Malaria, Malaria Transmission, Morbidity and Mortality

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background Information

Malaria, a disease caused by parasites of the genus of Plasmodium is one of the deadliest diseases and has proved to be a significant threat to human health especially in children under the age of five. It is estimated that 247 million cases of clinical malaria occur every year and nearly 1 million deaths due to malaria are reported each year most of them in children in equatorial Africa (WHO, 2008). Most of the malaria related deaths are caused by infection by Plasmodium falciparum, accounting for up to 91% of all malaria cases worldwide (WHO, 2008),with children under the age of five being the most affected with an estimate of 88% (WHO, 2008).

Immunity to malaria develops slowly and wanes when an individual moves away from malaria endemic area (Langhorne et al, 2008) and it has been suggested that immunity to malaria in children living in endemic areas is acquired only after a few infections (Gupta et al, 1999). In Kenya, the pattern of malaria transmission vary across the country with the lake Victoria region experiencing high transmission while the highlands of western Kenya experiencing low transmission intensities (WHO, 2005).

Endemic Burkitt’s lymphoma (eBL) is a cancer that mainly affects children aged between 2 and 15 years of age (Mwanda et al, 2004). It accounts for up to 74% of all childhood malignancies in Africa and it is the most prevalent pediatric cancer in Kenya (Makata et al; 1996, Mwanda, 2004). eBL is prevalent within the malaria holoendemic regions where there is chronic and intense transmission of P. falciparum malaria (Rainey et al, 2007). Infection with both holoendemic malaria and Epstein Barr virus (EBV) are two cofactors that have been implicated in the pathogenesis of eBL (Rochford et al, 2005) but the precise mechanism by which these two agents lead to the pathogenesis of eBL is not well known.

Programmed death-1 (PD-1) is a cell surface molecule that is mainly expressed by activated B cells, CD4+ T cells, CD8 T cells and myeloid cells (Riley, 2009). PD-1 binds to its ligands programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2) and the engagement of PD-1 to its ligands transduces a signal that inhibits T cell proliferation, cytokine production and cytolytic activity (Freeman et al, 2000; Latchman et al,2001). PD-L1 and PD-L2 are upregulated upon activation or interferon-gamma (IFN-γ) treatment on monocytes and dendritic cells (Freeman et al, 2000). PD-1 and its ligands are negative regulators of T cells as invitro treatment of T cells with anti CD3 resulted in impaired T cell proliferation and IFN-γ production (Freeman et al, 2000).

Many studies have reported the upregulation of PD-1 in both murine and primate viral infections. In murine models, lymphocytic choriomenengitis virus specific CD8+ T cells express high levels of PD-1 and that in vivo blockade of this pathway reverses the “exhausted” CD8+ T cells and reduces the viral loads (Barber et al, 2006). In human, it has been shown that PD-1 is upregulated on HIV specific CD8+ T cells and that blocking this pathway lead to an increased T cell proliferation and effector functions (Trautmann et al, 2006; Petrovas et al, 2006).

1.2 Problem Statement

Repeated challenge of the immune system following persistent infection leads to T cells that become progressively dysfunctional and not effective in mediating immune functions (Wherry and Ahmed, 2004). Persistent viral infections have been reported to be associated with functionally impaired T cells, showing reduced proliferative potential and effector functions (Wherry et al, 2003), and it has been suggested that this might be the reason for the inability of the host to eliminate persisting pathogens (Barber et al, 2006). Such T cells have been termed as “exhausted” and have a reduced immunologic function. PD-1, a cell surface immune inhibitory molecule, is reportedly up regulated on the surfaces of exhausted cells in individuals having viral infections such as HIV, HCV, HBV in human, and in murine disease models, but its expression in healthy children with diverse P. falciparum transmission and exposure histories or those clinically presenting with endemic Burkitt’s lymphoma has not been reported.

1.3 Justification

The reason why children from malaria endemic regions have a higher prevalence of endemic Burkitt’s lymphoma (eBL) compared to children from regions where malaria is unstable is not fully understood but studies by Moorman et al, (2007), have implicated the elevated viral loads in children having acute clinical malaria and interference in the B cell subset as a possible mechanism for the pathogenesis of eBL.

Plasmodium falciparum malaria causes a complex pattern of immunopertubation with modulation of CD8+ T cells having consequences in the immune responses to other infection like EBV. The mechanism by which malaria leads to eBL is still unknown but expansion of B cell and suppression of specific T cell immunity have been proposed as two possible mechanisms (Rochford et al, 2005). By comparing IFN-γ and IL-10 responses in healthy children from two regions of western Kenya with differing malaria transmission patterns, Moorman et al, (2007) reported that there was an age related loss of specific T cell IFN-γ responses to EBV lytic and latent HLA class-1 restricted epitopes in children from malaria holoendemic region compared to children from epidemic prone area. Another study also reported a reduced level of EBV specific T cell responses in adults from a malarious region compared to another group of adults from a non malarious area of Papua New Guinea (Moss et al, 1983). Together, these two studies provide evidence that holoendemic malaria suppresses EBV specific immunity and also explains its role as a co-factor in the pathogenesis of Burkitt’s lymphoma.

It is reported that in viral infections such as HIV, HBV, HCV and LCMV, the expression of PD-1 is upregulated on virus specific T cells and that these cells progressively loose their immune functions during the course of the infection (Barber et al, 2006; Trautmann et al, 2006; Urbani et al, 2006). Langhorne et al, (2008), suggested that P. falciparum parasites induce the exhaustion of T cells and that parasitic infections have also exploited the PD-1-PD-L pathway to attenuate the immune system and establish chronic infection (Smith et al, 2004, Terrazas et al, 2005). It is also reported that T cell exhaustion is essential to the pathogenesis of Hogkins lymphoma (Yamamoto et al, 2008).

This study therefore aims at determining the expression of PD-1 in healthy individuals from two epidemiologically distinct areas of western Kenya that differ in transmission intensity of malaria and in children presenting with clinical Burkitt’s lymphoma.

1.4 Objectives of the Study

1.4.1 General objective

To investigate the activation induced exhaustion of lymphocyte populations in children from two areas with differential malaria transmission pattern and in children presenting with clinical endemic Burkitt’s lymphoma.

1.4.2 Specific objectives

1. To determine the frequency of PD-1 expression in lymphocyte subsets in children from two areas with differential malaria transmission patterns and in children with endemic Burkitt’s lymphoma.
2. To determine and compare the plasma levels of soluble PD-1 in the three study populations.
3. To compare the plasma levels of Th3 and Th3 cytokines in the three study populations.

1.5 Null hypotheses

There are no differences in the expression of PD-1 on lymphocyte subsets in children from divergent P. falciparum regions and in children presenting with endemic Burkitt’s lymphoma.

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Malaria, Malaria Transmission, Morbidity and Mortality

Malaria is a vector borne disease that is caused by protozoan parasites of the genus Plasmodium and is transmitted from person to person by the bites of an infected female Anopheles mosquito. There are five species of Plasmodium that cause malaria to humans; Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovaleand Plasmodium knowlesi(White, 2008). Malaria affects over three billion people worldwide (Snow et al, 2005), with about 247 million people reporting clinical cases of malaria yearly (WHO, 2008). It is estimated that 86% of all clinical cases of malaria occur in tropical and subtropical regions of Africa where malaria transmission is holoendemic with children under the age of five being the most affected, reporting an annual mortality of 881,000 with Africa reporting 90% of all the deaths (WHO, 2008). In sub-Saharan Africa, most severe cases of malaria are attributed to P.falciparum.

In addition to acute infections with malaria and deaths in Africa, malaria also contributes significantly to anemia in children and pregnant women that may lead to adverse birth outcomes such as abortion, stillbirth, premature delivery and low birth weight, hence increasing overall child mortality (WHO, 2005). Malaria endemicity in Africa is defined on the basis of parasite prevalence into mesoendemic, holoendemic and hyperendemic regions. In holoendemic areas, there is stable transmission of malaria that is characterized by recurrent exposure to the infection throughout the year (Snow et al, 1997).

2.1.1 Plasmodium life cycle

Malaria is transmitted to the human host by the bite of an infected Anopheline mosquito which inoculates motile sporozoites into the blood stream (Deans and Cohen, 1983). The sporozoites travel to the liver where they invade the hepatocytes and rapidly divide asexually by schizogony to produce merozoites. These merozoites reinvade other liver cells while others enter the blood stream and invade the erythrocytes. After the merozoites have invaded the host erythrocytes, they mature and continue to divide asexually to become schizonts which contain several nuclei. Depending on the species, each intraerythrocytic expansion burst infection cycle results 20-30 new merozoites (Pouniotis et al, 2004). The erythrocytes burst and release toxins throughout the body bringing about fever and chills that is characteristic of malaria. Some merozoites in the erythrocytes enter a sexual phase where they develop into microgametocyte and macrogametocyte. Mature macrogametocyte taken into the midgut of the Anopheles mosquito escape from the erythrocyte to form macrogametes. Microgametocytes exflagellate, each forming eight haploid motile microgametes after a few minutes in the mosquito’s midgut. The microgamete moves quickly to fertilize a macrogamete and form a zygote. Within 18 to 20 hours, the non motile zygotes transform into motile ookinetes that traverse the midgut epithelium and reach the extracellular space between the midgut epithelium and the overlaying basal lamina and transforms into an oocyst. Within 10 to 24 days after infection, depending on the Plasmodium species and the ambient temperature, thousands of sporozoites are released into the hemocoel and the motile sporozoites invade the salivary gland epithelium. When an infected mosquito bites a susceptible host, the Plasmodium life cycle begins again (Perlmann and Troye-Blomberg, 2002; Todryk and Welther, 2005).

2.1.2 Immunity to malaria

Malaria transmission intensity influences the course of development of immunity to the parasite. Children under the age of five years living in areas of stable malaria transmission initially suffer severe malaria but following repeated exposure to the infection, immunity to the parasite develops and the disease becomes less severe and this immunity limits high density parasitemia later in life (Perlmann and Troye-Blomberg, 2002). In areas of low malaria endemicity, both children and adults suffer from malaria and parasitemia since there is less repeated exposure to the parasite (Snow et al, 1997).

Immunity to malaria develops slowly and always wanes quickly when an individual moves away from the malaria endemic region (Langhorne et al, 2008). This suggests that the generation and maintenance of effector and memory cells require continued exposure to the malarial antigens.

The contribution of T-cell subsets and their cytokines to the development of natural immunity is essential both in regulating antibody formation and in inducing antibody-independent protection (Winkler et al., 1999). Cell mediated immunity involves inhibition of parasite growth and development in the hepatocytes by CD8+ cytotoxic T cells, macrophage activation by NK cells and production of IFN-γ for enhanced clearance of parasitized erythrocytes (Tsuji and Zavala, 2003). It has been suggested that IFN-γ production by T-cells and Nitric Oxide produced by macrophages has anti parasitic effect and that the Nitric Oxide has been shown to kill P. falciparum and P. chabaundi in vitro at high concentration (Balmer et al, 2000), however, studies in murine malaria suggest that Nitric Oxide is not required for parasite killing (Favre et al, 1999).

2.2 EBV and Burkitt ‘s Lymphoma

Epstein Barr virus (EBV) is a gamma-herpes virus that belongs to the family of Gammaherpesviridae (Babcock et al, 1998). EBV is estimated to infect 90% of adult population worldwide and in Africa, it has been suggested that by the age of three, about 80% of the children are EBV seropositive (Biggar et al, 1978). Primary infection occurs horizontally during childhood through the saliva and this coincides with the period at which maternal immunity diminish (Biggar et al., 1978). After primary infection, EBV establishes a life long latent infection and rarely causes disease unless the host-virus immune balance is upset (Donati, 2005).

EBV has been associated with a number of malignancies and cancer. The most common association is with Burkitt’s lymphoma. Burkitt’s lymphoma is a distinct form of Non-Hodgkin’s lymphoma and it is the most common paediatric cancer in equatorial Africa and it accounts for 74% of all childhood malignancies (Burkitt, 1983). Burkitt’s lymphoma can be classified as endemic, sporadic or HIV associated (Donati, 2005). Sporadic Burkitt’s lymphoma accounts for 20-30% of non Hodgkin’s lymphoma in children in developed countries. It affects the abdominal region and can be detected at any age. Endemic Burkitt’s lymphoma is almost exclusively found in Africa (Donati, 2005), affects mainly the facial skeleton in children aged between 2 to 9 years and is the most common paediatric cancer in equatorial Africa (Orem et al, 2007). In Kenya there is evidence that there is uneven geographical distribution in the incidences of endemic Burkitt’s lymphoma (Rainey et al, 2007). In young adults, BL manifest as acute infectious mononucleosis (AIM) that is characterized by rapid expansion of virus specific CD8 T cells in peripheral blood (Callan et al, 1998).

EBV and holoendemic malaria are two agents that have been implicated in the etiology of Burkitt’s lymphoma. Malaria causes a complex pattern of immunomodulation accompanied by polyclonal lymphocyte activation leading to increased numbers of circulating EBV infected B cells and so the viral loads (Whittle et al, 1984). The highest density parasitemia is observed in children of age between 6-11 months old and it is at this age that primary EBV infection is likely to occur (Rochford et al, 2005). It has been reported that acute malaria causes impairment of EBV specific T cell immunity (Gunapala et al, 1990) and that this impaired EBV specific T cell responses is indicated by the loss of IFN-γ mediated killing of virus infected cells (Moss et al, 1983). The immunosuppression of EBV specific T cell immunity and expansion of latently infected B cell pool are two possible mechanisms that have been proposed to explain how holoendemic malaria impacts on EBV latency and how this increases the risk to endemic Burkitt’s lymphoma (Rochford et al, 2005).

2.3 Programmed Death-1 (PD-1)

Programmed death-1 (PD-1) is a cell surface protein, a member of the CD28/cytotoxic T cell antigen-4 (CTLA-4) family of T cell receptors that negatively regulates antigen receptor signaling. This inhibitory effect has been shown to be effective both in CD4+ and CD8+ T cells (Carter et al, 2002). PD-1 (or CD279) was initially cloned as a molecule that was over expressed in cells undergoing cell death (Ishida et al, 1992) and hence named programmed death-1. Although PD-1 was initially known to be a death receptor due to its preferential over expression by dying cells, further studies have shown that its expression is associated with negative lymphocyte activation (Agata et al, 1996; Vibhakar et al, 1997).

PD-1 binds to PD-L1 (CD274) and PD-L2 (CD273) that are both members of the B7 homologues. The engagement of PD-1 to its ligands results in both inhibition of T cell activation and cytokine production (Freeman et al, 2000; Latchman et al, 2001).

2.3.1 Structure of PD-1

Structural and biochemical analyses have shown that PD-1 is a monomer both in solution and on cell surfaces (Zhang et al, 2004). PD-1 is a 288 amino acid type I transmembrane protein that is encoded by Pdcd1 gene on chromosome 1 in humans. It is composed of one immunoglobulin super family domain, a 20 amino acid stalk that separates the IgV domain from the plasma membrane, a transmembrane domain (Keir et al, 2008). It also has two tyrosine molecules that are located in its cytoplasmic tail. There is an intra cellular domain of approximately 95 residues containing an immunoreceptor tyrosine based switch motif (ITSM) and immunoreceptor tyrosine based inhibitory motif (ITIM) located at the C terminal end (Parry et al, 2005). It has been shown that an intact ITSM is essential in mediating the inhibitory effects of PD-1 in human T cells and a mutation in the ITSM interferes with the ability of PD-1 to limit T cell expansion and cytokine production (Chemnitz et al, 2004).

PD-L1 is a 290 amino acid type I transmembrane protein encoded by Cd274 on human chromosome 9 while PD-L2 is also a type I transmembrane protein that is encoded by PdcdIg2 gene (Keir et al, 2008).

2.3.2 PD-1 signaling

The signaling pathways by which PD-1 exerts its effect are just beginning to be understood. PD-1 does not transduce an inhibitory signal when cross linked alone but only does so when engaged simultaneously with either a T cell receptor (TCR) or B cell receptor (BCR) (Freeman et al, 2000). The cytoplasmic domain of PD-1 contains two tyrosine molecules, ITIM and ITSM (Parry et al, 2005) both of which can be phosphorylated upon receptor engagement. Upon receptor engagement, the tyrosine residue located at the ITSM is phosphorylated and rapidly recruits Src homology region 2 domain containing phosphatase (SHP-2) to the PD-1 cytoplasmic domain (Parry et al, 2005; Chemnitz et al, 2004). This leads to dephosphorylation of effector molecules activated by the TCR or BCR signaling (Latchman et al, 2001) leading to a reduction of TCR/CD28 signals. Some of these effector molecules include ZAP 70 and CD3ζ in T cells and Syk and phosphatidylinositol-3-kinase (PI3K) in B cells. PD-1 signaling inhibits Akt phosphorylation, glucose metabolism and expression of gene survival protein Bcl-xL by preventing CD28 mediated activation of PI3K (Chemnitz et al; 2004., Parry et al, 2005).

2.3.3 Expression if PD-1 and it’s ligands

PD-1 is expressed on activated CD4+ T cells, CD8+ T cells, B cells, natural killer cells, dendritic cells and monocytes and is induced on T cells after activation in vitro (Agata et al, 1996). PD-1 is rapidly upregulated on activated T cells and diminished on memory T cells after antigen clearance (Zhang et al, 2004). PD-L1 is expressed on resting B cells, T cells and dendritic cells (Latchman et al, 2001) and it’s expression is up regulated upon activation by both type I and type II IFN’s. It is also expressed on a wide range of non hematopoietic cells and at immunoprivilleged sites such as the placenta and the eye (Sharpe et al, 2007). The expression of PD-L1 has been reported on many solid tumours and high levels of PD-1 expression have been associated with poor prognosis of the disease (Latchman et al, 2001). PD-L2 is inducibly expressed on dendritic cells, macrophages and mast cells.

2.3.4 PD-1 expression in viral and parasitic infections

During an acute viral infection CD8 T cells undergoes an expansion phase resulting in the generation of effector CD8 T cells that participate in viral clearance (Wherry and Ahmed, 2004). This is followed by a death phase where 90-95% of the effector CD8 T cells die (Kaech et al, 2002) and the remaining 5-10% of the effector CD8 T cells differentiate further to generate a pool of long lived memory CD 8 T cells and these are maintained for long period of time in the absence of antigen stimulation (Lau et al, 1994; Murali-Krishna et al, 1999). These maintained number of memory CD8 T cells are highly functional and provide an important component of protective immunity (Wherry and Ahmed, 2004). On the other hand, in chronic infections, functional effector CD8 T cells are generated during early stages of the infection but they loose their function during the course of the infection (Wherry et al, 2003). This loss of function is referred to as “exhaustion” (Zajac et al, 1998) and is a defining characteristic of many chronic infections and factors such as the availability of CD4 T cell help, level of antigen exposure and the duration of exposure determines the level of exhaustion (Freeman et al, 2006).

Exhaustion comprises of a range of dysfunctions from mild to extreme and occurs in a hierrachial manner with functions such as IL-2 production and proliferative potential being lost first while other functions such as IFN-γ production occurring later. Wherry et al, (2003) identified three levels of exhaustion in virus specific CD8 T cells; mild (partial exhaustion I) where there is little IL-2 production and TNF-α production starts to be impaired and lytic capacity starts reducing, to moderate (partial exhaustion II) consisting of modestly defective IFN-γ production, little IL-2 or TNF-α production and cytotoxicity, to severe or extreme exhaustion (full exhaustion) where the CD8 T cells lack all the effector functions i.e. IFN-γ, IL-2 or TNF-α production and cytotoxic activity. Impaired proliferative potential is a key feature of exhaustion and it has been shown that it occurs when other functions of the T cells such as cytokine production and cytotoxicity are intact. Freeman et al., (2000), found out that the proliferative potential of T cells decreased alongside the loss of these functions while apoptosis increased and as the antigen load increased or CD4 help decreased, the virus specific T cells became more exhausted.

Lymphocytic choriomenengitis virus (LCMV) is a natural pathogen in mice and has been used to elucidate the function of PD-1 and it’s ligands in immunity and infection. There are two strains of LCMV that can cause either an acute or chronic infection in mice; the Armstrong strain causes an acute infection that is cleared within 8-10 days after infection while clone 13 causes chronic infection that overwhelms the immune system (Wherry et al, 2004). Surprisingly, these two strains differ in only two amino acids in their entire genome (Matloubian et al, 1993). During an acute infection, Barber et al, (2006) found out that PD-1 was briefly expressed on early effector CD8+ T cells but was rapidly downregulated. On the other hand, during chronic infection, there was continued expression of PD-1 on LCMV specific CD8+ T cells and that the high levels of PD-1 expression were sustained during the infection (Barber et al, 2006). PD-L1 was also upregulated on infected cells suggesting that this ligand together with PD-1 may be involved in regulation of T cell function during chronic LCMV infection.

The blockade of the interaction between PD-1 and its ligand has been shown to rejuvenate the exhausted T cells and restore their function. Barber et al, (2006) treated chronic infected mice with blocking antibody specific for PD-L1 and monitored T cell responses and viral control. They found out that in contrast to untreated mice, a higher percentage of virus specific CD8 T cells from chronically infected mice had expanded and had an increased ability to produce IFN-γ and TNF-α. PD-L1 blockade also resulted in a striking reduction in viral loads in mice treated with PD-L1 specific antibody while the untreated mice still had high levels of the virus that was maintained even after the anti-PD-L1 was stopped (Barber et al, 2006).

In mice infected with Schistosoma mansoni, there was increased expression of PD-1 on splenic CD4+ and CD8+ T cells compared to naïve T cells and the macrophages expressed high levels of PD-L1 (Smith et al, 2004). Similarly, during Taeniacrassiceps infection in mice, a high percentage of CD4+T cells express PD-1 and both PD-L1 and PD-L2 were upregulated on macrophages (Terrazas et al, 2005). These two studies suggest that parasitic infections may also exploit the PD-1-PD-L pathway to downregulate specific anti-parasitic immunity and establish a chronic infection.

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Study Area

This study was conducted at Kanyawegi and Mosoriot villages and at new Nyanza provincial general hospital (Appendix 2). Kanyawegi is an area of holoendemic malaria in the lowlands of western Kenya. Mosoriot is an area of unstable P.falciparum transmission situated 150 kilometers northeast of Kisumu in the highlands of Rift valley province.

3.2 Study Population

3.2.1 Inclusion criteria

Inclusion criteria into the study included children aged from six months to eighteen years who are residents of the two study area, body temperature of ≤37.5oC and may be parasitemic but asymptomatic. Children having eBL but have not started chemotherapy were included. Adults (>18 years) control were also included.

3.2.2 Exclusion criteria

Individuals who have Hb of less than 5g/dL, parasitemia with fever or evidence of another etiology of fever and individuals who are generally unwell due to other unconfirmed health conditions.

3.3 Ethical Considerations

Approval for the study was obtained from the Kenya Medical Research Institute National Ethical Review Committee and Ethical Review Board of University of Massachusetts Medical School, USA. Written informed consent was obtained from participants and parents or guardians of all study participants. Qualified phlebotomists collected venous blood under sterile conditions in order to minimize the risk of infection and discomfort.

3.4 Study Design

This study was a cross sectional study involving a total of 100 individuals (<1, 1 – 5, 5 – 9, 9 – 14, >18 years) from both the high (Kanyawegi) and low (Mosoriot) malaria transmission areas. Twenty children presenting with eBL were enrolled to determine PD-1 expression in different lymphocyte subsets.

3.5 Sample Size Calculation

Sample size was calculated based on G-power software. In order to achieve a statistical power of 80%, a significance of α=0.05 with a large effect size, a total of 64 age matched individuals were be enrolled (see figure below).

3.6 Blood Sample Collection microscopy and complete blood count (CBC)

Venous blood (2 – 5ml from children and 8 – 10ml from adults) was drawn by venipuncture into 10ml heparinized vacuitainer and transported within two hours to the University of Massachusetts/KEMRI laboratory located at Centre for Global Health Research (CGHR) in Kisian and processed on the same day. P. falciparumdiagnosis was determined by microscopic examination of Giemsa-stained thick and thin blood smears. A coulter counter was used to quantify the hematological indices of the study participants.

3.7 Peripheral Blood Mononuclear Cell Isolation

PBMCs were separated from sodium heparin anticoagulated whole blood by standard Ficoll-Hypaque density gradient centrifugation. In this procedure, the anti-coagulated blood was layered carefully onto 5mL of Ficoll-paque (GE Healthcare, Sweden) in a 15mL tube and then centrifuged at 450×g for 30 minutes. Plasma was transferred into Sarstedt tubes (Sarstedt, Germany) and stored at -800C while the PBMCs was collected using a sterile 10mL pipette and transferred into a 15mL tube. The cells were washed by adding sterile 1× PBS, pH 7.0, without calcium or magnesium, to bring a total volume in the tube to 12mL followed by centrifugation for 15 min. at 350×g at room temperature. The supernatant was aspirated off, the pellet broken by gentle flicking of the tube and then washed again as described above and centrifuged for 10 min at 350×g. The supernatant was aspirated, the pellets broken by gently flicking the tubes and the cells resuspended in 1mL of sterile 1 × PBS, pH 7.0. 10µL of 0.4% Turk’s solution was used to dilute the cells in a 1:1 ratio to aid in visualizing the cells under a microscope and using a Haemocytometer to calculate the yield using the formula (cell count in 1ml = (# cells counted in 5 squares)×5×2×104). The calculations of the cell count were carried out in Microsoft Excel sheets.

3.8 Staining for Flow Cytometry

Half a million cells was aliquoted into appropriately numbered 5mL polystyrene tubes (Becton Dickinson USA). Appropriate monoclonal antibodies specific for different surface molecules were added (Appendix 1). The tubes were then be vortexed and incubated at room temperature for 20 minutes in the dark. After incubation, 2mL of cold flow buffer was added to each tube and vortexed gently and then be spun at 450×g for 5 minutes at 40C. The supernatant was aspirated off and 0.5mL of 2% Paraformaldehyde added to all the tubes, vortexed gently and incubated in the dark for 20 minutes at room temperature.

3.9 Flow Cytometry Acquisition and Analysis

Data was acquired within 24 hours using CELLQuestPro software on a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). The fluorescent intensity measurement was done using logarithmic amplifiers whereas the forward scatter and side scatter measurement was made using linear amplifiers. Flowjo software (Tree star Inc, USA) was applied to collect data for compensation and processing of data before statistical analysis.

3.10 Soluble PD-1 ELISA

A 96-well microplate was coated with 100µL of anti human PD-1 capture antibody, sealed and incubated overnight at room temperature. After a three steps wash (0.05% Tween 20 in PBS pH 7.2), non specific binding was blocked for one hour using 300µL of blocking buffer (1% BSA in PBS pH 7.2). These were then washed twice and 100µL of standards or the samples added to the wells, covered using a plate sealer and incubated for two hours at room temperature. After the incubation period, the plates were washed twice with wash buffer, 100µL of biotinylated goat anti human PD-1 detection antibody added to each well, covered using a plate sealer and further incubated for two hours at room temperature followed by addition of 100µL of streptavidin HRP to each well. HRP activity was detected using 3,3′,5,5′-tetramethylbenzidine (Organon Teknika) in h3O2 and the reaction stopped by adding 2M sulfuric acid (h3SO4). The optical density was determined at 450 nm (Anthos 2001 reader, Anthos Labtec Instruments, Salzburg, Germany).

3.11 Th3 and Th3 Cytokine Testing by Bioplex Suspension Array System

A 1.2 µm 96 well Millipore filter plate was pre wetted with 100µL/well of PBT (PBS pH 7.4, 1% BSA, 0.05% Tween 20) and aspirated using a vacuum manifold. Using a multichannel pipetor 50 μL of the 2 x concentrated working microsphere mixture were aliquoted into the appropriate wells of the filter plate, then the samples or the standards will be added to the appropriate wells. This reaction will then be gently mixed using a multichannel pipettor and the filter plate covered and incubated for 30 minutes