Loss of balance between protective and proinflammatory synovial tissue T-cell polyfunctionality predates clinical onset of RA

Written by Achilleas Floudas, Declan Sweeney, Tracy Madigan, Maryanne Murphy-Lyons, Ursula Fearon

The window for effective therapeutic intervention in rheumatoid Arthritis (RA) is limited. Additionally, current T cell specific therapies for the treatment of RA are broad and affect all T cells irrespective of their contribution to disease pathogenesis and progression, therefore, not differentiating between protective and pathogenic T cell responses. 1-2 More refined and targeted therapeutic approaches will improve disease progression while limiting unwanted side-effects and toxicity. Importantly, characterisation of T cell responses in the synovial tissue of at-risk individuals will help identify prognostic markers and extend the window for effective therapeutic intervention. Ex-Th17 T cells with the capacity to produce multiple pro-inflammatory cytokines simultaneously have previously been identified in the synovial fluid of patients with RA, these polyfunctional T cells are also resistant to inhibition by regulatory T cells (Treg). 3-4

Our understanding of polyfunctional T cells that share characteristics with multiple T cell subsets and transcend previous dogmas of well-defined polarised T cells has increased significantly, however, several key questions remain unanswered. While, the implications of polyfunctional T cell responses extend beyond autoimmunity, response to pathogens, anti-cancer responses and encompass vaccine development, characterisation of polyfunctional T cells is technically challenging and requires new data exploration, visualization and analytical approaches. 5 Polyfunctional CD4 T cells are molecularly distinct from monofunctional cells however, several questions regarding the temporal cytokine production dynamics, transcriptional control and stimulation requirements of polyfunctional T cells remain unanswered. 6 Early studies suggest T cell polyfunctionality is a result of sequential cytokine release with T cells obtaining polyfunctionality status gradually and maintaining simultaneous cytokine secretion for a short period of time. 7 SARS- CoV-2 specific T cell IL-2, TNF-α and IFN-γ cytokine polyfunctionality is dependent on viral antigen specificity and contributes to viral clearance. 8 Importantly, T cell receptor (TCR) dependent and TCR independent stimulation resulted in similar cytokine secretion patterns over time, suggesting that both TCR mediated signals and bystander T cell activation could be important for the emergence of polyfunctional T cell states. 7

Recent studies have highlighted the importance of deleterious polyfunctionality in autoimmune inflammation. Highly polyfunctional T cell responses have been identified in the synovial tissue of psoriatic arthritis patients and their frequency correlates strongly with disease severity. 9 Increased T cell plasticity across multiple T cell subpopulations and polyfunctionality of Th cells have recently been identified in children with Down syndrome associated arthritis, an aggressive form of erosive polyarticular inflammatory arthritis. 10

Despite accumulating evidence regarding the importance of T cell polyfunctionality in autoimmunity, little is known about the potential contribution of polyfunctional T cells to RA disease progression and the timing of their emergence in relation to the onset of clinical inflammation. Additionally, polyfunctional T cell responses can be highly heterogeneous raising the potential for immunoregulatory as well as proinflammatory states of polyfunctionality. 11 We have recently performed extensive characterisation of peripheral blood and synovial tissue T cell subpopulations with emphasis on polyfunctional T cell responses in RA patient, IAR and HC synovial tissue biopsies. Synovial tissue RNAseq analysis with pathway enrichment analysis, revealed an enrichment in T cell activation and differentiation pathways prior to clinical inflammation. Importantly, we have characterised synovial polyfunctional T cell responses that pre-date the clinical onset of RA with distinct and potentially protective polyfunctionality in HC synovial biopsies. In addition, we identified highly polyfunctional synovial CD4+CD8 dim T cells that correlate with disease severity in RA patients.

While the influencers of synovial tissue polyfunctionality remain elusive, the enrichment of common T cell activation and differentiation pathways in IAR and RA patient synovial tissue biopsies indicate their potential as candidate regulators of polyfunctional T cell responses. Further characterisation of the synovial pathways identified could lead to the development of methods for the therapeutic manipulation of pathogenic polyfunctional T cell responses.

T cell polyfunctionality contributes early in the pathogenesis of RA and raise the possibility of future therapeutic interventions targeting CD4+CD8dim T cells and the balance between protective and pathogenic polyfunctional T cells.

Why challenging previous dogmas in rheumatology research is important

Collaborative, interdisciplinary research is the only way to increase our understanding of the immunological mechanisms that underline synovial inflammation in order to bring more effective, targeted treatments to the patients. Patients have to be in the centre of research as partners, guides supporters and guardians of the path to actionable knowledge. Below is but a glimpse of evidence that support efforts in challenging current dogmas of immunological research in order to refine the currently “exploratory” treatment regimens of arthritis.

My Arthritis (RA) Journey (by Declan Sweeney)

The first week in September 2014 was like previous years, new school bags and lunch boxes for the boys, the usual assortment of ‘window envelopes’ dropping through the post-box, but against that Manchester City had won the Premiership, and I had managed to knock a couple of shots of my handicap! Life was good, what could possibly go wrong?!

Later that week, I became aware of my neck being quite stiff, and over the next number of weeks found it increasingly difficult to get out of bed in the morning. After numerous blood tests, and X- Rays at our nearest Hospital, I was advised to see a Rheumatologist.

The first meeting was a bit of a blur, trying to process the words “I’m afraid you have Rheumatoid Arthritis, which is going to get worse” He wasn’t joking, and so began my long downward journey into a World of acute pain, frustration, and false dawns which was to last for over four years.

What you soon discover with RA is that unlike an infection where you are prescribed an antibiotic and recover, RA drugs are a completely different kettle of fish! Finding the right drug to suit you is like playing a game of ‘lucky dip’! In my case I was not so lucky, as it takes some three months to determine whether the drug in question is working, and all this time your joints, particularly your hands and feet are swelling to a point where you cannot even hold a toothbrush.

It’s hard to convey the change it brings, not just for the individual but for the whole household. Laughter is in short supply, my three boys became withdrawn and my wife took on the role of carer, helping me to dress, and driving me to Hospital for three-day stints hooked up to a drip to control the inflammation. By this stage it had become so acute that my Rheumatologist was injecting each space between my fingers and toes.

Over the next four years, I was prescribed a plethora of strange sounding drugs, such as Methotrexate, Enbrel, Rituximab and numerous others, coupled with large doses of prednisolone increasing my normal weight from 83kg to over 100kg. Thankfully in 2018 I was prescribed Orencia, which I self-inject weekly, along with other RA drugs and a reduced dose of steroids.

This has been Life changing, and has enabled me to lead a somewhat normal life again, returning to golf, swimming and cycling. With the love and support of my Family, I have managed to stay mostly positive throughout this difficult period. Indeed, even with the occasional ‘flare up’, I take one look at the plight of the Ukrainian people and realise just how lucky I am!

The end user RA (by Tracy Madigan)

In product development there was always a process or roadmap that was followed:

  1. Decide what the end user needs
  2. Design the end product
  3. Create the end product
  4. Test the end product
  5. Release the product to the end user

Only at step number 5 was the end user ever really involved in the product. Now swap the words for end user for patient and product for drug. This roadmap is familiar in lots of industries and many of these are either changed or changing. Why?

When the lifecycle of any product starts with all stakeholders investing their time, experiences and skills the project will always be more successful.

If you can harness the skills and experiences of the right patients at the very beginning you will find that there is power in people, especially in people so invested in the success of the product. We may be patients in the clinical sense but we are also accomplished and successful people who manage all sorts of challenges everyday and while we may not understand the full scope of the “science bit” you have no idea what we can offer along a long journey where the outcome is unknown. For the researchers I feel that the emotions and experiences we offer provide perspective, focus and a more patient orientated end product. Being involved gives us hope for future generations and a feeling that there is value in our experience.

Arthritis (RA) as a part of my life (by Maryanne Murphy-Lyons)

The birth of our third child in October 2015 was a very exciting time for my husband Niall and I; our little boy Harry was welcomed adoringly into our family by our two other children James and Sarah who were aged six and two respectively. Harry was a great baby, he was instantly very good at breastfeeding and sleeping, almost perfect! Unfortunately, within four to six weeks of Harry’s birth, I started to feel unwell. While I was familiar with the usual post-partum aches and pains and tiredness that are common with a newborn baby, this was different and unrelenting.

Every day I was noticing something different, swollen knee, ankle, my hands were feeling hot and swollen. I was noticing the stiffness was getting worse and increasing every day at an alarming rate. I made an appointment with my GP and I mentioned that I was feeling unwell. He was extremely attentive and showed great care to me as I cried, explaining that I was feeling so awful and was struggling with lifting and holding Harry. I didn’t understand why he was testing me for the rheumatoid factor at that time.

My GP rang me a few days later to inform me that my blood results were clear and that the rheumatoid factor in my blood was negative. But my symptoms of hot, painful swollen joints and joint stiffness continued. My GP said to come back to him in one week, which I did. It was almost Christmas and l was feeling more and more anxious and in a lot of pain.

In the following weeks, lots of family and friends came to help me manage baby Harry and my other two children. I consulted with various alternative therapists to try alleviate my symptoms, acupuncture, reflexology, osteopathy to name a few! Indeed more than one person suggested to me that stopping breastfeeding would help my joints and alleviate my symptoms. Thankfully, I did not do this. I consulted with Dr Jack Newman, a world-famous expert in Canada on breastfeeding, who absolutely reassured me that I could continue breastfeeding, despite my joint pain and stiffness.

I started to ‘google’ more on serum negative arthritis and started to realise that my GP was worried I had rheumatoid arthritis. I reassured myself that this was not the case as my blood test was negative and I had no family history of RA. I became determined not to have rheumatoid arthritis! Nonetheless, I was struggling to manage the pain and stiffness in my joints, while looking after Harry and my other two children.

My appointment with a rheumatologist a few weeks later confirmed exactly what my GP had suggested to me, that I had developed rheumatoid arthritis. He explained that the rheumatoid factor could be negative; I did not know that you do not need to have a family history of RA to develop it. The rheumatologist also explained to me that women are two to three times as likely as men to develop RA and that post-partum onset is not uncommon.

Reassuringly, he advised me that I could continue breastfeeding. He prescribed medication for me called a DMARD. I found the following weeks very difficult, as I tried to accept that I had RA. I continued to try lots of alternative therapies and alternative diets and I didn’t start the medication that was recommended. I know now that this is not advisable and there is little scientific evidence to support the effectiveness of alternatives like herbs or acupuncture and strict diets for RA. I found it difficult to cope and worried so much all the time about my future living with this condition.

I sought an opinion of another rheumatologist during this time and he confirmed what my GP and the first rheumatologist advised about my post-partum onset RA. I agreed after this consultation to commence the recommended medication and a course of short term steroids. During this period of time, we discovered to our surprise and delight that, we were having another baby.

Pregnancy affects people with autoimmune disease differently and RA is no exception to this. Many people enjoy full periods of disease remission during pregnancy. My symptoms improved greatly during my early pregnancy and I felt better than I had in months! However, remission from my RA did not last long and my symptoms slowly started to return. My fears and anxieties about RA caused a lot of distress to me at this time and I was very worried about having another baby and coping with the pain and stiffness and a post-partum flare-up. While pregnancy can give short-term remission from active disease in RA, post-partum flare-ups are very common.

During this time, I was referred to Prof. Doug Veale and Louise Moore, an Advance Nurse Practitioner in rheumatology in Our Lady’s Hospice Harold’s Cross, both of whom have a special interest in rheumatology and reproductive health. This referral was hugely important as it was the beginning of a turning point in the management part of rheumatoid arthritis.

I was 30 weeks pregnant and was very worried about coping with another new baby and RA. However, with some education and consultation with Professor Veale and Louise Moore, I was reassured that post-partum flare-ups can be very well managed and I found reassurance that she would be in touch me four to six weeks after my baby’s birth. My RA symptoms were managed with a low dose steroid for the rest of my pregnancy.

Samuel was born just in time for Christmas! We had a wonderful busy Christmas with our little new baby boy.

However, as expected within four to six weeks of Samuel’s birth, I started to have a post-partum flare-up. I was in a much better place psychologically this time as I had the support I needed and I was not in as much pain due to the medication. I was only taking steroids at this time which offered a temporary relief from RA symptoms and are not a long-term solution to managing RA.

When Samuel was about five months, I was finished breastfeeding him and my symptoms of RA were still active. The following eight to nine months were difficult, as I tried various combinations of medication to manage my RA. There are a number of different medications available to manage RA and it can be trial and error to find the right combination. It can also take a number of months to see the benefit of the medications. Unfortunately, side effects are also common and it is a careful balance of medication and side effects that make the process of finding the correct medication important. What I have leant from that process as a patient is that you must be compliant and cooperative and be an active participant in your own care.

This is something that took me a while to learn, as I was not the most compliant patient and this did not help managing my RA. Keeping a diary of your symptoms, medication benefits and side effects all assist your doctor/nurse to provide you with the best care possible. I also found during this time that it is important to develop a relationship with your pharmacist, as they are another source of information and support. Professor Veale and Louise Moore were very knowledgeable and up to date in the range of medications available to manage Rheumatoid Arthritis and were able to prescribe a biologically medication that suited me.

I have always been a very active person and was used to exercising, but I started exercising more frequently as I found it helped my joint stiffness and also helped my coping skills. After a number of months and a couple of different medication changes, a suitable medication was found that suited me and alleviated my symptoms with minimal side effects. I was able to slowly wean-off my steroids. During this period, I also attended other professionals such as a physiotherapist and occupational therapist. This was also a very important step for me, they taught me invaluable skills and shared great insights with me on managing pain, stress and fatigue, all common and reoccurring symptoms of RA.

As I learnt more about RA, I grew more confident in managing my life with the condition, as opposed to viewing it as something separate. Becoming an active participant in your own care is very important and, in my view, it has been one of the key factors in me living well with RA. It does take some energy and emotional management and I do still get ‘tired’ of RA, but I no longer fear it and when I am in periods of pain or fatigue I can recognise these symptoms and self-manage them.

Becoming an active participant as opposed to a passive recipient in my own care gave me the confidence to learn my self-management skills. Exercise, living a healthy, balanced, but busy life with work and four young children and my RA are the norm for me now!

RA no longer impacts my life in the way it used to, it is now just part of my life.

A. Example of synovial tissue biopsies isolated by arthroscopic surgery.

B. SPICE algorithm visualization of synovial tissue CD4 T cell cytokine expression and frequency of polyfunctional CD4 T cells for Healthy controls, individuals at risk (IAR) and patients with Rheumatoid Arthritis (RA).

C. Enrichment plots of RNAseq data pathway analysis for significantly upregulated gene pathways for IAR vs HC, Dot size represents number of differentially regulated genes per pathway, color intensity represents significance and x axis is indicative of the pathways fold enrichment change. D. Representative imaging flow cytometry of Rheumatoid Arthritis patient synovial fluid CD4+, CD8+ and CD4+CD8+ DP T cells. F. FLIM images and cumulative data of RA patient flow sorted peripheral blood CD4+ and CD4+CD8+ DP T cells.

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