ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2018

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1455

Vγ9Vδ2 T cells - response to P.falciparum-derived phosphoantigensand potential for use in colon cancerimmunotherapy 

CHENXIAO LIU

ISSN 1651-6206ISBN 978-91-513-0311-6urn:nbn:se:uu:diva-347682

Dissertation presented at Uppsala University to be publicly examined in B7:113a,Biomedicum, Husargatan 3, Uppsala, Wednesday, 16 May 2018 at 13:15 for the degree ofDoctor of Philosophy (Faculty of Medicine). The examination will be conducted in English.Faculty examiner: Professor Susanna Cardell (University of Gothenburg).

AbstractLiu, C. 2018. Vγ9Vδ2 T cells - response to P. falciparum-derived phosphoantigens andpotential for use in colon cancer immunotherapy . Digital Comprehensive Summaries ofUppsala Dissertations from the Faculty of Medicine 1455. 53 pp. Uppsala: Acta UniversitatisUpsaliensis. ISBN 978-91-513-0311-6.

Vγ9Vδ2 T cell is the dominant circulating γδ T cell subset in humans, can expand massivelyupon malaria infection and are cytotoxic to cancer cells. Vγ9Vδ2 T cells are stimulated byphosphoantigens, primarily isoprenoid pyrophosphates like isopentenyl pyrophosphate and(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP). Vγ9Vδ2 T cell from humanblood were studied for proliferation, response to blood-stage malaria parasites and during coloncancer progression. Vγ9Vδ2 T cell proliferation was stimulated by media from P. falciparum-infected erythrocytes from all asexual blood stages - rings, trophozoites, schizonts and rupturingschizonts as well as sexual stage gametocytes assessed by the protocols we developed toobtain pure cultures of all stages. Further, we demonstrated that the molecules that stimulatedthe Vγ9Vδ2 T cell proliferation are phosphoantigens that are released from intact infectederythrocytes. This does not require schizont rupture. Interestingly, the parasites consumed allthe iron ion of hemoglobins during their development from the ring to the rupturing schizontstage. We found that an Anopheles gambiae immune cell line responds to HMBPP by activationof MAPK and PI3K signaling pathways. Moreover, transcription of dual oxidase and nitricoxide synthase was upregulated by addition of HMBPP in the midgut of Anopheles gambiaewhich increases cell tolerance to oxidative stress. A range of small isoprenoid pyrophosphateswere found to stimulate proliferation of Vγ9Vδ2 T cells from PBMCs as was the isoprenoidmonophosphate DMAP. However other isoprenoid monophosphates and alcohols did not. Wefound that cryopreserved unexpectedly increase the proliferation ability of HMBPP–stimulatedPBMCs. To test the cytotoxicity of Vγ9Vδ2 T cells against adherent colon cancer cell lines, aflow cytometry-based assay was developed. Using the assay we found that proliferated Vγ9Vδ2T cells are cytotoxcitic to various cancer cells and that HMBPP increases cytotoxicity towardsadherent colon cancer cells. In a clinical study we found that Vγ9Vδ2 T cells could not always beproliferated from colon cancer patients and that the inflammatory homing receptor CXCR3 wasexpressed at higher levels in colon cancer patients than the control group. Moreover, at cancerstadium 4 a lower frequency of Vγ9Vδ2 T cells was more common than in the other groups.

Chenxiao Liu, Department of Medical Cell Biology, Box 571, Uppsala University, SE-75123Uppsala, Sweden.

© Chenxiao Liu 2018

ISSN 1651-6206ISBN 978-91-513-0311-6urn:nbn:se:uu:diva-347682 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-347682)

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Lindberg B, Merritt EA, Rayl M, Liu C, Parmryd I, Olofsson B, Faye I. (2013) Immunogenic and antioxidant effects of a pathogen-associated prenyl pyrophosphate in Anopheles gambiae. PLoS One 8, e73868

II Liu C, Emami N, Pettersson J, Ranford-Cartwright L, Faye I, Parmryd I. Vγ9Vδ2 T cells proliferate in response to phosphoanti-gens released from erythrocytes infected with Plasmodium falcipa-rum at asexual and gametocyte stages. Under revision.

III Liu C, Skorupinska-Tudek K, Parmryd I. A flow cytometry method for Vγ9Vδ2 T cell cytotoxicity towards adherent cells and potentiat-ing Vγ9Vδ2 T cell proliferation and cytotoxicity. Submitted.

IV Liu C, Birgisson H, Parmryd I. Characteristics of Vγ9Vδ2 T cells in colon cancer progression. Manuscript.

Reprints were made with permission from the respective publishers.

Contents

Introduction ..................................................................................................... 9T lymphocytes ............................................................................................ 9T lymphocyte differentiation ...................................................................... 9αβ T lymphocyte differentiation in blood .............................................. 9Vγ9Vδ2 T lymphocyte differentiation ................................................. 10Antigens for Vγ9Vδ2 T cells ............................................................... 10Antigen presentation to Vγ9Vδ2 T cells .............................................. 12The role of interleukins in activation and differentiation and of Vγ9Vδ2 T cells .................................................................................... 12

The mosquito as a host for malaria parasites ............................................ 13Malaria parasites and Vγ9Vδ2 T cells ...................................................... 14

Introduction in malaria ......................................................................... 14Vγ9Vδ2 T cells activation by P. falciparum infected erythrocytes ..... 14The life-cycle of P. falciparum in humans .......................................... 14Transmembrane transport in infected erythrocytes .............................. 16Possibility of transmembrane export of pyrophosphates in P. falciparum and infected erythrocytes ................................................... 16

Vγ9Vδ2 T cells recognition of cancer cells and cytotoxicity to cancer cells ........................................................................................................... 17Immunotherapy using Vγ9Vδ2 T cells cells to treat cancer focusing on colon cancer .............................................................................................. 18

Introduction to colon cancer ................................................................ 18Immunotherapy using Vγ9Vδ2 T cells ................................................ 20

Aims and questions ....................................................................................... 22

Methodological considerations ..................................................................... 23P. falciparum culturing and synchronization ........................................... 23Peripheral blood mononuclear cells (PBMCs) preparation ..................... 23Selection of PBMCs samples ................................................................... 24Vγ9Vδ2 T cells culture ............................................................................. 25Cytotoxicity assay ..................................................................................... 26Proliferation assay .................................................................................... 29

Results ........................................................................................................... 30Paper I ....................................................................................................... 30

Antigenic molecules to Vγ9Vδ2 T cells are released from P. falciparum-infected erythrocytes at the ring stage ............................... 30HMBPP increases phosphorylation of the MAPK/Erk activated proteins p38, JNK and the transcription factor FOXO in an Anopheles gambiae immune cell line. ................................................................... 31HMBPP induces changes in the transcription of genes related to the immune response in Anopheles gambiae ............................................. 31

Paper II ..................................................................................................... 32Vγ9Vδ2 T cells response to HMBPP ................................................... 32Simultaneous acquisition of synchronized cultures at ring, trophozoite, schizont and merozoite stages .............................................................. 32Media from parasite-infected erythrocytes at all asexual blood stages induce proliferation of Vγ9Vδ2 T cells ............................................... 32Purity of the synchronized cultures ...................................................... 33The phosphoantigens in culture media are not due to broken infected erythrocytes .......................................................................................... 33Media collected from uninfected cultures cannot cause the proliferation .............................................................................................................. 33Phosphatase treatment stops the proliferation ...................................... 34

Paper III .................................................................................................... 34Cryopreservation of PBMCs for proliferation of Vγ9Vδ2 T cells ....... 34Isoprenoid pyrophosphates length C5-C20 and monophosphate DMAP stimulate Vγ9Vδ2 T cells ..................................................................... 35HMBPP strengthens the cytotoxicity of Vγ9Vδ2 T cells towards colon cancer cells ........................................................................................... 35

Paper IV .................................................................................................... 36The frequency of Vγ9Vδ2 T cells in colon cancer patients ................. 36The capability of Vγ9Vδ2 T cell proliferation in colon cancer patients .............................................................................................................. 36CXCR3 and CCR5 expression in Vγ9Vδ2 T cells in colon cancer patients ................................................................................................. 36Vγ9Vδ2 T cells differentiation in colon cancer patients ...................... 37

Summary and conclusion .............................................................................. 38Paper I ....................................................................................................... 38Paper II ..................................................................................................... 38Paper III .................................................................................................... 38Paper IV .................................................................................................... 39

Acknowledgments ......................................................................................... 40

References ..................................................................................................... 42

Abbreviations

AMP Antimicrobial peptide APC Antigen presenting cell ATP Adenosine triphosphate BTN3A1 (CD277) Butyrophilin 3A1 CAR Chimeric antigen receptor CCR C-C chemokine receptor CD277 Cluster of differentiation 277 CD3 Cluster of differentiation 3 CDP-ME 4-diphosphocytidyl-2-C-methylerythritol CSFE Carboxyfluorescein succinimidyl ester CXCR CXC chemokine receptor DMAPP Dimethylallyl pyrophosphate DMSO Dimethyl sulfoxide DOXP 1-deoxy-D-xylulose 5-phosphate DUOX Dual oxidase EC50 Half maximal effective concentration EtOH Ethanol FACS Fluorescence-activated cell sorting FMO Fluorescence minus one FOXO Forkhead box class O FPP Farnesyl pyrophosphate GPP Geranyl pyrophosphate HMBPP (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate HMG-CoA 3-hydroxy-3-methylglutaryl-CoA IFN-γ Interferon-γ IL Interleukin IPP Isopentenyl pyrophosphate JNK c-Jun N-terminal kinase MAPK/ERK Mitogen-activated protein kinases/extracellular signal-

regulated kinases MEcPP 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate

MEP 2-C-methylerythritol 4-phosphate MHC Major histocompatibility complex MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide NBP Nitrogen-bisphosphate NOS Nitric oxide synthase P. falciparum Plasmodium falciparum PBMC Peripheral blood mononuclear cell PGN Peptidoglycan PHA Phytohaemagglutinin PI-3K Phosphoinositide 3-kinase TCM cell Central memory T cell TCR T cell receptor TEM cell Effector memory T cell TEMRA cell Effector memory RA T cell (terminally differentiated

cells) TGF-β Transforming growth factor beta TN cell Naïve T cell TNM Tumor, the number of regional lymph nodes and dis-

tant metastasis TRAIL Tumor necrosis factor-related apoptosis-inducing lig-

and

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Introduction

T lymphocytes αβ Τ cells, a type of lymphocyte of the adaptive immune system, are the majority of T cells in humans. αβ Τ cells are classified to CD4+ T helper cell and CD8+ cytotoxic T cells. αβ Τ cells are activated by specific short pep-tides presented by MHC (major histocompatibility complex) of antigen pre-senting cells. MHC I can mediate a cytotoxic response of CD8+ T cells and MHC II presentation stimulates CD4+ T cells [1]. CD28 signalling provides the co-stimulation for the activation. The other subset of T lymphocytes in humans are γδ T cells that have features of both adaptive immune response and innate immune response [2]. MHCs are not antigen presenting molecules to γδ T cells. Two major groups of γδ T cells are Vδ1+ T cells and Vδ2+ T cells [3]. Vγ9Vδ2 T cells (it was once named Vγ2Vδ2 T cells) is the dominant population in circulating γδ T cells in hu-mans [4]. The range is 3 to 5 % in percentage of Vγ9Vδ2 T cells in circulat-ing γδ T cells [5, 6]. Circulating γδ T cells and/or Vγ9Vδ2 T cells respond to various microbial infections of bacteria and protozoal parasite including Plasmodium falciparum (P. falciparum) infection [7]. γδ T cells can reach 30-40% of T lymphocytes in peripheral blood during acute P. falciparum infection [8]. The subset is found in higher primates exclusively but the Vγ9 gene and/ or Vδ2 genes exist in various Placentalia [9].

T lymphocyte differentiation

αβ T lymphocyte differentiation in blood αβ T cells differentiate through naive T cells (TN), central memory T cells (TCM), effector memory T cells (TEM) to terminally differentiated T cells (TEMRA) in human peripheral blood. TN cells have not encountered the cog-nate antigen. After stimulation of antigen, TN cells differentiate to TCM cells that respond to cognate antigens faster and stronger than TN cells [10, 11]. TCM cells differentiate into TEM cells that produce much IFN-γ and CD8+ TEM cells produce perforin with stimulation of antigens [11]. The CD8+ TEM can differentiate into terminally differentiated T cells also known as effector

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memory RA (CD45RA) T cells (TEMRA). The TEMRA cells produce most per-forin and granzyme by stimulation of antigens [11]. CD27, a member of tumor necrosis factor receptor superfamily, is expressed much in the surface of TN cells and TCM cells. CD45 is a protein tyrosine phosphatase, receptor type C, that is expressed in all leucocytes and plays an important role in con-trolling T cell receptor signalling [12]. CD45RA, an isoform of CD45, is expressed on TN and TEMRA. Chemokines directs the cells move to the necessary site. For instance, they attract memory T cells to inflammatory site. C-C chemokine receptor type 7 (CCR7), a homing receptors that induces the cell to migrate to secondary lymph nodes, is highly expressed in TN cells and TCM cells [13]. When the naive CD8+ T cells develop to memory T cells, the CCR7 that is expressed on T cells is down-regulated but the inflammatory chemokine receptors, CCR5, CXCR3 (CXC chemokine receptor 3) and CXCR6 are up-regulated [14].

Vγ9Vδ2 T lymphocyte differentiation The classification for differentiation of αβ T cells can also be used for Vγ9Vδ2 T cells. The Vδ2 TN phenotype cells start to proliferate at low con-centration of IPP while the TCM phenotype cells need higher concentration for proliferation [15]. Much IFN-γ was found in Vδ2 TEM phenotype cells and much perforin was found in Vδ2 TEMRA phenotype cells collected from inflammatory sites [15]. The pattern of expression of chemokine receptors (CCR2, 5, 6, 7 and CXCR3) in the 4 phenotypes is similar to it of αβ TN, TCM, TEM, TEMRA in lymph nodes and inflammatory site [15].

Antigens for Vγ9Vδ2 T cells Vγ9Vδ2 T cells respond to a group of non-peptidic antigens - phosphoanti-gens that are organic phosphates. Two typical phosphoantigens are isopen-tenyl pyrophosphate (IPP) that is an intermediate in both the mevalonate and the non-mevalonate pathway and (E)-4-Hydroxy-3-methyl-but-2-enyl pyro-phosphate (HMBPP) that is an intermediate in the non-mevalonate pathway for isoprenoid synthesis (Figure 1). IPP and HMBPP are five-carbon iso-prene unit providers in isoprenoid synthesis. The mevalonate pathway exists in most eukaryotes, some bacteria and archaea [16-18], the non-mevalonate pathway, an alternative pathway for isoprenoid synthesis, is found in many bacteria, apicomplexan protozoa, chloroplasts (of plants) and photosynthetic eukaryotes [18] (Figure 2).

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IPP HMBPP

Figure 1. The structures of IPP and HMBPP (Created with ACD/3D Viewer software)

Figure 2. Isoprenoid synthesis. The mevalonate pathway (right with purple arrows) is a metabolic pathway in humans and provides precursors for isoprenoid synthesis. The pathway begins from acetyl-CoA. Through acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), the HMG-CoA reductase catalyzes the HMG-CoA to yeild mevalonate. The reaction is the rate-limiting step of the pathway. Mevalonate is converted to 5-phosphmevalonate by catalysis of mevalonate kinase that is the second essential enzyme in the pathway. After phosphorylation of mevalonate, the 5- carbon IPP is yielded by decarboxy-lation of mevalonate -5 pyrophosphate. IPP is converted to 5-carbon dimethylallyl pyrophos-phate (DMAPP). 1 IPP and 1 DMAPP are synthesized to 10- carbon geranyl pyrophosphate (GPP) and 1 GPP and 1 IPP are synthesized to 15- carbon farnesyl pyrophosphate (FPP) [19]. The non-mevolante pathway (left with green arrows) begins from pyruvate and glyceralde-hyde 3-phosphate to 1-deoxy-D-xylulose 5-phosphate (DOXP). In the second step, DOXP is

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converted to 2-C-methylerythritol 4-phosphate (MEP). This reaction is the committed step [20] so the non-mevalonate pathway is known as MEP pathway. MEP is converted to 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME). Through 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-MEP) and 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate (MEcPP), HMBPP is synthesized. HMBPP is reduced to IPP or DMAPP. (Adapted from author’s licentiate thesis).

Artificially synthesized bisphosphonates and bacteria-produced 2 to 5- car-bon alkyl chain alkylamines with a single primary amine induce accumula-tion of intracellular IPP by inhibition of farnesyl pyrophosphate synthase in the mevolonate pathway of the humen cell cytoplasm. The accumulated IPP and other pyrophosphates activate Vγ9Vδ2 T cells [21-23] (Figure 1).

Antigen presentation to Vγ9Vδ2 T cells A lot is unknown about the activation of Vγ9Vδ2 T cells. In 1995, a study showed the cell contact is necessary for phosphoantigen-media activation [24]. However, traditional antigen presenting molecules, MHCs, are not required for TCR signalling [17, 24]. In 2012, it was reported that the trans-membrane protein Butyrophilin 3A(BTN 3A) was involved in the activation of Vγ9Vδ2 T cells [25]. Later, it was shown that BTN3A1 acts as the antigen presenting molecule but the exctracellular portion of it binds to phosphoantigens with low affinity [26]. The intracellular B30.2 domain high-affinity binds to phosphoantigens directly and induce activation of Vγ9Vδ2 T cells [27]. By nuclear magnetic resonance (NMR) and X-ray crystallography, it has been shown phosphoantigens bind to theB30.2 do-main selectively. Some other negatively charged molecules also can bind to the domain but only phosphoantigens induce the conformation of B30.2 domain [28]. It is believed that some proteins contributing to the BTN3A1 signalling have not yet been discovered [29, 30]. In summary, more research is needed for the role of BTN 3A1 in antigen presentation. Moreover, it has been reported accumulated intracellular IPP is exported to out of cells by the ATP-binding cassette transporter in dendritic cells [31]. It has been reported HMBPP can be uptake into cells by some energy-consuming way [32].

The role of interleukins in activation and differentiation and of Vγ9Vδ2 T cells Interleukins are a group of molecules acting as signals for communication among populations of leukocytes [33]. Interleukins (ILs) induce proliferation and differentiation of Vγ9Vδ2 T cells in the presence of phosphoantigens. IL-2 induces proliferation of Vγ9Vδ2 T cells. IL-2 is considered a T cell growth factor [34] but has been found IL-2 suppresses CD4+ and CD8+ T cells by interaction with T regulatory cells [35]. IL-2 can be produced by αβ

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T cells, natural killer cells and dendritic cells [35]. Based on our results (Pa-per IV) and other studies, Vγ9Vδ2 T cells develop to effector memory T cells with stimulation of phosphoantigens and IL-2 but cannot develop to terminally differentiated cells [3, 15]. IL-15, a T cell growth factor with similar structure to IL-2, is another cyto-kine for Vγ9Vδ2 T cells [36]. The IL-2 receptor and IL-15 receptor share 2 subunits of 3, as a result, they have several similar functions in stimulation of cells but also play some distinct roles in the adaptive immunity response [37]. One of the two subunits, the common cytokine receptor γ-chain, is shared by IL-2, IL-4, IL-7, IL-15 and IL-21 receptors [38]. It has been re-ported that IL-15 helped Vγ9Vδ2 TEM cells to differentiate to TEMRA [39]. IL-12, a proinflammatory cytokine strengthens the Vγ9Vδ2 T cells activation by phosphoantigens and IL-2 or IL-15. IL-21, expressed in T helper cells and natural killer T cells mainly, is a cytokine for polarization of Vγ9Vδ2 T cells to central memory follicular B helper T (TFH) like cells in the presence of phosphoantigens [40, 41]. IL-4 polarizes Vγ9Vδ2 T cells to T helper type 2 like cells with the help of anti-IL-12 antibody and IL-2 [42]. Vγ9Vδ2 T cells can be polarized to terminally differentiated T helper type 17 like cells by addition of IL-1, IL-6, IL-23 and transforming growth factor beta (TGF-β) [43] and T regulatory like cells by addition of TGF-β and IL-15 [44].

The mosquito as a host for malaria parasites

The Anopheles gambiae is a crucial host of P. falciparum. The parasites develop and fertilize in the mosquitoes. Briefly, the mosquito takes gameto-cytes into the midgut by blood meal. In the midgut lumen, gametocytes de-velop to gametes. Gametes fertilize and develop to zygotes. Further, the zy-gotes transform to ookinetes and the ookinetes invade and pass through the epithelium of the midgut. Ookinetes transform to oocysts once the parasite reach the basement membrane of the epithelium. The oocysts begin to pro-duce sporozoites and release sporozoites to the hemolymph. The sporozoites migrate to the lumen of salivary gland and reach the humans by mosquitoes biting [45]. The mosquitoes defence against the parasite through both humoral immunity and cell-mediated immunity. The humoral immunity includes complement-like immune response and release of antimicrobial peptides (AMPs). The cell-mediated immunity includes phagocytosis. Nitric oxide and oxidative-mediated damage is also important in the defence [45, 46]. The hemocytes are crucial and play multiple roles such as phagocytosis, nitric oxide and oxidative-mediated damage and release of AMPs in insects immune system [47]. The hemocytes are classified to 3 types of cells - granulocytes, oeno-

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cytoids, and prohemocytes according to different functions in mosquitoes [48].

Malaria parasites and Vγ9Vδ2 T cells

Introduction in malaria Malaria is a deadly disease caused by infection of mosquito-born parasitic protozoans. There were 212 million cases and 429 000 death due to malaria globally in 2015 according to World Malaria Report 2016 [49]. 99% of the deaths were caused by P. falciparum so P. falciparum is the most dangerous among parasitic protozoans. Though some special drugs such as artemisinin cyanidin, chloroquine, and primoquine have been found, many drug re-sistance mutations have also been found [50-54]. Interestingly, the fitness of drug-resistant parasites to the hosts and no drug environment is less than that of wild- type [55]. The symptoms of malaria include fever, trembling, respiratory distress, joint pain and headache [56]. Periodic fever is the typical symptom of malaria and period in infection of P. falciparum is approximately 48h. The erythrocytes rupture when merozoites are released in the blood cell cycle (Figure 2). Due to synchronizing development of most parasites, following the burst of many erythrocytes, the fever occurs soon [57].

Vγ9Vδ2 T cells activation by P. falciparum infected erythrocytes HMBPP, IPP and DMAPP are synthesized in the apicoplast of P. falciparum by the non-mevalonate pathway and this is the only isoprenoid synthesis in the parasite [58]. The lysate from P. falciparum in the asexual blood cycle activated Vγ9Vδ2 T cells or γδ T cells. Phosphoantigens released from lysed parasites were suggested to induce the activation [59-61]. It has been report-ed that schizont rupture-released molecules stimulate γδ T cells [62] or Vγ9Vδ2 T cells [63, 64]. Later, the molecules are suggested to be phospho-antigens released due to schizont rupture [65]. In addition, the stimulation of γδ T cells by proteins released from gametocytes has been shown [66].

The life-cycle of P. falciparum in humans The life cycle of P. falciparum is complicated. Briefly, female Anopheles gambiae transmit sporozoites to humans. In the human host, the sporozoites develop to merozoites in liver cells and are released to human peripheral

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blood. The merozoites infect erythrocytes and develop. After passing the ring stage and the trophozoit stage, the parasite develops from a merozoite to a schizont. The schizont ruptures and releases 16 merozoites [67]. Soon the infected erythrocyte ruptures and merozoites are released to human blood. The development from one merozoite to sixteen merozoites is called as asexual stage (asexual cycle). It has been reported that the period of the asexual blood cell cycle is approximately 48h in parasites [61] (Figure 3). At the ring stage, some parasites redirect to the sexual stage by development to mature micro (male) and macro (female) gametes through 5 stages. The mosquito takes gametes into the midgut by a blood meal.

Figure 3. Blood cell cycle of P. falciparum. The cycle starts from the invasion of a merozoite to an erythrocyte. After the invasion, the parasite is at the merozoite stage. The merozoite becomes flat and develops to the ring stage. With Giemsa staining, the parasite shows a ring form. The parasite at the ring stage develops to the trophozoite stage. The troph-ozoite becomes bigger and has irregular shape. In the stage, the number of free ribosomes increases and it means the synthesis of proteins increases. The trophozoite develops to the schizont. In the schizont, the division of nucleus is visible and the parasite has more free ribosomes, mitochondria, plastids and lipid vacuoles. 16 merozoites are released after schi-zont rupture and the merozoites are released from erythrocyte. During the ring stage, some parasites redirect to micro (male) and macro (female) gametocytes. (Adapted from author’s licentiate thesis).

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Transmembrane transport in infected erythrocytes Compared with uninfected erythrocytes, the permeability of plasma mem-brane in infected erythrocytes increases a lot for many small molecules such as amino acids, purines and some vitamins [68]. The transport of the mole-cules is due to expression of parasite-associated channels in the plasma membrane of infected erythrocytes [69]. Whether the channels transport pyrophosphates is unknown.

Possibility of transmembrane export of pyrophosphates in P. falciparum and infected erythrocytes IPP, DMAPP and HMBPP are synthesized in apicoplast by the non-mevalonate pathway in the parasites. It is very possible that IPP and/or DMAPP are exported from the apicoplast to the parasite cytoplasm for fur-ther isoprenoid synthesis but the mechanism is unknown [70]. It has been reported that intracellular IPP was transported out from nitrogenbisphos-phate (NBP)-treated dendritic cells by adenosine triphosphate (ATP)-binding cassette transporter A1 [31]. ATP-binding cassette transporters were found in the plasma membrane of P. falciparum [71]. The ATP-binding cassette transporter A1 is expressed in human mature erythrocytes [72]. Therefore, the IPP is likely to be transported from apicoplast to blood plasma. But whether HMBPP and other pyrophosphates can be exported is unknown. (Figure 4)

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Figure 4. Release of phosphoantigens from an infected erythrocyte. IPP, DMAPP and HMBPP are synthesized in the apicoplast by the non-mevalonate pathway. Some scientific evidence supports IPP is exported through membranes of apicoplast, parasite and infected erythrocyte. But whether HMBPP or other pyrophosphates can be released from through membranes of apicoplast, parasite and infected erythrocyte is unknown. In this sche-matic diagram the proportions have been distorted.

Vγ9Vδ2 T cells recognition of cancer cells and cytotoxicity to cancer cells Activated Vγ9Vδ2 T cells produce and secrete cytokines including interfer-on -γ (IFN-γ) and tumor necrosis factor -α (TNF-α) to kill cancer cells [73]. Moreover, activated Vγ9Vδ2 T cells produce tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [73, 74] and perforin/granzymes [73, 75, 76] for the killing. The mevalonate pathway is up-regulated in various cancer cells [77, 78] including colon cancer cells [79] by dysregulation of HMG-CoA reductase

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and other enzymes of the mevalonate pathway in cancer cells [77, 78]. As a consequence, the level of the pyrophosphates, intermediates of the mevalo-nate pathway, is high in the cancer cells. It has been reported the mevalonate pathway-upregulated cell line - Daudi cell stimulated Vγ9Vδ2 T cell receptor (TCR) [80]. In addition, Vγ9Vδ2 T cells did not respond to Daudi cells and a breast cancer cell line by inhibition of the synthesis of intracellular isoprenoid pyrophosphates [81]. TCR-mediated cytotoxicity was also shown in a melonoma cell line [82, 83]. To amplify the signal from the mevalonate pathway, cancer target cells were treated with NBPs for accumulation of intracellular isoprenoid pyrophos-phates [73, 84-86]. These studies show Vγ9Vδ2 T cells are cytotoxic to vari-ous NBPs-treated cancer cells. In addition, the cytotoxicity to NBP-treated target cells is mediated by the Vγ9Vδ2 TCR [73]. Natural killer group 2D (NKG2D), a transmembrane protein, is found to be expressed in γδ T cells, natural killer cells and activated CD8+αβ T cells in humans [87]. By binding to NKG2D ligands, NKG2D recruited DNAX-activating protein of 10  kDa (DAP10) to activate the phosphatidylinositide-3-kinase (PI-3K) pathway and Grb/Vav1 signalling in cells [88]. The activa-tion induces cytotoxicity. NKG2D ligands are rarely expressed in healthy cells but are expressed under cellular stress such as malignant transformation and infection [87]. NKG2D–mediated cytotoxicity in Vγ9Vδ2 T cells has been shown to be either co-stimulated by TCR mediated signalling or kill target cells independent of TCR mediated signalling [73, 82, 89]. Vγ9Vδ2 T cells were shown to be cytotoxic to colon cancer cells [89] and colon cancer stem cells [73].

Immunotherapy using Vγ9Vδ2 T cells to treat cancer focusing on colon cancer

Introduction to colon cancer Cancer is one of the mortal diseases in worldwide. There were 14,1 million new cases and 8,2 million death related to cancer in 2012 [90]. Colon cancer is also known as colorectal cancer. It is the third most common cancer in both sexes (9,7% of total cases) and about 690 000 patients died worldwide in 2012 according to the world cancer report 2014 [90]. In Europe, colon cancer is the second most common cancer in both genders and there are around 210 000 death in 2012 [90]. Chromosomal instability, CpG (cytosine nucleotide phosphate guanine nucleotide) island methylator phenotype, and microsatellite instability are causes of colon cancer at the level of genes [91].

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Colon cancer tumor grows in situ (intraepithelial) and invades into mucosal lamina propria, submucosa until the tumor penetrates through entire colon and reaches other organs. According to the invasion of primary tumor (T), the number of regional lymph nodes (N) where the tumor tissue metastasizes and distant metastasis (M) (TNM classification), the colon cancer is classi-fied to 4 stages nowadays [92](Table 1). Tumor deposits is another consid-ered factor in the TNM system. The previous Dukes classification that was created decades ago is still used for description of many colon cancer cell lines that were collected decades ago.

Table 1. The TNM classification [92] Table 1.1. Definitions of T, N, M Site of invasion of primary tumor (T) Furthest site or tissue of invasion TX Primary tumor can not be assessed T0 Primary tumor can not be found Tis Intraepithelial or lamina propria T1 Submucosa T2 Muscularis propria T3 Pericolorectal tissues T4a Visceral peritoneum T4b Nearby organs or other tissues Regional lymph nodes (N) The number of metastasis regional

lymph nodes NX The number can not be assessed N0 Not any N1a 1 N1b 2-3 N1c 0 but there are tumor deposits in

tissues N2a 4-7 N2b Above 7 Distant metastasis (M) Number of distant metastasized

organs/sites M0 0 M1b 1 M1 Above 1 Table 1.2. TNM classifications Stages T N M Dukes stages 0 Tis 0 0 I 1 0 0 A

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I 2 0 0 A IIA 3 0 0 B IIB 4a 0 0 B IIC 4b 0 0 B IIIA 1-2 1/1c 0 C IIIA 1 2a 0 C IIIB 3-4a 1/1c 0 C IIIB 2-3 2a 0 C IIIB 1-2 2b 0 C IIIC 4a 2a 0 C IIIC 3-4a 2b 0 C IIIC 4b 1-2 0 C IVA Any Any 1a IVB Any Any 1b

Immunotherapy using Vγ9Vδ2 T cells Immunotherapy means therapy by activation or inhibition of the immune response to treat disease. Fever-induced tumor regression is not a new find-ing and this phenomenon has been known for several hundred years at least. In the 19-century, some formal reports showed several cases in this field. In some very early reports, tumors regressed in malaria parasites infected pa-tients [93]. William Coley invented several inactivated bacteria vaccines (Coley’s toxins) to induce fever and inflammation in cancer patients in ex-perimental treatments [94-96]. Killed Serratia marcescens is one of the bac-teria [95] and it has the entire set of enzymes of the non-mevalonate pathway [97]. This suggests the vaccine can stimulate Vγ9Vδ2 T cells by bacteria-produced HMBPP. However, the efficacy of the toxins cannot be verified. The idea that using human immunity resists cancers has been developed. CAR (chimeric antigen receptor) T cell treatment to cancer is a breakthrough in immunotherapy. Using gene technology, patient autologous αβ T cells can be engineered to anti-cancer cells by introducing anti-cancer CAR. The en-gineered T cells are then adoptively transferred to patients for treatment [98]. By now, CAR T cell immunotherapy has been successfully used in some cancers treatment [98, 99]. Other immunotherapies for instance immuno-therapy by inhibition of immune checkpoints such as anti-PD1 (programmed death 1) is used for patients [98, 100]. Vγ9Vδ2 T cells kill a wide range of cancer cells. The adoptive human Vγ9Vδ2 T cells suppress tumour growth in murine cancer models with ad-ministration of NBPs [84, 101-103]. Many infiltrated Vγ9Vδ2 T cells were

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found in tumours of general primary carcinoma [85] and renal cell carcino-ma [76]. Zoledronate, one of the NBPs, is medically used for treatment of osteopenia including bone loss and fracture due to metastasis of tumors or/and due to hormonal therapy for breast and prostate cancers [104, 105]. BTN3A1, the potential antigen presentation molecule, was not downregulat-ed in colon cancer tumor tissue while some other BTN families were down-regulated [106]. This suggests that colon cancer tumor does not have re-duced capacity to activate Vγ9Vδ2 T cells through BTN3A1. Based on the findings described above, adoptive immunotherapy of Vγ9Vδ2 T cells with the combination of zoledronate is potent for treatment of cancer. The source Vγ9Vδ2 T cells or γδ T cells can be acquired not only from the patients themselves but also from haploidentical donors [107]. It has been shown that the immunotherapy using Vγ9Vδ2 T cells is effective for some cancer patients [108, 109]. Based on a meta study, the therapy is not corre-lated with severe adverse effect and has favourite effect on patients [110]. An adoptive immunothery-Immunicell®, is evaluated at phase II and III cur-rently. Interestingly, 22 subsets of leukocytes were identified in solid cancer tumors including colon cancer. Higher level of γδ T cells correlated with longer survival time with the most statistic significance among the 22 subsets [111].

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Aims and questions

1) To investigate whether Anopheles gambiae immune system responds to phosphoantigens likely to be present in P. falciparum-infected blood.

2) To investigate whether P. falciparum-infected erythrocytes release phosphoantigens that can induce proliferation of Vγ9Vδ2 T cells at all blood cycle stages.

3) To develop a protocol for simultaneous collection of pure cultures at all stages of P. falciparum-infected erythrocytes.

4) To investigate what small isoprenoid pyrophosphates, isoprenoid monophosphates and isoprenoid alcohols stimulate proliferation of Vγ9Vδ2 T cells.

5) To design a cryopreservation procedure for Vγ9Vδ2 T cells and tests if cryopreservation influences the proliferation.

6) To design a FACS (fluorescence-activated cell sorting) cytotoxicity assay for adherent target cells.

7) To characterise the frequency, proliferation capacity, differentiation and inflammatory homing potential of Vγ9Vδ2 T cells from colon cancer patients compared with age-matched healthy donors.

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Methodological considerations

P. falciparum culturing and synchronization The NF54 and FCR3 strains of P. falciparum were cultured by infection of type O erythrocytes in growth media with type AB serum. Type O erythro-cytes are compatible with serum collected from all types (A, B, AB and O) of blood [112] and type AB serum lack anti-type A or B antibodies so it is compatible with all types erythrocytes. 5% hematocrit was kept in the cul-tures verified by Giemsa stained smear checking. Cultures were tested for mycoplasma infection regularly. Plasmion is a kind of gelatin solution. Plasmion is used for synchronization of the parasite-infected erythrocytes asexual stages because infected cells at the late trophozoite sediment slower than infected cells at the ring and young trophzoite stages and uninfected cells. However, Plasmion is only useful in knob+ P. falciparum strains [113]. Knob-like protrusions are expressed in plasma membrane of knob+ P. falciparum infected erythrocytes and help cells to attach in endothelium of capillaries [114]. Sorbitol ruptures tropho-zoite and schizont infected erythrocytes by osmotic pressure because sorbitol diffuses into cells via the plasmodial erythrocyte surface anion channel ex-pressed by infected cells [115, 116]. By the combination of Plasmion and sorbitol protocols, highly pure synchronized cultures at the rupturing schi-zont stage, ring stage, trophozoit stage and schizont stage can be acquired (Paper II Figure 2 and Supplementary Tables 1,2 and 3). The purity was checked by microscopy.

Peripheral blood mononuclear cells preparation Peripheral blood mononuclear cells (PBMCs) were collected from peripheral blood or buffy coats by Histopaque density centrifugation (Figure 5). PBMCs include lymphocytes, monocytes and macrophages. It has been re-ported that the neutrophil, a kind of multi-lobed nuclei cell, inhibits activa-tion and proliferation of Vγ9Vδ2 T cells by generation of reactive oxygen species [117] and 3 serine proteases [118]. The 3 serine proteases reduces the expression of CD25 and BTN3a1. Reactive oxygen species downregulate the expression of CD25 and CD69. As a result, removing neutrophils is good for expansion of Vγ9Vδ2 T cells.

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Purified Vγ9Vδ2 T cells (Vγ9Vδ2 T cell lines) that are maintained by phy-tohaemagglutinin (PHA) and IL-2 [119]can proliferate with stimulation of phosphoantigens and IL-2 but the proliferation increases much in the pres-ence of PBMCs as antigen presenting cells [24]. In addition, a subset of memory αβ T helper cells has been reported to improve the proliferation of Vγ9Vδ2 T cells [120].

Selection of PBMCs samples Samples of PBMCs were isolated from blood of local healthy donors (under 67 years old expect one) or from buffy coats (donors’ age from 18 to 60) from the local blood center. The donors were from both genders. Immunose-nescence is found in the γδ T cells or Vδ2 T cells [6, 121, 122]. The absolute number of Vδ2 T cells and αβ T cells per unit of blood decreases by age (donor’s ages are from 20) but Vδ1 T cells do not [121, 122]. The expansion of Vδ2 T cells from old people (75-89) and very old people (100-104 years old) is lower than that from young people [121]. Gender may be an impact of frequency of Vδ2 T cells [122, 123]. Interestingly, one paper shows the number of Vδ2 T cells in females is significantly lower than it in male in Japanese donors [122]. However, another one shows the percentage of Vγ9Vδ2 T cells in CD3+ cells of female is significantly higher than it in male in American donors [123]. As a result, selection of age and gender-matched samples is crucial for some studies.

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Figure 5. PBMCs isolation and expansion of Vγ9Vδ2 T cells. Buffy coats were diluted at the ratio of 1:4 to 1:6 with RPMI-1640. Blood was diluted at the ratio of 1:1. 20-36 mL of diluted buffy coat or blood was added into 50mL Falcon tube under 10-13mL of His-topaque 1077. Samples were centrifuged at 400g, at room temperature for 30 min. A white ring, the PBMCs, was extracted between plasma and Histopaque. HMBPP solution at final concentration of 80 pM was added into a culture container and kept until the EtOH: NH3 solvent evaporated entirely to exclude the impact of the solvents to PBMCs. The PBMCs were washed in RPMI-1640 and added into culture container. IL-2 was added days 3, 5 and 7(and day 9 in patient samples). On days 11-12, the cells were harvested or analyzed. (Adapted from author’s licentiate thesis).

Vγ9Vδ2 T cells culture Phosphoantigens partly activate the MAPK/Erk (mitogen-activated protein kinase/extracellular signal-regulated kinases) and PI-3K signalling pathways of Vγ9Vδ2 T cells [124]. IL-2 was not added to PBMCs until to 3 days of incubation (phosphoantigen is added on day 0) to restrict proliferation of other types of cells. (Figure 5) 25U /mL IL-2 was chosen for proliferation to mimic the IL-2 concentration in PBMCs with stimulation of PHA [125]. Compared with some other protocols where they used 100-1000U/ml IL-2 [126-129], 25U /ml IL-2 is a more physiological concentration.

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Cytotoxicity assay Cytotoxicity assay based on FACS have been developed and improved for many years [130-133]. Compared with other typical methods (Table 2), the advantages of this method are quantitative analysis by counting absolute number of cells with multiple markers labelling in samples. The methods were designed for suspension target cells but some adapted methods were applied for adherent target cells [134]. For our study on the cytotoxicity of Vγ9Vδ2 T cells, we developed a new assay based on the old methods. 96-well, U-bottom plate was applied for the assay. Briefly, purified γδ T cells were mixed with CSFE-labelled colon cancer cells at different ratios in 96-well plates. After incubation, the mixed cells were transferred to FACS tubes and the dead cells were labelled with 7-AAD. The live and dead cancer cells were counted with a flow cytometer. Surprisingly, we find even using tissue-culture treated surface polystyrene; colon cells cannot adhere on the surface or adhere poorly during four hours of the incubation (Paper III). Table 2. Comparison of cytotoxicity assays Assay Brief descrip-

tion Advantages Disadvantages

Cr51 assay Target cells are labelled with Cr51. The per-centage of killed cells is estimated by measurement of radioactivity due to leakage from dead cells.

Suitable for both adher-ent and sus-pension cells.

Risky on ra-dioactivity. Cannot count absolute number of killed cells. Cannot label other markers.

MTT* assay

A colorimetric assay where the purple forma-zan reduced from MTT catalyzed by NAD (P)H*-

Suitable for both adher-ent and sus-pension cells.

Cannot count absolute number of killed cells Cannot label other markers. Cannot differ-

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*MTT - 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide *NAD (P) H- nicotinamide adenine dinucleotide phosphate

FACS analysis of T cell differentiation In our study, we have characterized the frequency, differentiation status and tumour targeting potential of Vγ9Vδ2 T cells in patient samples. To identify the Vγ9Vδ2 T cells from PBMCs and acquire the frequency, anti-CD3, anti- γ9 and anti- δ2 antibodies were applied. To acquire the differentiation status, anti-CD27 and anti-CD45RA were applied. Anti-CXCR3 and anti-CCR5 are used to estimate the tumour targeting potential. The samples were stained with 7-fluorophore conjugated antibodies. Due to spectral overlap, fluores-cence compensation was set according to fluorophore single staining con-trols [136]. Fluorescence minus one (FMO) controls means that the sample is stained with all fluorophores except one. FMO controls are used for de-termining gates and quadrants because compensation and other factors may cause the scatter of compensated background fluorescence of stained sam-ples spreads wider than the scatter of unstained sample (Figure 4B). It has been reported that there was no distinct border between CD45RA+ and CD45RA- Vγ9Vδ2 T cells [137]. We also find the border between the two populations is not clear as CD3+Gamma9-Delta2- population (Figure 6, A).

dependent oxi-doreductases and dehydro-genases is measured [135]. The dead cells cannot catalyze the reaction.

entiate cell death between target cells and effector cells.

Flow cytom-etry

Target cells are labelled with fluorescence dye and dead cells are la-belled also. The sample are tested with flowcytometry

Acquire absolute number of killed cells. Samples can be labelled with multiple markers

Since all tar-get cells need to be resus-pended in the sample, it is difficult for adherent cells.

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A

B

Figure 6. Gating for analysis of the PBMCs sample A, Comparison between CD45RA-population and CD45RA+population in Vγ9Vδ2 T cells and non-Vγ9Vδ2 T cells. PBMCs were labelled with antibodies to multiple cell surface markers. Vγ9Vδ2 T cells (Gamma9+Delta2+CD3+) cell and non-Vγ9Vδ2 T cells (Gamma9-Delta2-CD3+ cells) were quadranted to 4 populations by anti-CD27 and anti-CD45RA. Two distinct populations be-tween CD45RA- CD27-and CD45RA+ CD27- in non-Vγ9Vδ2 T cells but not in Vγ9Vδ2 T cells B, Comparison spreads of background fluorescence between unstained sample and FMO sample (minus Gamma9). Spread of background fluorescence in Gamma9 axis of unstained

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sample is less than the background of FMO sample. The quadrant should be higher than it in unstained control according to FMO control.

Proliferation assay The formula used for acquiring the absolute number of Vγ9Vδ2 T cells in each sample: number of total cells × percentage of Vγ9Vδ2 T cells in total cells × volume of cell suspension of sample. Fold increase is introduced to investigate whether Vγ9Vδ2 T cells proliferate under various conditions by comparison of absolute numbers of Vγ9Vδ2 T cells before and after stimula-tion. Some studies assess if the Vγ9Vδ2 T cells proliferate by comparison of the percentages of Vγ9Vδ2 T cells in total cells or lymphocytes [22, 138]. Our method is further compared with the absolute number of Vγ9Vδ2 T cells before and after growing. Some proliferation assays such as thymidine in-corporation assay cannot determine proliferation of subpopulation from PBMCs. Carboxyfluorescein succinimidyl ester (CFSE) proliferation assay can be used to estimate proliferation of Vγ9Vδ2 T cells [139]. Since CSFE is a fluorescence dye stable bond to cytoplasm, the intensity of CSFE demon-strates division times of cells. This way is convenient but indirect. In addi-tion, CSFE effects development and proliferation of lymphocytes [140](Table 3). Table 3. Comparison different proliferation assays Name Advantages Disadvantages FACS by comparison of percentages of target cells in total cells

This method illustrates the proliferation by FACS dot plots.

This method does not take into account the difference in the num-ber of cells between samples.

Combination of FACS and absolute number estimation

This method directly shows the number of cells from subpopula-tion increases or de-creases after culture.

Time consuming. There are many steps in the method.

CSFE assay It is convenient and an effective method.

It indirectly assesses the proliferation by cell division. It effects development and proliferation of lymphocytes.

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Results

Paper I

Antigenic molecules to Vγ9Vδ2 T cells are released from P. falciparum-infected erythrocytes at the ring stage To demonstrate that the Anopheles gambiae’s immunity system can encouter the phosphoantigens from infected erythrocyte blood, examining whether the blood possibly contains phosphoantigens is crucial. Very few parasites at the schizont or trophzoite stage are found in peripheral blood [141] since most of them accumulate in vesicular tissues of some organs such as adipose tis-sue, skeletal muscle and the heart [142]. If the phosphoantigens are released due to schizont rupture only [63-65], there are still questions. For example, are there many schizonts around the Anopheles biting site? To bypass the questions, we reported the parasites at the ring stage released molecules an-tigenic to Vγ9Vδ2 T cells in this paper. In detail, media were collected from infected cells synchronized to the ring stage and the media were added into PBMCs for stimulation. After incubation of 12 days with assistance of IL-2, the data were acquired with flow cytometry and cell counting.

Small isoprenoid pyrophosphates stimulate proliferation of Vγ9Vδ2 T cells but HMBPP has much higher antigenic bioactivity than other isoprenoid pyrophosphates [143, 144] in Vγ9Vδ2 T cell lines. In addition, the antigenic molecules are pyrophosphates according to the findings of our paper II. As a result, HMBPP should have crucial function in the proliferation. The con-centration of HMBPP can be estimated by “HMBPP unit based on the EC50” method. EC50 is defined as half maximal proliferation of Vγ9Vδ2 T cells in PBMCs at the certain concentration of IL-2 [145]. One unit of HMBPP is defined as the concentration of HMBPP at EC50. As a result, the lower con-centration at EC50, the higher the antigenic bioactivity. By comparison of dilution series and the HMBPP dose-response curve (Pa-per II, figure 1), the concentration HMBPP in media can be estimated rough-ly. Using 20× diluted media, there was less proliferation than it from the 4× diluted media. It suggests the concentration of HMBPP in the 4 × dilution is

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lower or close to 80pM. So the concentration in the media should be 320pM or lower.

HMBPP increases phosphorylation of the MAPK/Erk activated proteins p38, JNK and the transcription factor FOXO in an Anopheles gambiae immune cell line. MAPK/ERKand PI-3K pathways are activated by HMBPP in the absence of IL-2 in Vγ9Vδ2 T cells. The response is T cell receptor related [124]. In this paper, we find the homologous response in Anopheles gambiae. HMBPP - stimulation induces an increase of the phosphorylation of MAPK/Erk acti-vated proteins JNK (c-Jun N-terminal kinases), p38 and transcription factor FOXO (Forkhead box class O) in insect immune competent hemocyte-like cell line – 4a3B but does not upregulate phosphorylation of Erk. JNK is involved in the regulation of hydrogen peroxide detoxifying enzymes of Anopheles gambiae [146]. In addition, the transcription factor FOXO is involved in oxidative stress [147] and p38 is phosphorylated when hydrogen peroxide challenges [148]. Based on the findings, HMBPP plausibly upregu-lates the resistance to oxidative stress. This is further supported by our result. Our result shows the HBMPP-pretreated 4a3B cells give tolerance to 1mL hydrogen peroxide.

HMBPP induces changes in the transcription of genes related to the immune response in Anopheles gambiae To test if the HMBPP stimulates the immune response in Anopheles gambi-ae, 6 genes related to the immune response were selected to investigate if their transcription changes after adding HMBPP to a blood meal in Anophe-les gambiae. Transcription of five of the six genes changed significantly compared with control feed. The five genes were 3 AMP (antimicrobial pep-tide) genes and two genes implicated in H2O2/NO generation- dual oxidase (DUOX) and nitric oxide synthase (NOS). DUOX has an important func-tion in the midgut epithelial immunity of Anopheles gambiae [149]. Moreo-ver, HMBPP stimulation regulates the abundance of bacteria in the midgut. The number of bacteria in the midgut is related the parasite infection rate [150-152]. It has been reported that peptidoglycan (PGN) regulates tran-scription of AMP genes in Anopheles gambiae by PGN Recognition Protein LC [151]. Our paper also shows the PGN regulates transcription of two of the three AMP genes significantly but only HMBPP regulates DUOX and NOS.

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Paper II

Vγ9Vδ2 T cells response to HMBPP To find out the concentration of HMBPP that induces near maximum prolif-eration of Vγ9Vδ2 T cells, various concentrations of HMBPP were tested for expansion of Vγ9Vδ2 T cells in present of IL-2. Concentrations from 800fM to 8nM of HMBPP induce proliferation and the peak is from 8pM to 8nM. 80pM HMBPP was chosen as a positive control for subsequent experiments.

Simultaneous acquisition of synchronized cultures at ring, trophozoite, schizont and merozoite stages A new protocol has been developed for simultaneous acquisition of cultures of P. falciparum at all asexual blood stages by using Plasmion and sorbitol. Briefly, two subcultures are separated from a mother culture. Merozoite and trophozoite are synchronized from one subculture. Schizont and ring stage parasite are acquired from the other. The synchronized cultures are incubated in new media for several hours. The time of incubation cannot exceed the transformation time to the next stage. That means, after incubation, the syn-chronized cultures still were in the same stage had not developed to next stage. The media was collected by filtering out the erythrocytes and para-sites.

Media from parasite-infected erythrocytes at all asexual blood stages induce proliferation of Vγ9Vδ2 T cells Two phosphoantigens HMBPP and IPP are produced by apicoplast of P. falciparum. In Paper I, we demonstrate the antigenic molecules that were released to media collected from ring stage culture support Vγ9Vδ2 T cells proliferation. In Paper II, media collected from all blood cycle stages were tested if they support proliferation. Several batches of cultures and PBMCs from different donors were applied for the experiment. The data were gath-ered and analysed by generalized linear mixed model (GLMM) model. The model has been used for many medical studies [153]. We found a significant difference between our experimental groups and negative control group. There was no difference among the experimental groups. This result chal-lenges the traditional view that phosphoantigens are released to media by schizont rupture and the burst of erythrocytes. To investigate if parasites at ring, trophozoit and schizont stages release an-tigens to stimulate Vγ9Vδ2 T cells; some other possibilities must be consid-ered. In Paper II, we figured out 3 possibilities: How pure were the synchro-nized cultures? Investigate if the antigens were leaked from lysed infected

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erythrocytes? And do phosphoantigens released from uninfected erythro-cytes support proliferation?

Purity of the synchronized cultures More than 90% purity of schizonts should be acquired from Plasmion that is a commercial gelatin solution [113] and the purity of ring stage parasites in infected erythrocytes should be 95% after sorbitol lysis [154]. In our study, we checked the purity by microscopy and found there was around 90% puri-ty in the ring culture and the remaining parasites were at the trophozoit stage. Purity in the trophozoit culture was around 90% and the remaining parasites were schizonts. There was 100% purity in schizont culture. In mer-ozoit/schizont rupture culture, we find around 80% of parasites were at mer-ozoite/ruptured schizont stage. Importantly, the result shows almost no para-site at schizont rupture was mixed into the other 3 cultures (ring, trophozoite and schizont).

The phosphoantigens in culture media are not due to broken infected erythrocytes It is possible that infected erythrocytes are lysed during culture and release antigens to the culture’s media. To investigate this possibility, we measured the iron released from broken erythrocytes in some samples. By comparison between lysed erythrocytes control and samples, we found average level of rupture in total erythrocytes is 0,3-0,4 % while around 5 % erythrocyte were infected in the parasite cultures. The lysis rate in infected samples was not higher than the uninfected samples. These results strongly suggest the phos-phoantigens in culture media are not due to infected erythrocytes. The level of iron in media of the culture with the parasites that had under-gone schizont and erythrocyte rupture was not higher than media from other cultures. It suggests the parasites used almost all hemoglobin. In the infected cells, the parasites consume hemoglobin to construct their proteins and non-protein part, heme (with iron), is released from hemoglobin. The heme is toxic to parasite and the parasite converts it to the insoluble hemozion (with iron) [155]. At the late trophozoit stage, 61% iron of the host erythrocyte was found in hemozoin [156]. The hemozoin crystals were removed when we filtered the cultures to get the cell-free media.

Media collected from uninfected cultures cannot cause the proliferation Erythrocytes produce IPP and other pyrophosphates by the mevalonate pathway though the amount of pyrophosphates seems unlikely to support the

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proliferation because the much higher bioactivity of HMBPP. The result shows the Vγ9Vδ2 T cells cannot proliferate with stimulation by the media collected from uninfected erythrocyte cultures. Furthermore, Vγ9Vδ2 T cells do not proliferate with lysed uninfected erythrocyte but proliferate with the media from infected cultures.

Phosphatase treatment stops the proliferation Though no paper report (based on my knowledge) the Vγ9Vδ2 T cells rec-ognize peptidic antigens, other cells such as CD4+αβ T cell may release cytokines like IL-2 to induce proliferation of Vγ9Vδ2 T cells. These cells are likely to be stimulated by peptidic antigens from parasite. As a result, we tested if some proteins stimulated the Vγ9Vδ2 T cells indirectly. Heating (95oC, 10min) the media collected from the infected cultures could not stop the proliferation of Vγ9Vδ2 T cells. This suggests the antigenitic molecules are not proteins. After the media collected from infected cultures were treated by phosphatase (apyrase), Vγ9Vδ2 T cells did not proliferate. Apyrase catalyzes the pyro-phosphate hydrolysed to alcohols or monophosphates and lowers the anti-genic bioactivity (Paper III). This result strongly suggests the proliferation was induced by pyrophosphate-containing phosphoantigens.

Paper III In this study, we investigated if Vγ9Vδ2 T cells in PBMCs responded to various isoprenoid phosphates and addressed their response ability at differ-ent conditions. To address if the phosphoantigens strengthens cytotoxicity of Vγ9Vδ2 T cells to adherent cells, we developed a new FACS based assay.

Cryopreservation of PBMCs for proliferation of Vγ9Vδ2 T cells We are successful to expand Vγ9Vδ2 T cells from cryopreserved PBMCs with stimulation of HMBPP. This procedure can be used to preserve large numbers of PBMCs to save PBMCs preparation time and experimental ma-terial. Based on statistics, proliferation from cryopreserved Vγ9Vδ2 T cells is better than it of fresh samples from the same donors. In the cryopreserva-tion procedure, much DMSO (dimethyl sulfoxide) was used to protect cells from lysis due to crystals of water. DMSO affects permeability of the cell membrane and other function of cells [157, 158]. To investigate if DMSO affects proliferation, 0,1% DMSO was administrated to the PBMCs. There was no significant change in the phosphoantigens-mediated proliferation.

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DMSO has a negative effect in 3 of 4 freshly isolated samples but the posi-tive effect in 3 of 5 cryopreserved samples in HMBPP-responded prolifera-tion. Moreover, cryopreserving may lower percentage of regulatory cells in PBMCs that suppress the proliferation in some samples of PBMCs [159, 160]. Another explanation for the high proliferation in cryopreserved cells is that the healthiest cells survived the cryopreservation and that these cells proliferate fast.

Isoprenoid pyrophosphates length C5-C20 and monophosphate DMAP stimulate Vγ9Vδ2 T cells Except IPP and HMBPP, other length C5-C20 isoprenoid pyrophosphates stimulate proliferation of Vγ9Vδ2 T cell lines. Moreover, It has been report-ed that IPP has higher biological activity than DMAPP (5 carbons, GPP (10 carbons), FPP (15 carbons) and GGPP (20 carbons) [144, 161]. In this paper, we show DMAPP, GPP, FPP and GGPP can stimulate proliferation of Vγ9Vδ2 T cells in PBMCs collected from different donors. Cells can take up isoprenoid alcohols – DMA, G, F and incorporate them to prenylated proteins [162]. This finding suggests the alcohols can be phos-phorylated in cells. The rephosphorylated alchols bind can to B30.2 intracel-lular domain of BTN3A1 to induce activation of Vγ9Vδ2 T cell. None of isoprenoid alcohols stimulated Vγ9Vδ2 T cells in PBMCs in our experi-ments. Among the isoprenoid monophosphates, only DMAP shows immu-nogenicity.

HMBPP strengthens the cytotoxicity of Vγ9Vδ2 T cells towards colon cancer cells FACS based cytotoxicity assay shows the absolute number of live cells and injured cells after cell killing. To address it is only suitable for suspension cells, a flow-cytometry based method for assessing cytotoxicity towards adherent cells was developed. By microscopy, we demonstrated all the ad-herent target cells could be resuspended and collected after incubation. It has been reported that HMBPP increases the cytotoxicity of Vγ9Vδ2 T cell to-wards suspension target cells [163]. By using our newly developed assay, we find the HMBPP improves cytotoxicity to adherent colon cancer cell lines (HT29 and SW620) at different ratios.

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Paper IV Immunotherapy for colon cancer using patient themselves Vγ9Vδ2 T cells is a potiential therapy. To acquire status and more information of Vγ9Vδ2 T cells in the patients, we characterised the frequency, proliferation capacity, differentiation and homing potential of Vγ9Vδ2 T cells from colon cancer patients.

The frequency of Vγ9Vδ2 T cells in colon cancer patients Frequency or pencentage of circulating γδ T cells or/and Vγ9Vδ2 T cells is found to be lower than healthy donors in some cancers [164-166]. First, we investigated if the impairment occurs in colon cancer. We compare the per-centages of Vγ9Vδ2 T cells in lymphocytes between age-matched donors and colon cancer patients at stage I, II, III and IV (Table 4). The average The mean percentage of age-matched control group (3%) was higher than it of the four groups of patient samples (1,5%-2,5%).

The capability of Vγ9Vδ2 T cell proliferation in colon cancer patients Except the lower frequency, it has been reported that the capability of prolif-eration is impaired in cutaneous primary melanoma [164]. In this study, we investigated if the capability of proliferation in colon cancer patients was lower than the control. We found IL-2 and HMBPP induce proliferation of Vγ9Vδ2 T cells in all groups but some samples did not respond to HMBPP. As a result, we grouped the samples to non-responder, low responder, medi-um responder and high responder. The number of high responders in the control group was higher than it in the colon cancer stage I, III and IV groups. The number of non-responders in control group is lower than all patient sample groups. Based on these results, it is suggested that the Vγ9Vδ2 T cell proliferation is imparied in colon cancer patients. But the impairment does not appear to be related to the stage of colon cancer.

CXCR3 and CCR5 expression in Vγ9Vδ2 T cells in colon cancer patients The inflammatory chemokine receptors, CXCR3 and CCR5 are related to direct the infiltrated T cells to tumors [167-169]. Vγ9Vδ2 T cells express the two chemokine receptors and upregulate them after differentiation. To inves-tigate the infiltration potential of Vγ9Vδ2 T cells, we tested the expression of the two receptors in cancer samples compared with age-matched samples.

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After proliferation with stimulation of HMBPP with help of IL-2, there was a significant increase in CCR5 expression of Vγ9Vδ2 T cells and 90% the cells expressed CCR5 in all groups. Interesting, the expression of CXCR3 in all patient sample groups except stage IV was significant higher than control group after proliferation. Moreover, expression of CCR5 before proliferation in control group is lower than it in the four cancer groups. These results show the Vγ9Vδ2 T cells in colon cancer patients was potential to infiltrate tumors and that this capacity increases when they are stimulated by phosphoantigens.

Vγ9Vδ2 T cells differentiation in colon cancer patients We characterised differentiation status of the Vγ9Vδ2 T cells in patient samples. We found a significant increase in the percentages of TEM after proliferation while a decrease in the percentages of TN and TEMRA in all sample but one exeption. TEM cells and TEMRA cells play main role in cytotxicity. We find the combined TEM cells and TEMRA cells population significantly increased after proliferation in all samples. In summary, the Vγ9Vδ2 T cells of patients can differntiate to cytotoxic cells to cancer.

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Summary and conclusion

Paper I We address how Vγ9Vδ2 T cells, as well as Anopheles gambiae immunity cells, respond to phosphoantigens released from malaria parasites. In detail, we found that the phosphoantigen HMBPP increases phosphorylation of the MAPK/Erk proteins causing their activation. The HMBPP increases the re-sistance of oxidative stress by upregulation of the transcription of dual oxi-dase and nitric oxide synthase. We also have shown that Vγ9Vδ2 T cells were substantially stimulated by media collected from synchronized para-site-infected erythrocytes at the ring stage.

Paper II We found that Vγ9Vδ2 T cells were stimulated and proliferated by media collected from infected erythrocytes at all blood stages, i.e. both asexual and sexual stages. Moreover, the stimulation was independent of schizont rup-ture. Our result suggests that the parasites used all hemoglobin during their development from the ring to the schizont stage.The molecules stimulating Vγ9Vδ2 T cell were determined to be phosphoantigens. Phosphoantigens did not appear in the media due to the lysis of erythrocytes assessed by measur-ing the iron level in the media. These findings demonstrate phosphoantigens are released from parasites during all stages of blood cell cycle and suggest that phosphoantigens are transported through several membranes when they are released.

Paper III A protocol for proliferation of Vγ9Vδ2 T cells from cryopreserved samples was designed and we find the cryopreservation does not abrogate the prolif-eration response. Actually, it was higher than the proliferation from freshly isolated samples. Although many cells were lost during cryopreservation, no significant change in percentages of δ2+ cells CD3+ cells was found. DMSO

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was tested if it enforces the proliferation but the DMSO did not show a sig-nificant effect. We also show that fresh media should be used for prolifera-tion. Most small natural pyrophosphate intermediates of isoprenoid synthesis induced proliferation but most corresponding monophosphates did not with the exception is the monophosphate DMAP. Vγ9Vδ2 T cells respond to stimulation of phosphoantigens and phosphoantigens enforce cytotoxicity to cancer cells. An assay was developed for testing the cytotoxicity of HMBPP-stimulated Vγ9Vδ2 T cells against adherent colon cancer cell lines.

Paper IV In this study, we characterised the frequency, proliferation capacity, and inflammatory homing potential of Vγ9Vδ2 T cells from colon cancer pa-tients. The patients were classified to 4 groups (stage I-IV) according to TNM systems. We found the impairment of capability of Vγ9Vδ2 T cells proliferation in all patient sample groups. In addition, the percentage of Vγ9Vδ2 T cells in total lymphocytes in all patient groups was lower than control group. The expression of CXCR3 and CCR5 in patient samples is not lower and even higher than it of control group. These results suggest the Vγ9Vδ2 T cells of patients have potential to infiltrate tumor. After prolifera-tion, the percentage and number of TEM cells increase in both control and patient groups, the result suggests the Vγ9Vδ2 T cells have potential to be used for adoptive immunotherapy.

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Acknowledgments

Many people helped me to finish the thesis. I am grateful to my supervisor Ingela Parmryd. You gave me many good suggestions on science. Thank you for your encouragement, patience and support. Thanks to my co-supervisor Sebastian Barg for your nice suggestions. Thanks to Erik Gylfe and Anders Tengholm for your nice suggestions and organization of the Thursday meetings. Thanks to Jeremy Adler for help with image processing. Thanks to my members of my group Jan Saras, Lijun Zhao, Yonas Hus-sein, Dhanya Grero, Parham Ashrafzadeh and Warunika Aluthgedara for supporting my experiments. Thanks to Gunilla Westermark for nice support to PhD students and organ-ization of Brain pub. Thanks to Nils Welsh, Erik Gylfe, Shumin Pan, Camilla Sävmarker, Göran Ståhl and Carl Lundström for your work in administrative issues. Thanks to my co-authors of papers Eleanor A. Merritt, Melanie Rayl, Berit Olofsson, Jean Pettersson, Karolina Skorupinska-Tudek. Noushin Emami, Bo G. Lindberg, Ingrid Faye, Ingela Parmryd, Lisa Ranford-Cartwright and I discussed a lot of science on the papers. I learned a lot interesting knowledge in malaria parasite and mosquito. Helgi Birgisson who gave me an opportunity to do the research on colon cancer. Thanks to all blood donors in my research and thanks to Caroline Riesen-feld, Eva Jensen and Sara Abu Hamdeh Bui-Quy for helping me to collect the samples in hospital. Thanks to to Jing Cen, Daniel Espes, Andreas Ejdesjö and Johan Staaf for helping me to do experiments. Thanks to Björn Åkerblom for the teaching tasks. Thanks to Bo Hellman and Eva Grapengiesser for the organization of cof-fee breaks. Thanks to Qian, Jian, Peng, Ye, Misty, Nikhil, Marie, Johan, Sara, Ida, Carl, Hanna, Jing, Xiaohong, Beichen, Xuan, Hjalti, Nadine, My, Gon-zalo, Antje, Azazul, Karin, Levon, Alenka, Vishal, Omar, Parvin, Love, Hongyan. We spent nice time and helped each other in and out of the de-partment. Thanks to Emma, Kailash, Yunjian, Oleg, for your nice sugges-tions in science and technology.

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The work in the thesis supported by China Scholarship Council, EU research infrastructures project, Wenner-Gren Foundations, the Swedish Research Council, AFA Insurance, Clas Groschinsky’s Foundation. Special thanks to my parents

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1455

Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)

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