely to make chemokines at later times, chemokines are not sequentially made by early cytokine-secreting cells. The IFNγ early secreting cells kept secreting IFNγ and expressing chemokines at the late T cell activation times. Even though the IFNγ producing cells have a preference for making chemokines, further calculation of the percentage of IFNγ producing cells in the chemokine producing population, based on cytokine positive and negative cell ratio in sorting, showed that most chemokine producing cells did not produce IFNγ during early T cell activation. Of note, sorting process may give some unknown impacts to cytokine and chemokine expression even in cold temperature. Improvement in experiment method will be the next step for tracking the sequential secretion. The heterogeneous human effector/memory CD4 T cells produce cytokine and chemokine during the immune response. The measurement of chemokine expression should be included in the calculation of total responded T cells. The systematic analysis of the virus specific T cell response may be a strategy to measure the vaccine response rate and predict the induction of immune memory [111, 114]. The analysis of cytokine expression has been widely used to measure T cell function, differentiation and heterogeneity during the immune response, but the secretion of chemokines was seldom analyzed. Chemokines and their receptors are important for effector cell homing [43, 115]. The analysis of cytokine production after influenza vaccination did not show significant changes in cytokine responses

30

[85]. The preexisting memory to cross-reactive epitopes may mask elevated responses from immunization. The increased CCL1, CCL3 and CCL4 response observed at late in vitro time points at day 7 after vaccination revealed the T cell functional change after vaccination. Such elevation was antigen specific, because the secretion of chemokines was similar at different time points in response to SEB. The measurement of chemokine expression facilitates the detection of the T cell response after influenza vaccination. Whether the increase in chemokine expression is due to the activation of pre-existing memory CD4 T cells or the new generation of effector T cells is still unknown. In summary, our study indicates that the patterns of cytokine and chemokine gene expression in activated T cells are different. Due to the different secretion kinetics of cytokine and chemokine by different effector T cells, the measurement of chemokine expression at late time points is highly recommended for assessing T cell function.

31

Figure 3-1 Secretion profiles of human CD4 T cell. Sorted human effector/memory CD4 T cells were stimulated with anti-CD3 and anti-CD28 antibody. (A) Chemokines and cytokines that reach a detectable level in 96 hours. The dashed line indicates the concentration of 20ng/ml. (B) The detailed secretion kinetics of chemokines and cytokines with more than 20ng/ml concentration. The concentration of each time points is normalized to the highest value.

32

Figure 3-2 Secretion of cytokine and chemokine in human CD4 T cell. Sorted effector/memory CD4 T cells were stimulated with anti-CD3 and anti-CD28 antibody for 96 hours. The concentration of secreted cytokines and chemokines in the supernatant were measured by multiplex assay at different time points. The concentration ratio of 8 hours to 96 hours and 24 hours to 96 hours is plotted on the graph. Plots of chemokine are circled.

33

Figure 3-3 Analysis of CD4 T cell

cytokine

and

secretion.

Schematic

analysis

of

chemokine for

cytokine

the and

chemokine profiles of human CD4 T cell stimulated with antiCD3 and anti-CD28 antibody for the indicated time point. The secretion

of

cytokines

and

chemokines was analyzed by intracellular staining or Elispot. (A) The CD4 T cells were gated as CD14-CD3+CD4+CD8-CD56-, further gating effector/memory cells with CD45RAlow and then analyzing

their

cytokine

chemokine expression. summary

of

and

(B)The

intracellular

and

Elispot assays. The open and filled symbol represent Human naive and effector/memory CD4 T cells respectively.

34

Figure

3-4

Kinetics

of

cytokine

and

chemokine mRNA expression in human CD4 T cells after TCR stimulation. Human naive (open symbol) and effector/memory (filled symbol) CD4 T cells were stimulated with anti-CD3 and anti-CD28 antibody for the indicated time points. Cytokine and chemokine mRNA expression were tested by Real-time PCR.

35

Figure 3-5 Induction of cytokines and chemokines by different antigens. Human PBMCs were stimulated with 2009-2010 inactivated influenza vaccine or SEB for the indicated time points. Cytokine and chemokine secretion was tested by intracellular cytokine staining. The expressing cell numbers were normalized to the maximum frequency. (A) Secretion curves of different stimuli, each curve represents one test subject. (B) The half maximum secretion time was calculated based on the secretion curve and plotted as cytokines vs chemokines. Each point represents one test subject stimulated with anti-CD3 and anti-CD28 antibody (diamond), SEB (rectangle) or influenza vaccine (triangle).

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Table 3-1 The half maximum secretion time of each cytokine and chemokine was calculated based on the secretion curve from different stimuli. The average value from 5 subjects was calculated.

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Figure 3-6 Tracking the chemokine secretion in cytokine producing cells. PBMC with or without PBSE labeling were both stimulated with 1µM SEB for 8 hours. CD69high cytokine positive and negative cells, and CD69low effector /memory CD4 T cells were sorted from PBSE labeled population by cytokine secretion assay. The sorted cells were put back into culture with PBSE unlabeled SEB stimulated PBMC. Cytokine and chemokine secretion were tested after 36 hours culture by intracellular staining.

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Figure 3-7 Secretion of chemokines in sorted cytokine producing cells. (A) The representative plot of cytokine and chemokine secretion in sorted cytokine positive or negative cells in mixed culture. The sorted cells were gated from the host population by PBSE+ and further gated the cytokines or chemokines with CD69. (B) The ratio of cytokine and chemokine production between each sorted cytokine positive and negative CD69high effector/memory CD4 T cells. (C) The percentages of each chemokine producing cells which were secreting each individual cytokine at early T cell activation were calculated based on the percentage of cytokine producing cells at early activation and percentage of chemokine production at late intracellular staining.

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Figure 3-8 Kinetics of cytokine and chemokine secretion during influenza vaccination. Blood was drawn from healthy donors before, 7 days and 28 days after inactivated influenza vaccination. Isolated PBMCs were stimulated ex vivo with influenza vaccine or SEB for the indicated time points. Cytokine and chemokine secretion was tested by intracellular cytokine staining. The secreting cells were normalized to 1 million CD4+ T cells. (* P < 0.05)

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Chapter IV: Ki-67 expression reveals strong, transient influenza specific CD4 T cell responses after adult vaccination 4.1 Introduction The human respiratory pathogen influenza virus is a significant threat for all age groups [63]. The risk of influenza infection and the associated morbidity and mortality can be reduced by vaccination with partially purified subunit vaccines (TIV) or live attenuated viruses (LAIV) containing the predicted seasonal strains of A/H3N2, A/H1N1 and B viruses. When matched, antibodies are unquestionably effective against influenza infection [116]. However, CD4 and CD8 T cells assist resolution of infection in mice, and may play a similar role in humans [79, 84, 117]. CD4 T cells provide help for B cell antibody responses and may also contribute to local inflammatory responses. In adults, TIV or LAIV vaccination does not significantly alter CD4 or CD8 T cell cytokine expression responses [85, 86]. Almost all adults have detectable IFNγ memory T cell responses to influenza due repeated exposure to infection and/or vaccination. Thus preexisting memory to cross-reactive epitopes may mask elevated responses from immunization [89]. Nevertheless, vaccination or infection may reshape the repertoire or effector functions [90, 91] of the influenza response. We have now measured the kinetics of expression of three cytokines (IL-2, IFNγ, TNFα) and two chemokines (CCL3, CCL4) by T cells responding to TIV and LAIV vaccination. Simultaneous measurement of Ki-67, a nuclear antigen expressed during and recently after proliferation [118, 119], revealed an unexpectedly vigorous but transient T cell response to influenza vaccine, suggesting that vaccination may substantially reshape the CD4 T cell response, and providing a method for evaluating the quality of T cell vaccine responses.

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4.2 Results The lack of significant adult memory T cell responses to TIV and LAIV [85, 86, 89] could be due to balanced expansion and contraction phases. We considered the possibility that influenza vaccine does in fact induce a T cell response, but the expansion and contraction phases are overlapped for primary and secondary responses so that no net response is observed between pre- and post-vaccination responses. To explore this possibility, we tested in vitro cytokine responses in daily samples to detect potential transient responses, and measured Ki-67 expression to determine whether a subset of influenza-specific T cells proliferated after vaccination. CD4 T cells respond to TIV vaccination CD4+CD8-CD45RAlow

memory/effector

cells

expressing

CD69,

cytokines,

chemokines and Ki-67 were identified by ICS (Figure 4-1A). In a representative subject who received TIV vaccination (Figure 4-1B) at day 0, influenza antigens stimulated significant numbers of memory CD4 T cells to express cytokines and chemokines in vitro. After vaccination, the frequency of circulating influenzaresponsive cells decreased from days 1 to 3, possibly due to sequestration of influenza-responsive T cells from the circulation to lymphoid tissues. The frequency of influenza responsive CD4 T cells then increased on days 4 to 6, probably reflecting the return of vaccine-responsive cells into the circulation after proliferation in lymphoid tissues. The immune response returned to base level around days 9 to 10 (Figure 4-1B). Ki-67 is expressed selectively by proliferating T cells, but not induced in vitro within 10-hour stimulations [119, 120]. Thus Ki-67 expression should identify cells that recently proliferated in vivo. In agreement with this, Ki-67 expression was low among influenza-responsive T cells before vaccination, increased markedly at days 46 and returned to lower levels by days 9-10 (Fig. 4-1).

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In the overall response of multiple subjects to TIV influenza vaccine or peptide antigens, cytokine and chemokine expression increased modestly during the expansion phase (days 4-6), and returned to pre-immunization values after ten days, although only CCL3 and CCL4 reached statistical significance in H1N1 peptideresponsive cells (Figure 4-2). The expression of CCL3 reduced during the late phase in H1N1 peptide responsive cells whereas the levels of other cytokines and CCL4 were remained. The initial drop and subsequent rise in the cytokine response seen in one subject (Figure 4-1) occurred in some but not all subjects (Figure 4-2). More striking changes occurred in influenza-specific Ki-67+ cytokine or chemokineexpressing cells, which increased in all subjects for which day 0 and day 4-6 data were available (Figure 4-2). The number of Ki-67- cells producing IFNγ, IL-2, TNFα or CCL3 did not significantly increase after vaccination, only cells producing CCL4 were significantly increased during the expansion phase under H1N1 peptide stimulation. Ki-67+ cells comprised 10-30% of circulating influenza responsive CD4 T cells expressing IFNγ, IL-2 or TNFα, and nearly 20-50% of cells expressing CCL3 or CCL4 during the expansion phase of the response, peaking at about day 5 (Figure 43). As expected, vaccination did not change the low levels of Ki-67 expression in SEB-responsive CD4 T cells (Figure 4-3) or in the total memory CD4 T cell population (Figure 4-4). Frequency of Ki-67 positive cells was similar in memory CD4 T cells before and after vaccination despite ex vivo stimuli. Thus influenza TIV vaccination selectively induced Ki-67 expression in the influenza-specific but not bulk CD4 memory T cell populations. CD4 T cells responds to LAIV vaccination. The CD4 T cell response was also measured after LAIV vaccination. Influenza virus stimulated significant numbers of memory CD4 T cells to express cytokines and

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CCL4 in vitro. In a representative subject, after vaccination, number of circulating influenza-responsive CD4 T cells declined on days 1-4, increased on days 5-9, and returned to base after 10 days (Figure 4-5A). This pattern was similar to the CD4 T cell response after TIV vaccination. There was a 1-2 day delay in expansion of CD4 T cell responding to LAIV compared with TIV vaccination. But the duration of CD4 T cell expansion phase was longer after LAIV immunization. In the overall response of multiple subjects to LAIV influenza vaccine, there was a two to three fold increase in the number of cells expressing cytokine and chemokine during the expansion phase (days 5-9) in some but not all subjects. The increased IL2, IFNγ and TNFα responses reached statistical significance between days 7 to 10 after vaccination (Figure 4-5B). This increase was influenza specific and was undetected for SEB stimulation (Figure 4-6). Similar to TIV vaccination, the most striking changes were observed in influenza specific Ki-67+ cytokine-expressing cells. In some but not all subjects, the number of Ki-67+ influenza responsive T cells dramatically increased from days 6 to 9 after vaccination (Figure 4-5), particularly on days 7 and 8 after LAIV vaccination (Figure 4-5B). Ki-67+ cells comprised 20-85% of circulating influenza responsive CD4 T cells expressing IFNγ, IL-2 or TNFα during the expansion phase of the response, peaking at about day 7 (Figure 4-7). The low levels of Ki-67 expression in SEBresponsive CD4 T cells did not change in IL-2 or TNFα-expressing cells, but increased slightly in IFNγ-expressing cells at days 6 to 10 after vaccination (Figure 47). 4.3 Discussion Although previous reports [85, 86] suggest that adult TIV influenza vaccination does not significantly alter the overall number of influenza-reactive CD4 memory T cells, we have now shown a striking increase in Ki-67+ cells during the response, in some

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subjects reaching more than 30% of the cytokine-expressing cells at 4-6 days after TIV vaccination. These apparently paradoxical results can be reconciled if it is assumed that the vaccine actually induces a vigorous memory cell proliferative response, but that this response is balanced by a rapid contraction phase, resulting in little change in overall numbers. This is supported by selective Ki-67 expression in the influenza-responsive but not total or SEB-stimulated populations. The increase of Ki-67+ cells was more dramatic after LAIV vaccination. In some subjects more than 80% of cytokine expressing cells were Ki-67 high at 6-9 days after immunization. The high proliferative response and prolonged expansion phase may contribute to the significant elevating of overall influenza responsive T cell after LAIV immunization. Ki-67 expression before vaccination is low, consistent with the low rate of homeostatic renewal of influenza-specific resting memory CD4 T cells [121], and does not change after short term ex vivo stimulation [120]. Thus Ki-67 expression is a measure of in vivo T cell proliferation, even when combined with short-term in vitro stimulation. The percentage of proliferating T cells is probably underestimated by the transience of Ki-67 expression [122], thus some circulating CD4+Ki-67- T cells may have previously expressed Ki-67. Ki-67 expression is also increased in antigen-responsive cells after tetanus toxoid vaccination [123]. The expansion of Ki-67+ cell is 1 to 2 days later in LAIV vaccination comparing with TIV. Such delayed proliferative response may be contributed by difference in antigen delivery [124, 125]. Inoculated virus are captured and processed through epithelial cells and macrophages in the respiratory tract before presenting [126]. Viral antigens pass through several steps and barriers before reaching lymphoid tissues after LAIV immunization; while intramuscular injected proteins can rapidly be distributed. Lack of antigen presenting may be one of the major reason for absence of cellular and antibody responses after LAIV immunization in some subjects. The delayed antigen delivery contributed to the different kinetics in the T cell proliferation response

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between LAIV and TIV vaccination. In addition, the duration of the CD4 T cell proliferative expansion is longer after LAIV vaccination. The constant provision of antigen from virus clearance will prolong the proliferative response. Moreover, protection induced by cold adapted influenza vaccine (CAIV) was observed in the absence of a serum antibody but mucosal IgA response [127]. Generation of local antibody response may be more dominant in LAIV elicited protection. Significant increase of T cell response was observed in children after LAIV not TIV immunization [85], suggesting that T cell mediated immunity may contribute to the efficacy of CAIV. The much higher percentage of Ki-67+ influenza responsive T cell, detected in circulation after LAIV vaccination, may result from egress of most antigen exposured effect T cells to the extralymphoid tissues [128]. The tissue homing of influenza responsive T cell will be important for the generation of protection and elevation of mucosal antibody responses to LAIV vaccination [129], while lymphoid localization has essential role in the generation of serum antibody responses to TIV vaccination. The unexpectedly large proliferative and contraction responses to influenza vaccine implied by our results are consistent with substantial reshaping of immune memory responses by influenza vaccination, because the final population could be derived from: minority populations in the original memory; further differentiation of original populations; or naïve responses to new epitopes. CD4 T cell specificities could change substantially, e.g. emphasizing cross-reactive specificities, or adding new specificities. Effector functions could also change. Although influenza memory comprises predominantly Th1 cells, responses to new epitopes (J.M. Weaver, F.E. Lee and T.R. Mosmann, unpublished) may be biased towards the Thpp (uncommitted IL-2+ TNFα+ IFNγ- CD4 T cells) responses induced by protein vaccines [130]. As the selective expression of multiple Th1 cytokines can correlate with different protective outcomes [131-133] such effector function changes could significantly influence the effectiveness of the vaccine response.

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In conclusion, we detected significant T cell responses after TIV or LAIV immunization. The expression of Ki-67 is transiently increased in influenza responsive T cells at days 4 to 6 for TIV and days 6-9 for LAIV after vaccination. This increase is antigen specific with little bystanding response. The Ki-67 measurement could be chosen as a tool evaluating the efficacy of influenza vaccines.

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Figure 4-1 Identification of influenza-specific CD4 T cells by flow cytometry. PBMC from TIV immunized subjects were analyzed by in vitro antigen stimulation and ICS. (A) Gating strategy to identify cytokine, chemokine and Ki-67 expressing cells. (B) Kinetics of cytokine and chemokine expression in one subject.

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Figure 4-2 Transient increase in Ki-67+ cytokine-expressing T cells after TIV immunization. Cytokine expression was analyzed as in Figure 4-1, and the numbers of cytokine+ Ki-67+ T cells were averaged for each subject for days 4-6 and 9-10. (*P