Science & Business Management

Sjögren’s Syndrome ‘There is more to it than meets the eye’

Rogier Peters, 0365955

University medical Center Utrecht, The netherlands

Dept. Of Rheumatology & Clinical Immunology

Contents Introduction ............................................................................................................................................. 3

The Immune System ............................................................................................................................ 3

IL-7 ................................................................................................................................................... 4

T cells ............................................................................................................................................... 4

B cells ............................................................................................................................................... 5

Sjögren’s Syndrome ............................................................................................................................. 5

Symptoms ........................................................................................................................................ 5

Prevalence ....................................................................................................................................... 5

Social Implications ................................................................................................................................... 6

Market Analysis ....................................................................................................................................... 7

Market Potential ................................................................................................................................. 7

Partners ............................................................................................................................................... 7

Products on the Market ...................................................................................................................... 8

Palliative Medicines ......................................................................................................................... 8

Current Research & Products .......................................................................................................... 8

The Role for IL-7 .............................................................................................................................. 9

Research Background ............................................................................................................................ 10

“IL-7 driven T cell and B cell activation in primary Sjögren’s Syndrome (pSS)” ................................ 10

Conclusion ......................................................................................................................................... 12

Works Cited ........................................................................................................................................... 12

Introduction This essay will focus on the use of my research project for society. The research project itself focused

on the effect of the signaling molecule interleukine-7 (IL-7) on lymphocytes and tried to determine

whether B cells can be activated in a IL-7-induced T cell-dependent manner and hereby enhance the

immune-pathology in patients with primary Sjögren's Syndrome (pSS). I will try to show why research

in this field is both useful and necessary to our society. To make this clear I will first have to have you

understand the complex mechanisms behind Sjögren’s Syndrome and the research I have been

working on. To do so I will first explain briefly: the immune system, the involvement of IL-7, the

function of T cells, and of B cells. Secondly, I will discuss Sjögren’s Syndrome, the symptoms that are

involved, and the prevalence. Thirdly, I will discuss the social implications of pSS and lastly, I will

provide a market analyses for current products/therapies available against pSS and how my research

on IL-7 contributes. At the end of this essay you will find a summary of my research on IL-7 and pSS

patients.:

“IL-7 driven T cell and B cell activation in primary Sjögren’s Syndrome (pSS)”

The Immune System The immune system protects us from foreign intruders like bacteria, fungi or otherwise, and

disturbed body own (cancerous) cells. The immune system can be divided into three main barriers 1)

is the physical barrier i.e. skin and

mucus preventing foreign

intruders from entering the body,

2) the innate system i.e. the

primary response to foreign

intruders, and 3) the adaptive

system i.e. the specific immune

reaction. This system contains

many different cells, all with their

own and mostly unique purposes.

Figure 1 demonstrates the

schematic process that occurs

when the immune system

encounters an antigen. An

antigen is a molecular structure Figure 1 A Schematic overview on the immune system (5).

that is part of and specific for a certain intruder (e.g. the “skin” of a bacteria) that can be recognized

by the immune-system. A certain type of cell within the immune-system called the macrophage, is

able to recognize and “eat” the antigen and shows it to the environment (Becomes an Antigen

Presenting Cell; APC). This cell triggers the immune cells by activating the T helper cells. Depending

on the kind of intruder that has entered the body, the T helper cell decides what response is best

suited. The T helper cell may choose to activate B cells (lower left of figure 1) or T cells (lower right of

figure1). These two cell types (T cells and B cells) are of special interest regarding Sjögren’s

Syndrome. The typical feature of these T cells and B cells is that they normally are able to

discriminate between body own cells and foreign pathogens and/or disturbed cells. With this ability

they are able to attack pathogens and/or clean disturbed body own cells when necessary or, and this

is just as important, leave body-own cells alone. Hence, when this discrimination does not occur

accurately, auto-immunity may occur. Auto-immunity is the phenomenon that immune cells

(lymphocytes) are attacking body own cells causing all kind of problems. There are many auto-

immune diseases as a means of complexity and severity, where pSS is just one of them (1).

IL-7

IL-7 is a member of the IL-2 family (hormone-like signaling molecule) and a potent T cell activating

cytokine. IL-7 can be found in the bloodstream and is produced by epithelial cells in lymphopoietic

tissue such as bone marrow, the thymus, the spleen, and the gut (2). IL-7 is involved in T cell

homeostasis and contributes as a growth-factor to early T cell development and T cell maturation (3).

Increased levels of IL-7, both systemically and locally, have been reported in several auto-immune

disorders such as juvenile idiopathic arthritis, rheumatic arthritis, spondyloarthritis psoriasis and

psoriatic arthritis (2). Recently our department showed that IL-7 is found in high levels within pSS

patients.

T cells

Generally speaking, cytotoxic T cells are “specific” for the cleaning of

cellular abnormalities, whereas B cells are more “specific” in cleaning

humoral abnormalities, for example during a bacterial infection.

Cytotoxic T cells, as shown in figure 2, are able to find disturbed body

own cells (e.g. cancer cells or virally infected cells) and eradicate

them. When T cells do not distinguish between normal cells and

disrupted body own cells, this might lead to severe problems and

abnormalities within an individual and is called auto-immunity.

Recently our department demonstrated these cytotoxic T cell to be

highly activated in pSS patients (4; 5).

Figure 2 Schematic overview of Cytotoxic T cell (Tc) attacking infected cell (27).

B cells

B cells, once activated and transformed to plasma cells, are able

to produce antibodies (a kind of molecular label) that recognize

and bind specific pathogens (e.g. bacteria, figure 3). When a B

cell gets over activated and/or loses its ability to discriminate

between pathogens and body own material, antibodies against

body own tissue may be produced, causing all kinds of problems.

Antibodies related to pSS that are produced by these B cells have

very strongly been implicated in the progression of pSS. Also,

both the T cells and B cells have been found in very high

frequencies and highly activated in lip biopsies from pSS patients

indicating a clear role for these cells (6).

Sjögren’s Syndrome Sjógren’s Syndrome can be described as follows: “Primary Sjögren’s Syndrome (pSS) is a chronic

inflammatory auto-immune disorder that manifests lesions of the exocrine glands characterized by

lymphocyte infiltration” (7). What it basically means is that body-own cells i.e. B cells and T cells, that

are normally there to protect you from foreign intruders (e.g. bacteria), turn against the body. These

cells are becoming auto-immune, and start attacking body-own tissue. In case of Sjögren’s Syndrome

those (auto-immune) cells (mainly) start attacking the glands.

Symptoms

How symptoms vary per patient depends on how the immune system reacts upon its own tissue and

cannot be predicted upfront. Patients are diagnosed by a specialized rheumatologist via a blood-test,

tear-measurements and/or lip-biopsies1. Sjögren’s syndrome itself is incurable, still most symptoms

can more or less be suppressed. Helpful tools could be artificial tears or the drinking of lots of water,

however the disease is progressive and the symptoms are permanent requiring life-long treatment.

In more severe cases the practitioner might subscribe immune-suppressors i.e. corticosteroids or

hydroxychloroquine, to reduce inflammation and suppress the auto-immune cells, causing the

symptoms to decline (8).

Prevalence

The prevalence of pSS, despite its unfamiliarity, is nothing less than approximately 1% of the western

world adult population with a male to female ratio of 1:9. Patients suffering from pSS experience

1 For more information go to: http://www.sjogren.nl/www/node/14 (dutch website)

Figure 3 Schematic view of plasma B cell cell attacing a germ (28).

fatigue, keratoconjunctivitis sicca (which is the clinical term form for dry eyes2), xerostomia (which

means dry mouth3), muscle and joint problems. They often display hypergammaglobulinemia

(deregulation of the immune system4), and they have an increased risk of developing B cell

lymphomas” (a type of cancer which has been indicated to relate with pSS5) (7).

Concluding, pSS is not that uncommon and plays a significant role in many patients’ lives, affecting

our society not to the least. The tools and treatments available focus on the suppression of

symptoms or help weaken it, but do not cure the disease itself. To help treat these patients, coming

up with new medicines and more effective therapies requires further investigation and research.

Social Implications Every year billions are spent on research and the development (R&D) for various treatments. In

Holland alone the total R&D expenditure was 1.7 billion euro (in 2003) of which 653 million euro is

accounted for by the academic medical centers (9). Total healthcare costs (in 2008) in the

Netherlands were € 79.2 billion (10). Apart from the moral obligation that we have to help patients

and those in need of special care, scientific research plays a pivotal role in our economic system (11).

When looking at Sjögren’s Syndrome, there are many questions that arise from within society

regarding these patients diagnosed with pSS (12). It is known what pSS is in terms of definition and

diagnosis and we know it is not lethal. More life-affecting questions, like what does it mean for a

patient to have pSS?, are answered by communicating the experience from patients. Also, there are

many websites available distributing information and stories from fellow patients (12; 13; 14; 15).

However, the more fundamental, underlying questions like how do you get pSS?, how does pSS

develop over time?, and which mechanisms are involved?, remain uncertain. Therefore, many

researchers and research groups all over the world are trying to find answers regarding pSS. Some

are looking for answers from a neurological point of view and considering the neurological field to

provide useful knowledge (16), while others are approaching it from an immunological standpoint, as

did we. Different approaches call for different mindsets and forthcoming questions. Currently there

are no products or medicines on the market as a result from our research. This is because we are

standing at the beginning of unraveling the secrets of pSS. In my opinion, the immunological

approach will be the most promising, given our latest results. Blocking IL-7 could be a great target for

developing new therapies. However, since our results were inconclusive, more research is needed.

2 The phrase "keratoconjunctivitis sicca" is Latin, and its literal translation is "dryness of (the eye) cornea and conjunctiva"

For more information go to: http://www.merck.com/mmhe/sec20/ch230/ch230d.html 3Xerostomia reflects its Greek origins from "xeros" (dry) + "stoma" (mouth). For more information go to:

http://www.sjogrens.org/home/about-sjogrens-syndrome/symptoms/dry-mouth 4 http://emedicine.medscape.com/article/332125-overview

5 http://bloodjournal.hematologylibrary.org/cgi/content/full/90/2/766

Market Analysis

Market Potential As described earlier, the prevalence of pSS is nothing less than approximately 1% of the western

world adult population with a male to female ratio of 1:9. The exact figures on the prevalence on the

rest of the world is not known, however, the total world population is approximately seven billion

(17). The world age distribution is as following (18):

Age (yrs.) proportion Ratio Male/Femal (bn.)

0-14 27% 0,94/0,88

15-64 65,3% 2,23/2,19

>65 7,7% 0,23/0,29 This means that there are 2,46 bn. adult males and 2,48 bn. adult females on the world. With the

knowledge that every 1 in 1000 men get pSS and every 1 in 100 females (based on data from the

western world), there is a potential market of approximately 2.46 million men and 24.8 million

women suffering from pSS worldwide. There are currently no numbers on the severity within

patients, but not all patient will need medication. However, pSS is a progressive disease which affects

many people and thus a medicine that could heal or cure this disease has great global potential.

Partners There are several partners involved in our study against pSS. First of all there is a heavy competition

between different academic universities, competing for prestigious purposes i.e. the increasing

notoriety of the department or research group, and consequently the ability to get more funds. As

one of the basic rules within the academic field is: The one who shows results and publishes, gets

more funding. So departments are not very eager to share the results with other (competing)

universities. Nonetheless, our department works in collaboration with the University of Wageningen

and the Radboud University in Nijmegen. Results from our department will be discussed among

those parties and vice versa, however not to the fullest. There are clear political reasons for the

parties to be a bit hesitant not to share everything one knows (i.e. first come, first served). Although

this sounds a bit paradoxical, working together while not sharing every bit of information, still

Utrecht University is one of the few Universities who collaborate on their search for treatments

against pSS. Secondly, this research was partly sponsored by Amgen, a pharmaceutical company that

focuses on the production of antibodies for therapies (19). The results found by our group will be and

have been communicated with Amgen. They may decide, based upon the results, to stop sponsorship

or to continue sponsorship. Amgen provides our group with the necessary tools i.e. IL-7R blockers,

and equipment to proceed with the ongoing investigation on pSS. All possible treatments as a result

from this research will be for Amgen as well as the intellectual properties. Amgen, nor any other

pharmaceutical currently have an IL-7 based therapy or any other product on the market against pSS.

Lastly, the results will be communicated to the Reumafonds. This will not be, nor has it been detailed

information on the ongoing research, but rather the overall progress on the research. The in-depth

information is of no use to the patients, however progress on the investigation will be

communicated. The Reumafonds is always trying to keep patients informed on the latest progress. In

addition the department of Clinical immunology & Rheumatology is trying to keep their patients

informed with annual meetings regarding the research progress. Not only this is done to keep them

informed, but also to show patients that are willing to help the research by giving blood, results in

actual progress.

Products on the Market

Palliative Medicines

Currently there are no products available on the market that can cure pSS. There are however,

products that can suppress or diminish the symptoms of patients. As the disease is progressive and

symptoms differ from patient to patient, different drugs may help to lower the symptoms. For

example, pSS patients that suffer from joint or muscle pain can get a Non Steroid Anti Inflammatory

Drugs (NSAIDs). These drugs, as the name suggests, are anti inflammatory and relieve the patients

from pain. A well known NSAID often prescribed for joint or muscle pain, for example in patients with

arthritis or pSS, is ibuprofen (20). Another symptom that occurs in patients with pSS is the reduction

in saliva and mucus in for example the nose and eyes. Patients can get drugs that stimulate the

production of saliva and mucus such as Evoxac ( induces production of saliva), and artificial tears (14).

Another way to help patients is to slow down the progression of the disease by the use of

corticosteroids or Disease Modifying Anti Rheumatic Drugs (DMARDs). Corticosteroids and DMARDs

are a collection of many different drugs, but most of them work by slowing down the immune-

system so to say as they suppress the cells involved. These drugs however, have major side-effects

and may cause severe problems to the kidneys and the liver (21).

Current Research & Products

The palliative drugs improve dryness and diminish symptoms, but fail to affect the course of the

disease. There is a critical need for new drugs that helps patients suffering from pSS that have severe

organ involvement, and have higher risk of developing lymphoma’s. Currently, research is being done

on pro-inflammatory cytokines within Sjögren’s Syndrome. Pro-inflammatory cytokines are a cluster

of signaling molecules that are involved in inflammation and are found to be deregulated in pSS

patients (22). Potential targets such as TNF-α inhibitors and IFN-α have been proven to be partially

successful in rheumatic arthritis patients, however failed to achieve fully beneficial effects in clinical

trials for pSS patients (23; 24). Recently, N. Roescher et al. (2010) provided an overview on the

research against Sjógren’s Syndrome (SS) as shown in table 1. They demonstrate many different

targets to be researched, however none of them have resulted in a therapy/product yet. Some

targets such as IL-6, and IL-12/Il-23 are in the clinical trial phase III, which basically means that if they

pass this stage there is an enhanced likelihood of these targets to become actual therapies. Though

these results look promising, they have been proven successful for rheumatic arthritis (RA) and

systemic lupus erythematosus (SLE) only. How these targets will be useful for pSS patients remain to

be investigated.

Concluding, there are currently no disease affecting therapies or products available for pSS patients.

There are many researchers working on possible targets against pSS as cytokines, especially

interleukins, show promising results in other pSS-like diseases such as RA and SLE. Since the field of

research against pSS is very promising, but stands at the beginning, new developments are kept

strictly confidential. Therefore, it is very hard to find any information on the development of

potential new products, by pharmaceuticals, against pSS.

The Role for IL-7

As described above, interleukins show promising results for possible targets against pSS. We found

substantial indications for the involvement of IL-7 in the pathogenesis of pSS. Blockers against the

receptor for IL-7 (IL-7R) could very well be potential products to the market. IL-7R blockers prevent

IL-7 from binding to the target (immune) cells, diminishing auto-immune effects, and has been

proven feasible and effective in both ex-vivo and in-vivo studies (25). Also, since anti IL-7 can be

produced in a generic manner and is not restricted to individual immunological specifics (as is often

the case with antibodies), it is suitable for production on a large scale, lowering the costs of

Tabel by N. Roescher et al. (2010)

production. This makes the production and the realization of IL-7R blockers as a therapy both

technically possible and highly feasible. Before this can be realized there are several things that need

to be done. The current results will be used by my supervisors within the division Rheumatology &

Clinical immunology that are constantly working on translational research. They work closely with

the Reumafonds (a Dutch rheumatology fund) and are constantly trying to improve research on pSS

(26). Based on our findings regarding the effect of IL-7, they will work towards more research

questions, further unraveling the secrets of pSS. In the end the focus lies with creating new therapies

for clinical trials in collaboration with suitable partners such as Amgen for in-vivo use, ultimately

leading to save and feasible therapies for pSS patients.

Research Background

“IL-7 driven T cell and B cell activation in primary Sjögren’s Syndrome

(pSS)” As described above, T cells and B cells are part of the adaptive immune system and have been

implicated in auto-immune reactions in general and within pSS patients. Our objective was to

determine the effect of interleukine-7 (IL-7) on T cells and B cells. The aim of this study was to know

whether B cells could be activated in an IL-7-induced T cell-dependent manner and hereby enhance

the immune-pathology in patients with primary Sjögren's Syndrome (7). Thus, can IL-7 activate T cells

of pSS patients and hereby activate B cells? To do so, we first needed to show that B cells, indeed, do

not express (reveal on the cell’s surface) the receptor for IL-7, so that we could prove that any B cell

activation was not caused by IL-7 directly. Secondly, we needed to show that T cells do express the

receptor for IL-7 and to what extent i.e. the level/number of receptor on the cell’s surface, and that

those cells are indeed activated by it. The next thing we wanted to discover is if IL-7 activated T cells

are able to activate B cells and how this would compare to healthy controls.

Our focus on future treatment lies in blocking elevated IL-7 levels or the receptor for IL-7 on

cells to restore immunological harmony and in order for us to incorporate results for possible future

treatments, we wanted and needed to know if and, if so, how the mechanism would differ from

healthy controls. If the mechanism would remain the same in patients as in healthy people and only

IL-7 levels differ, IL-7 blocking would be a perfect approach. However, if the mechanisms would show

a difference between healthy controls and patients, different approaches would be advised. Also,

despite the fact that the receptor for IL-7 might not be found on B cells, and in order to subscribe

possible activation to T cells, we needed to determine that IL-7 indeed does not activate B cells on its

own.

Blood was obtained from fifteen patients, diagnosed with pSS, and ten age and gender-

matched healthy controls. Their blood was studied for its lymphocyte (T cell and B cell) activation

upon IL-7 stimulation. A common way to determine the activation of certain cells is via the detection

of specific activation markers and co stimulatory-markers. Those markers will be up-regulated (i.e.

expressed at higher levels on the cells’ surface) and can then be labelled for detection via FACS

analysis. FACS analysis is a method to count the number of positive stained receptors on the cells’

surface. After three days of culturing (T cells [first activated with IL-7] + B cells and nutrition for the

cells in a Petri dish), the expression of activation-markers and co stimulatory-markers on T cells and B

cells from peripheral blood mononuclear cells (PBMC) and T/B co-cultures was assessed by FACS

analysis. PBMC are a cluster of lymphocytes that include the T cells and B cells, subtracted from the

blood. To further determine the level of activation, lymphocyte proliferation-assays were performed

four days after culturing. T cells and B cells that become activated tend to proliferate (multiply) and

during this process their DNA will be copied. DNA is like the blue print for building a copy cell. When

those cells are activated and proliferate, radioactive labeled tritium (that was added to the cultures)

will be incorporated and enables us to detect it (7).

As we hypothesized, high levels of the receptor for IL-7 on T cells in pSS patients were shown,

whereas B cells show no receptor for IL-7 . IL-7 stimulated T cells and B cells in PBMCs showed

elevated marker-expression on both of these cells in pSS patients. IL-7 induced activation of T cells

but not B cells in a T and B cell co-culture and was hampered compared to IL-7-induced activation in

PBMCs from pSS patients. This means that there are certain factors in PBMC that stimulate both cells

even more. Interestingly, co-cultures stimulated with a mix of IL-7 and TLR-7L (the ligand for TLR-7,

found on B cells) synergistically proliferated. We stimulated the B cells with additional TLR-7. TLR-7 is

a common part of a certain bacteria that triggers the B cells to become activated. So what this result

means is that when we culture IL-7 activated T cells and TLR-7L activated B cells together, their

activation is more than their individual summation. This means that these cells indeed interact upon

one another (7). We could not determine significant differences between the results from patients

and healthy controls, due to a lack of suitable controls and the low n number. Future research should

focus on increasing the n number, meanwhile facilitating more options to detect activation markers

like cytokines. All in all we found lots of evidence for the role of IL-7 in pSS, however more research

is needed to fully understand its function within pSS. Also, research on the effects of an IL-7 blocker

and the translation to in-vivo research is needed in order to facilitate a possible treatment. This

research paves the way for finding new ways for pSS treatments, however, there is still a long road to

take before therapies as such could be realized.

Conclusion To our knowledge, we were the first to show that IL-7 causes T cell and B cell activation of patients

with pSS. IL-7 activation of T cells and TLR-7 activation of B cells from healthy controls and pSS

patients synergistically increase proliferation of co-cultured T cells and B cells. Together these data

indicate that IL-7 contributes to the immune-pathology in pSS by causing T cell and B cell activation.

Blocking IL-7 or the receptor for IL-7 may provide for potential new therapies in the future, however,

more research is needed (7).

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