Immune Modulation

By Valerie Chew (Team Lead), Lee Yun Hua, Lim Chun Jye, Phuong Nguyen H, Samuel Chuah

Despite recent success in cancer immunotherapies, the dynamics between tumor-microenvironment (TME), tumor cells and the systemic immune system remain elusive. Our immunological analysis aims to identify and characterise important immunological components in hepatocellular carcinoma (HCC) for their potential clinical implications. Our approach consists of seamless integration of high dimensional mass cytometry by time-of-flight (CyTOF), Next-generation sequencing and multiplexed immunohistochemistry (Vectra) analysis to identify and understand the multiple immune subsets residing in the tumor, its adjacent non-tumor liver tissues and peripheral blood in patients with HCC. We recently described an immune gradient of progressively more suppressive immune subsets from the periphery, the adjacent non-tumor tissues to tumor¹. Exhausted and immunosuppressive immune subsets such as T cells with higher expression of PD-1, CTLA-4, Lag-3 and Tim-3; the regulatory-T cells; and tumor-associated macrophages demonstrated preferential enrichment within the TME. Further functional and transcriptomic characteristics of these cells showed that their phenotypes are profoundly related to the microenvironment where they reside. Likewise, HCC tumors with different etiologies, i.e. those with chronic hepatitis B infection versus non-viral-related HCC, showed distinct tumor microenvironments². Importantly, the immunomics in the peripheral blood could serve as a powerful biomarker to predict HCC patients who will response to selective internal radiation therapy (SIRT)³. The current approach provides a holistic analysis and mapped the immune landscape of tumor in unprecedented detail. It serves as a platform for the design of immunomonitoring and immunotherapy for cancers.

References:
1.Chew, V. et al. Delineation of an immunosuppressive gradient in hepatocellular carcinoma using high-dimensional proteomic and transcriptomic analyses. Proceedings of the National Academy of Sciences of the United States of America 114, E5900-E5909 (2017).

2. Lim, C.J. et al. Multidimensional analyses reveal distinct immune microenvironment in hepatitis B virus-related hepatocellular carcinoma. Gut (2018).

3. Chew, V. et al. Immune activation underlies a sustained clinical response to Yttrium-90 radioembolisation in hepatocellular carcinoma. Gut (2018).

By Leong Jing Yao (Team Lead), Lai Liyun, Phyllis Chen

The embryonic development of the immune system is a finely controlled and calibrated process. We seek to understand, in particular, how the ontogeny of T cells occurs across early and mid gestation in healthy foetus. Currently we are performing high-dimensional interrogating of single cell phenotyping with CyToF on fetal circulatory and tissue samples. We have observed parallel and redundant control in both T cell effector and regulatory compartment across the gestation period and anatomical sites. Translational insights into T cell development could address challenges experienced in in-uterine stem cell therapy, where fetal rejection is an issue. The gestational therapeutic window and the level of T cell immune-competence is pertinent to understanding how therapeutic modulation can be better achieved.

By Yeo Kee Thai (Team Lead), Amanda Lim, Camillus Chua, Leong Jing Yao, Martin Wasser, Poh Su Li, Sharifah Nur Hazirah, Yeo Joo Guan

Preterm infants’ immune system exhibits distinctly different, rather than deficient, function compared to term infants. There is a current lack of understanding on how the immune system of preterm infants develop and how antenatal and postnatal events may shape immune development. Understanding this complex development of the preterm immune system will require a holistic understanding of the preterm immunome of at birth and in early life.
We have developed a biobank of maternal, infant and placental specimens and we are using the immunomics platforms developed at TII for high-dimensional interrogation of the preterm immune system in order to elucidate key cell subsets and important pathways that are important in this process.

By Bhairav Paleja (Team Lead), Ahmad Lajam, Camillus Chua, Guo Dianyan, Lai Liyun, Nursyuhadah Sutamam, Sharifah Nur Hazirah

Kidney transplantation significantly improves the survival of end stage renal disease patients. The outcome after kidney transplant have been shown to be affected by race and ethnicity. In the United States, long term allograft survival rates are highest among Asian recipients, followed by Caucasians and lowest for African-Americans. Both, immune and non-immune mediated factors have been accounted for these ethnic discrepancies. African-Americans have been shown to have higher frequencies of lymphocytes and exhibit higher levels of cellular immune response than other ethnic groups. In comparison, Asian recipients show low incidence of acute or chronic rejection but higher rates of death due to infection after kidney transplant. This suggests significant differences in immunological phenotype between Asian and other races. In the current study, we will perform immune profiling of kidney transplant recipients from different racial populations, including Chinese from Singapore and Caucasians/African-Americans from the United States.

By Bhairav Paleja (Team Lead), Ahmad Lajam, Camillus Chua, Lai Liyun, Pavanish Kumar, Sharifah Nur Hazirah

Osteoarthritis (OA) is a degenerative multifactorial joint disease and a significant cause of joint pain and disability. Age, joint trauma and obesity have been identified as major risk factors for OA. Although traditionally OA has been considered a pathological response to abnormal joint loading and mechanics, the involvement of low-grade inflammation is now recognised as a common finding. However further thorough understanding of the immune mechanisms involved is needed for unravelling pathogenesis and yield therapeutic targets that can modify the course of OA. In the current study, we aim to explore the immunological signatures in defining inflammatory phenotypes of knee OA using high throughput transcriptomics and immunomics approach.

​By Bhairav Paleja (Team Lead), Ahmad Lajam, Camillus Chua, Lai Liyun, Pavanish Kumar, Sharifah Nur Hazirah

Systemic sclerosis (SSc) is a multisystem connective tissue disease characterised by excessive extracellular matrix deposition, microvasculopathy and autoimmunity. Excessive collagen accumulation results in fibrosis of several organs such as skin and lungs. Pulmonary arterial hypertension (PAH) and interstitial lung diseases are major causes of death in SSc. The extent of organ involvement, severity of disease, progression of disease and prognosis is heterogenous in each patient. This necessitates identification of biomarkers that can help in diagnosis and prognosis of the disease. Furthermore, identifying biomarkers will also aid in understanding of disease pathogenesis in SSc. A variety of immunological abnormalities of T and B cells have been detected in SSc, however there is lack of systematic studies of functional relations between immune cell subsets and their role in pathogenesis of SSc. This lack of mechanistic knowledge hampers targeted intervention. The current study aims at an in depth analysis of systemic immune composition in SSc, using high dimensional mass-cytometry based immunophenotyping, with the potential to delineate mechanisms of pathogenesis and identify diagnostic and/or therapeutic targets.

​By Leong Jing Yao (Team Lead), Camillus Chua, Pavanish Kumar, Phyllis Chen, Sharifah Nur Hazirah, Yeo Joo Guan

We have previously published and identified two dichotomous dysregulated populations within the circulating Teff (CPLs) and Treg (iaTreg) compartments in juvenile idiopathic arthritic patients, that are inflammatory, antigen experienced, correlating with disease activity and exhibiting similar TCR oligoclonality with synovial T cells. Their commonality in immune-phenotype despite being in two functionally distinct compartments, signal the possibility of a common pathogenic origin during active disease manifestation. In this study, we seek to determine the common micro-environment transcriptomic drivers influencing dysregulation in both Teff and Treg compartments during active disease in JIA patients.

By Yeo Joo Guan (Team Lead), Camillus Chua, Poh Su Li

The pathogenesis of Systemic Lupus Erythematosus (SLE) involves multiple derangements that perturb the fine balance between immunity and regulation. Traditional investigational approaches focusing on the pathogenic or protective role of individual cell type or molecule independently are inadequate for the study of a complex, multifactorial disease like SLE. The lack of a holistic understanding of the lupus immunome is a critical unmet need. Simultaneously, there are other important knowledge gaps pertaining to the role of B regulatory cells in SLE where there is a lack of specific cell surface and transcriptional factor markers akin to the forkhead box protein P3 (Foxp3) found in T regulatory cells.

The over-arching hypothesis is that abnormalities in multiple components of the immune system contribute to lupus pathogenesis. Specifically, we hypothesise that the immune regulatory component is dysfunctional (both quantitatively and qualitatively) during lupus flare. To address such unmet needs, we aim to use a high dimensional approach using mass cytometry to unravel the contribution of the regulatory and inflammatory immunome to lupus pathogenesis simultaneously. This will be achieved with the characterisation of 37 immune markers at the single cell level using mass cytometry, followed by an unbiased and unsupervised analysis with a machine learning custom software based on dimensional reductions followed by automated cells classification and clustering. Subsequently, hierarchical prioritisation of the cell populations based on its strength of association with disease activity for downstream mechanistic characterisation with fluorescence based flow cytometry sorting of the target cells of interest for full transcriptome analysis and in-vitro functional study will be done.

This approach has the immediate dual translational potential of identifying immune cell subsets relevant for clinical prognostication and the elucidation of disease mechanisms which will subsequently provide important information for the development of a multi-pronged immunotherapeutic strategy against SLE.

​By Leong Jing Yao (Team Lead), Lai Liyun, Phyllis Chen

The afflicted joints of juvenile idiopathic arthritic patients are characterised by high infiltration and expansion of a restricted T cell clonatype repertoire. This antigenic driven process limits the affliction to specific joints. It is unknown whether the presence of multiple affected joints within an individual are underscored by a common disease mechanism. We have in collaboration with Femke’s lab in Utrecht University, worked on the immunological phenotyping of disparate joints within each individual to answer this question. CyToF results indicate common immunological processes in the joints of the same individual and driven by a common antigenic force.

By Leong Jing Yao (Team Lead), Camillus Chua, Lai Liyun, Pan Lu, Phyllis Chen, Sharifah Nur Hazirah, Yeo Joo Guan

Juvenile idiopathic arthritis (JIA), the most common cause of childhood chronic arthritis, comprises of 7 distinct subtypes classified by the number of joints involved, the presences of systemic or cutaneous involvements, HLA-B27 or autoantibody (rheumatoid factor, RF). Despite the significant therapeutic advancement made with biological Disease Modifying Anti-Rheumatic Drugs (DMARDs) such as anti-TNFA, leading to dramatic improvement in disease outcomes, JIA etiology remains largely unclear. However, in recent years, the field has witnessed not only advances in our understanding of JIA pathogenesis, but also attempts to address several clinical unmet needs.

In particular, one core issue is in relation to therapeutic management and disease resolution with anti-TNFA. Up to 50% of patients with JIA achieve clinical remission with long-term anti-TNFɑ treatment. While short-to-medium term treatment is well tolerated, long-term anti-TNFɑ therapy can elicit serious adverse effects. Drug withdrawal in patients who attain clinical remission is further complicated by the fact that 50-80% patients relapse upon therapy discontinuation. This phenomenon indicates that a substantial proportion of patients who attain clinical remission on medication, continue to experience subclinical inflammation and persistence of disease. Conversely, patients who achieve disease resolution at the clinical and subclinical levels could be spared long-term drug effects. As such, there is a clinical need to address how discontinuing anti-TNFɑ therapy can be safely implemented, and a scientific need to understand how disease persistence or resolution occurs.

By Pavanish Kumar (Team Lead), Amanda Lim, Bhairav Paleja, Camillus Chua, Sharifah Nur Hazirah

Epilepsy affects more than 50 million people worldwide and onset typically occurs in childhood. Epileptic seizures are initiated unprovoked in affected patients, arising from uncontrolled electrical activity in the brain. Conventional anticonvulsants that largely target ion channels only improve seizure control and cannot directly prevent epileptogenesis. Refractory epilepsy, or Drug-refractory epilepsy (RE) is defined as a failure of 2 appropriate and adequate anti-epileptic drugs to control seizures. Drug-refractory epilepsy (RE) occurs in 30% of epilepsy cases. RE is associated with notable cognitive, behavioural and socio-economic difficulties. Autoimmune encephalopathy (AIE) is an increasingly recognized cause of encephalopathy in children. AIE is often triggered by infections, and patients develop seizures as part of their clinical presentation. Some AIE disorders are associated with pathogenic neuronal auto-antibodies. Patients who recover from AIE often remain affected by epilepsy, which in many cases is refractory. Although AIE responds well to immunotherapy and children with epileptic encephalopathies and refractory seizures are treated with steroids, there is little evidence for the efficacy of corticosteroids in RE as a whole. The mechanism of action for corticosteroids in AIE is unclear; the therapeutic effects are postulated to be due to direct neuronal effects, metabolic changes and immune-modulation or suppression. Here we are working to unravel the specific immune mechanism that contributes to refractory epilepsy pathogenesis. Deep understanding of the immunome in patients from refractory epilepsy will lead to development of better diagnostic, prognostic and therapeutic tools.