First weekly journal club presentation in lab. Used the format downloaded from school's website. :P Very informal presentation. I don't exactly thrive on presentations but I do enjoy them. #firstLabPresentation

1. Immune Signatures
in Lupus Patients
A Paper Review
Presented by –
Su Htwe Cho
Division of Medicine
Wolfson Institute for Biomedical Research
15/04/2016

2. Systemic Lupus Erythematosus:
Background
• Autoimmune disease
• Diagnosis - antibodies to dsDNA, ssDNA, phospholipids,
ribonucleoproteins, cardiolipins, SSA/Ro.
• Symptoms – marlar rash, rashes, arthritis, lupus nephritis, fever,
malaise, etc.
• Two types – discoid & systemic, due to ICs depositing in skin, joints,
kidneys, etc.
• Disease activity differs between individuals, unpredictable
• Periods of remission & flares intermittently (cumulative damage)

3. • DAI – using any of 6 composite scores (SLEDAI, BILAG, ECLAM, LAI, SLAM, SLAQ)
• Classification (of SLE) – 4 out of 11 criteria (85% sensitivity, 95% specificity)
1. Serositis - Pleurisy, pericarditis on examination or diagnostic electrocardiogram (ECG) or imaging
2. Oral ulcers - Oral or nasopharyngeal, usually painless; palate is most specific
3. Arthritis - Nonerosive, 2 or more peripheral joints with tenderness or swelling
4. Photosensitivity - Unusual skin reaction to light exposure
5. Blood disorders - Leukopenia (< 4 × 10 3
cells/µL on >1 occasion), lymphopenia (< 1500 cells/µL on >1 occasion),
thrombocytopenia (< 100 × 10 3
cells/µL in the absence of offending medications), hemolytic anemia
6. Renal involvement – Based on presence of proteinuria (>0.5 g/day or 3+ positive on dipstick testing) or cellular casts
(including red blood cells [RBCs], hemoglobin, granular, tubular, or mixed) [88]
or based on the opinion of a
rheumatologist or nephrologist [88]
7. Antinuclear antibodies (ANAs) - Higher titers generally more specific (>1:160); must be in the absence of medications
associated with drug-induced lupus
8. Immunologic phenomena - dsDNA; anti-Smith (Sm) antibodies; antiphospholipid antibodies (anticardiolipin
immunoglobulin G [IgG] or immunoglobulin M [IgM] or lupus anticoagulant); biologic false-positive serologic test
results for syphilis, lupus erythematosus (LE) cells (omitted in 1997 revised criteria)
9. Neurologic disorder - Seizures or psychosis in the absence of other causes
10. Malar rash - Fixed erythema over the cheeks and nasal bridge, flat or raised
11. Discoid rash - Erythematous raised-rimmed lesions with keratotic scaling and follicular plugging, often scarring
SystemicLupusErythematosus:
Criteria&DAI(MedScape,Sep2015)

4. • (paeds) 158 patients, 48 HC, 924 visits, 1412 days, 24 components of
SLEDAI
• Sample – 2:1 ratio
• Treatments –
• No Treatment (NT)
• Hydroxychloroquine (HC)
• Oral Steroids (OS +/- HC)
• Mycophenolate mofetil (MMF +/- HC/OS)
• Cyclophosphamide &/or methylpred. IntraVenous +/- HC/OS
Study Trial Design:

5. Whole Blood Fingerprint
• Global SLE Signature
• all samples compared (15386 transcripts)
• Prevalent IFN signature (784 of 924 samples – 84.8%)
• Linear Mixed Models – fixed disease effects & random effects
• Differentially Expressed Transcripts (DETs)
• Modular analysis – 260 modules (co-expressed, var. Immunological
conditions)
• Sorted cell pop. – two public data-sets
• SLE modular fingerprint (IFN, neutrophil, plasmablast)

6. Plasmablast Signature:
• Plasmablast – source of anti-dsDNA antibodies
• Reduced by ALL therapies, but most sig. by MMF and CIV
• MMF – inhibition of inosine 5’-monophosphate: mature B cells –
plasma cells
• Inclu. race & treatment in the model – 3501 DETs
• DA1 (SLEDAI: 0-2) , DA2 (SLEDAI: 3-7) , DA3 (SLEDAI >7)
• DA3 : DA1 – 486 transcripts (383:DA, 103 neg related with DA)
• Ethnicity effects – African-American
• higher plasmablast signatures: higher titre of anti-dsDNA & higher SLEDAI
• Anti-dsDNA titre: response to B cell depletion therapy
• Serum BAFF titre: response to anti-BAFF treatment

7. • Quantitative Set Analysis for Gene Expression (QuSAGE) – blood
modules as gene sets
• Pos transcripts – enriched for (IFN, plasmablast, cell-cycle,
neutrophil, histone, B cell modules)
• Plasmablast signature reproducibility > IFN response reproducibility
• Transcripts neg. correlated with DA – enriched for (NK
cell/cytotoxicity, protein synthesis, erythropoiesis)
• Genes found
• IFN-regulated (IFI6, IFI27, IFI27L1, DDX60L, SIGLEC1)
• Histone ( HIST2H2AAR, HIST2H4B)
• B-cell related (STAP1, LOC652126)
Plasmablast Signature

8. Race: A Contribution factor to DA
• DA of African-americans > DA of Caucasians
• Comparison & Analysis –
• Transcriptomes of Other Races VS (African-americans, Caucasians, Hispanic)
• 444 DETs
• Functional interpretation – enrichment of
• plasmablasts, cell cycle, erythropoiesis in AA
• neutrophil, myeloid lineage, inflam.-related molecules in Hispanics & Caucasians
• Comparisons of SLEDAI, anti-dsDNA Ab, C3, ESR –
• > SLEDAI, >ESR, >C3, >anti-dsDNA Ab in AA

9. Effect of Therapy on Blood Signatures
• Treatment – NT, HC, OS, MMF, CIV
• 622 DETs
• HC only: enrichment signature similar to NT
• OS & CIV: > neutrophil signature, > circulating neutrophils
• All treatments: < plasmablast signature
(strongest – MMF & CIV)

10. Lupus Nephritis, IFN signature, Treatments
• Neutrophils – IFN-primed, release IFN-genic DNA &
pro-inflam cytokines
• MMF Treatment
• extinguishes neutrophil sig. In Primary LN but not
Membranous LN
• might target upstream events in Primary LN (eg.
Plasmablast-derived auto-antiAb activating pDC &
neutophils)
• Combination Therapies might be better - with MMF
alone, pro-inflam. signatures (IL1A, IL1B, IL6R)
persisted
• > total & activated CD62L-low circulating neutrophils

11. • Correlation Test: DA & SLEDAI
• Five SLEDAI groups – No SLEDAI parameters (none) VS
• Alterations in serum parameters only (serology)
• Connective tissue +/- serology (skin/musculo-skeletal)
• Kidney +/- serology (renal)
• All combined (global)
• 3rd
Model: inclu. Treatment
• All comparisons – IFN, plasmablast, B cell modules
• Renal & global components – neutrophil, myeloid lineage, inflam. modules
Neutrophil Signature in Lupus Nephritis

12. Nephritis Classes, Treatments & Transcripts
• LN – six major types (I to VI)
• Severe classes – Proliferative (PLN, III & IV), Membranous (MLN & V),
Combinations (VI)
• Treatment – MMF (PLN + MLN)
• 4th
Model: inclu. Treatment + nephritis class
• Groups: no LN (I), mesangial nephritis (II), PLN (III & IV), MLN (V)
• Comparisons:
• NT-noLN VS NT-PLN (group 1)
• MMF-noLN VS MMF-PLN/MLN (group 2)
• MMF-PLN VS MMF-MLN (group 3)
• Results:
• Group 3 – no difference

13. • In silico Ingenuity Pathway Analysis (IPA) –
• MMF-PLN, MMF-MLN
• MMF-PLN
• Over-expression (FcRs, CR1, IL1B, IL6R, CTSC) – myeloid
lineage & inflam.
• Under-expressed ( CD19, EBF1, E2F5, GAS6, ADARB1) –
B cells & plasmablast
• MMF-MLN
• Over-expression (DEFA1, DEFA4, CAMP, RNASE2, LTF) –
activated neutrophils
• Over-expression ( MHC class 1, IRF9, PSMBs, OASL,
TRIM22) – IFN response
Nephritis Classes, Treatments & Transcripts

15. Personalized SLE Transcriptional ImmunoMonitoring
• Weighted gene co-expression network analysis (WGCNA) – individual focused
• Module/trait-correlation matrix (patient-specific)
• Module best correlated with SLEDAI was selected (SLEDAI-WGCNA module)
• EXAMPLE
• Patient: African-American, Female, Code: SLE-55
• 798 days – 13 times immuno-monitored
• 2 flares, > anti-dsDNA Ab & > neutrophil (both), > ESR (2nd
flare)
• Individual-specific 41 modules
• SLEDAI & Neutrophil % : myeloid lineage, inflam., IFN response
• Anti-dsDNA Ab titres : plasmablast, cell cycle, IFN modules
• ESR : erythropoiesis

17. • 80 patients (>= 5 visits) – 7 groups
• Groups based on five immune signatures
• SLEDAI, erythropoiesis, IFN response, myeloid
lineage/neutrophils, plasmablasts, lymphoid lineage
• PG3 – plasmablast modules, PG5 – IFN response &
myeloid lineage
• Positive Control – WGCNA module (correlate with
neutrophil %)
• eQTL – expression quantitative trait loci analysis
• 126/362 (35%) SNPs were acting in cis with an IFN-
inducible gene
**Patient Groups (Transcriptional correlates of SLEDAI)

18. • 80 patients (>= 5 visits) – 7 groups
• Criteria – patients with shorter follow-up’s
• 11457 transcripts (80 WGCNA SLEDAI modules)
• 149/3011 transcripts (erythropoiesis)
• 40/3011 transcripts (IFN response)
• 163/3011 transcripts (myeloid
lineage/neutrophils)
• 9/3011 transcripts (plasmablasts)
• 436/3011 transcripts (lymphoid lineage)
**These modules and stratification could be used on other patients as
well**
Molecular Stratification (Transcript Panel)

19. • Methods :
• WGCNA + SLEDAI => SLEDAI WGCNA
• Correlation matrix: SLEDAI WGCNA (y axis), whole blood (x axis)
• SNP analysis
• Benefits:
• Follow clinical traits overtime
• Analyze the profile and content of each WGCNA module
• Identify modules & transcripts best correlating with clinical traits
• Identify major module hubs through module membership quantification
• Result:
• Informed Personalized Therapeutic Interventions
Benefits of PSTIM

20. Conclusion
• Plasmablast signature – the most robust biomarker
• Neutrophils – active nephritis
• Patients stratified into groups based on IFN & plamabast sig.s that correlated
with DA -> immuno heterogeneity (Personalized Immuno-monitoring)
• Transcriptional sig. in groups– supported by SNP analysis (specific associations)
*SNP may affect neighbour genes’ expression*
• Pathways involved in familial SLE - immature B cell survival, early complement
cascade components, regulation of IFN production, and defects in cytoplasmic or
extracellular DNA degradation
• Group level sig. (IFN, Plasmablast) – failed in individual level (DA corr.)
• Failed to ID flare predictors – require standardized patient sampling

21. References
Romain Banchereau, Seunghee Hong, Brandi Cantarel, Jose Rossello-Urgell, Tracey Wright, Virginia Pascual. Personalized
Immunomonitoring Uncovers Molecular Networks that Stratify Lupus Patients. Cell 2016, 165 (1–15) Elsevier Inc.
Christie M. Bartels, Daniel Muller. Systemic Lupus Erythematosus (SLE) Workup [Internet]. Medscape 2016. Available from
[http://emedicine.medscape.com/article/332244-workup]
Thank You! It’s Q & A Time!