Exploration of the Peripheral Immune System in Subjects With New Onset T1 Diabetes (NOT1D)
Phase: NA
Conditions: Diabetes Mellitus, Type 1
Sponsor: GlaxoSmithKline

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The GlaxoSmithKline group of companies
OTX203158
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TITLE PAGE
Division: Worldwide Development
Information Type: Protocol Amendment
Title:
Exploration of the peripheral immune system in subjects with 
New Onset T1 Diabetes (NOT1D)
Compound Number:
NONE
Development Phase:
NA
Effective Date:
06-MAR-2017
Protocol Amendment Number:  03
Author (s):
Copyright 2017 the GlaxoSmithKline group of companies.  All rights reserved.  
Unauthorised copying or use of this information is prohibited.
PPD

2015N246275_03
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Revision Chronology
GlaxoSmithKline 
Document Number
Date
Version
2015N246275_00
2015-DEC-11
Original
2015N246275_01
2016-APR-04
Amendment No. 1

Objective "To assess the biochemical fingerprint derived from metabonomic 
analyses of peripheral blood in HVs and NOT1D subjects" & associated endpoint 
removed in Section 3. References to this objective and endpoint in Section 6.1; 
and Section 6.8 were removed accordingly. This objective and associated 
endpoint were removed because they should be addressed in separate studies in 
the future.

New exploratory objective (“To assess the phenotype and transcriptomic 
fingerprint of stromal cells from iLN core biopsy samples in HVs and NOT1D 
subjects”) and associated endpoint added in Section 3, Section 6.8 and Section 8.1 
were updated accordingly. This objective and associated endpoint were added to 
provide additional insights into the exploration of the peripheral immune system 
in subjects with New Onset T1 Diabetes, which were not envisaged during the 
finalisation of the original protocol.

The Questionnaire in Appendix 1 was modified to avoid any duplication with 
Informed Consent procedures and duplication of data collection in eCRF. The 
entire questionnaire has been moved from the Study protocol to the Study 
Reference Manual (SRM) and is therefore not present in the amended protocol 
anymore. 

All appendix numberings and in-text references to appendices have been updated 
to reflect that the Lymph Node Questionnaire is no longer located in Appendix 1 
of the Study Protocol.

Clarifying wording was added and typos were corrected in Section 6.2; Section 
6.7.5 and Section 6.8.

Appendix 1 - Abbreviations and Trademarks lists were updated.
2015N246275_02
2016-OCT-06
Amendment No. 2

Time and Event table: Duplicate assesment removed and updated (medical 
history/past current medical conditions and laboratory assessments). Clarified 
when telephone call for Follow Up Period 1 would be collected. Clarified genetics 
testing was optional in consenting subjects. Added SAE/AE review.

Added HbA1c to other screening tests and removed wording ‘levels’ to fasted C-
peptide.

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2015N246275_03
2017-MAR-06
Amendment No. 3

Extended the interval between the initial diagnosis and Day 1, from 6 weeks to 8 
weeks following recruitment challenges. 8 weeks is the upper limit for the 
NOTID population and does not affect the scientific rationale for the study.  The 8 
week change was updated throughout the protocol.

Clarified exclusion criteria, vaccination ≤ 28 days before day 1.

Interim Analysis: Added additional information for the Interim Analysis after the 
recruitment of 5 evaluable HVs and 5 evaluable NOTID subjects. This will 
facilitate the decision making of the study and prioritization of the exploratory 
assays.

Clarified fasting at screening in the Time and Event table and added footnote to 
Table 1 to clarify fasted glucose at screening and non fasted glucose on Day 1

P P D
P P D

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MEDICAL MONITOR/SPONSOR INFORMATION PAGE
Medical Monitor/SAE Contact Information: 
Role
Name
Day Time 
Phone 
Number and 
email address
After-hours 
Phone/Cell/
Pager 
Number
Fax 
Number
Site Address
Primary 
Medical 
Monitor 
Immuno-
Inflammation TAU 
GlaxoSmithKline
Medicines Research 
Centre
Gunnels Wood Road
Stevenage
SG1 2NY
Secondary 
Medical 
Monitor
Immuno-
Inflammation TAU 
GlaxoSmithKline
Medicines Research 
Centre
Gunnels Wood Road
Stevenage
SG1 2NY
SAE contact  
information 
= Medical 
monitor
Sponsor Legal Registered Address: 
GlaxoSmithKline Research & Development Limited
980 Great West Road
Brentford
Middlesex, TW8 9GS
UK
In some countries, the clinical trial sponsor may be the local GlaxoSmithKline Affiliate 
Company (or designee). If applicable, the details of the alternative Sponsor and contact 
person in the territory will be provided to the relevant regulatory authority as part of the 
clinical trial application.   
Regulatory Agency Identifying Number(s): Not applicable
PPD

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INVESTIGATOR PROTOCOL AGREEMENT PAGE
For protocol number 
I confirm agreement to conduct the study in compliance with the protocol, as amended by 
this protocol amendment.
I acknowledge that I am responsible for overall study conduct. I agree to personally 
conduct or supervise the described study.
I agree to ensure that all associates, colleagues and employees assisting in the conduct of 
the study are informed about their obligations. Mechanisms are in place to ensure that site 
staff receives the appropriate information throughout the study. 
Investigator Name:
Investigator Address:
Investigator Phone Number:
Investigator Signature
Date

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TABLE OF CONTENTS
PAGE
1.
PROTOCOL SYNOPSIS FOR STUDY OTX203158.................................................9
2.
INTRODUCTION....................................................................................................12
2.1.
Study Rationale ..........................................................................................12
2.2.
Brief Background ........................................................................................12
2.2.1.
Type 1 Diabetes Mellitus..............................................................12
2.2.2.
Lymph node biopsy immunophenotyping.....................................14
3.
OBJECTIVE(S) AND ENDPOINT(S) ......................................................................15
4.
STUDY DESIGN ....................................................................................................16
4.1.
Overall Design ............................................................................................16
4.2.
Type and Number of Subjects.....................................................................17
4.3.
Design Justification.....................................................................................17
4.4.
Benefit:Risk Assessment ............................................................................17
4.4.1.
Risk Assessment .........................................................................18
4.4.2.
Benefit Assessment.....................................................................22
4.4.3.
Overall Benefit:Risk Conclusion...................................................22
5.
SELECTION OF STUDY POPULATION AND WITHDRAWAL CRITERIA .............22
5.1.
Inclusion Criteria.........................................................................................22
5.2.
Exclusion Criteria........................................................................................24
5.3.
Screening/Baseline/Run-in Failures............................................................25
5.4.
Withdrawal/Stopping Criteria.......................................................................26
5.5.
Subject and Study Completion....................................................................26
5.6.
Treatment after the End of the Study ..........................................................26
5.7.
Lifestyle and/or Dietary Restrictions............................................................27
5.7.1.
Caffeine, Alcohol, and Tobacco ...................................................27
5.7.2.
Activity .........................................................................................27
5.8.
Concomitant Medications and Non-Drug Therapies....................................27
5.8.1.
Permitted Medications and Non-Drug Therapies..........................27
5.8.2.
Prohibited Medications and Non-Drug Therapies.........................27
6.
STUDY ASSESSMENTS AND PROCEDURES .....................................................28
6.1.
Time and Events Table...............................................................................29
6.2.
Screening and Critical Baseline Assessments ............................................34
6.3.
Pre-& post-biopsy Questionnaire ................................................................34
6.4.
T1D auto-antibodies and other auto-antibodies ..........................................34
6.5.
Fine needle aspirate & core biopsy of the inguinal lymph node...................34
6.6.
Wound assessment ....................................................................................35
6.7.
Safety .........................................................................................................35
6.7.1.
Adverse Events (AE) and Serious Adverse Events (SAEs)..........35
6.7.1.1.
Time period and Frequency for collecting AE 
and SAE information...................................................35
6.7.1.2.
Method of Detecting AEs and SAEs ...........................36
6.7.1.3.
Follow-up of AEs and SAEs........................................36
6.7.1.4.
Death Events..............................................................36
6.7.1.5.
Regulatory Reporting Requirements for SAEs............36

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6.7.2.
Pregnancy ...................................................................................37
6.7.3.
Physical Exams ...........................................................................37
6.7.4.
Vital Signs....................................................................................37
6.7.5.
Clinical Safety & Other Laboratory Assessments.........................37
6.8.
Biomarker(s)...............................................................................................39
6.9.
Genetics .....................................................................................................41
7.
DATA MANAGEMENT ...........................................................................................41
8.
STATISTICAL CONSIDERATIONS AND DATA ANALYSES .................................42
8.1.
Hypotheses.................................................................................................42
8.2.
Sample Size Considerations.......................................................................43
8.2.1.
Sample Size Assumptions ...........................................................43
8.2.2.
Sample Size Sensitivity................................................................43
8.2.3.
Sample Size Re-estimation or Adjustment...................................43
8.3.
Data Analysis Considerations .....................................................................43
8.3.1.
Analysis Populations....................................................................43
8.3.2.
Interim Analysis ...........................................................................44
8.4.
Key Elements of Analysis Plan ...................................................................44
8.4.1.
Primary Analyses.........................................................................44
8.4.2.
Secondary Analyses ....................................................................45
8.4.3.
Other Analyses ............................................................................45
9.
STUDY GOVERNANCE CONSIDERATIONS........................................................45
9.1.
Posting of Information on Publicly Available Clinical Trial Registers............45
9.2.
Regulatory and Ethical Considerations, Including the Informed 
Consent Process ........................................................................................45
9.3.
Quality Control (Study Monitoring) ..............................................................46
9.4.
Quality Assurance.......................................................................................46
9.5.
Study and Site Closure ...............................................................................46
9.6.
Records Retention......................................................................................47
9.7.
Provision of Study Results to Investigators, Posting of Information 
on Publically Available Clinical Trials Registers and Publication .................48
10. REFERENCES.......................................................................................................49
11. APPENDICES........................................................................................................51
11.1.
Appendix 1: Abbreviations and Trademarks................................................51
11.2.
Appendix 2: Genetic Research ...................................................................52
11.3.
Appendix 3: Definition of and Procedures for Recording, Evaluating, 
Follow-Up and Reporting of Adverse Events...............................................55
11.3.1.
Definition of Adverse Events........................................................55
11.3.2.
Definition of Serious Adverse Events...........................................56
11.3.3.
Recording of AEs and SAEs ........................................................57
11.3.4.
Evaluating AEs and SAEs............................................................57
11.3.5.
Reporting of SAEs to GSK...........................................................59
11.4.
Appendix 4: Collection of Pregnancy Information........................................60
11.5.
Appendix 5: Country Specific Requirements...............................................61
11.6.
Appendix 6: Protocol changes ....................................................................62
11.6.1.
Amendement No. 3......................................................................62
11.6.2.
Amendement No. 2......................................................................67
11.6.3.
Amendment No. 1........................................................................70

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1.
PROTOCOL SYNOPSIS FOR STUDY OTX203158
Rationale
This study will assess and compare the molecular immune profile of cells derived from 
the inguinal lymph nodes (iLN) in healthy volunteers (HVs) and New Onset Type 1 
Diabetes Mellitus (NOT1D) subjects, to better understand the immunological processes 
that may lead to beta cell destruction. It is hypothesised that early changes in the immune 
system in NOT1D subjects can be detected in immune cells from the iLNs that will be 
distinct from changes observed in peripheral blood derived immune cells, such as for 
example Peripheral Blood Mononuclear Cells (PBMCs). Novel biomarkers of disease 
activity may be identified, potentially supporting the early readout of therapeutic efficacy 
in future Type 1 Diabetes (T1D) trials.
Objective(s)/Endpoint(s)
Objectives
Endpoints
Primary
1) To assess the frequency and 
phenotype of leukocyte subsets in iLN 
and peripheral blood in HVs and 
NOT1D subjects

Absolute number and/or 
proportion of leukocyte subsets in 
iLN and peripheral blood
Secondary
1) To assess the frequency and 
phenotype of leukocyte subsets in iLN 
core biopsies and iLN fine needle 
aspirates in HVs and NOT1D subjects

Absolute number and/or 
proportion of leukocyte subsets in 
iLN core biopsies and iLN fine 
needle aspirates.
2) To assess the safety and tolerability 
of iLN biopsy as well as expectations 
and experience of the biopsy procedure.

Number of AEs and SAEs 
following lymph node biopsy 
procedure

Descriptive data obtained by a 
questionnaire on the acceptability 
of iLN biopsy in research setting.  
Exploratory*
1) To assess suppression activity of T 
regulatory lymphocytes from the iLN 
and peripheral blood in HVs and 
NOT1D subjects

Relative levels of T lymphocyte 
suppressive activity of cells from 
iLN and peripheral blood
respectively
2) To assess the frequency of cytokine-
producing lymphocytes in HVs and 

Proportion of lymphocyte 
populations producing pro-
inflammatory and/or anti-

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Objectives
Endpoints
NOT1D subjects
inflammatory cytokines in iLN 
and peripheral blood
3) To assess clonal expansion of T and 
B lymphocyte populations in HVs and 
NOT1D subjects

Comparison of TCR and BCR 
usage in lymphocytes in iLN and 
peripheral blood 
4) To assess the gene expression 
fingerprint of auto-antigen specific 
lymphocytes in HVs and NOT1D 
subjects

Gene expression levels of sorted 
auto antigen stimulated T 
lymphocyte populations in iLN 
and peripheral blood 
5) To assess the phenotype and 
transcriptomic fingerprint of stromal 
cells from iLN core biopsy samples in 
HVs and NOT1D subjects

Absolute number and/or 
proportion of stromal cell subsets
and gene expression levels of 
stromal cell subsets from iLN 
core biopsy samples 
* The Exploratory endpoints are listed in order of priority and the conduct of any experimental assays will 
be dependent on the material available (i.e. number of cells obtained) from iLN biopsies and peripheral 
blood.
Overall Design

A multi-centre, non-drug treatment study to compare differences in immune cells 
derived from iLN and peripheral blood of NOT1D subjects and HVs.

After informed consent, each subject will attend a screening visit followed by a study 
visit for iLN biopsy and peripheral blood collection (Day 1). Follow up phone calls 
after the study visit will be carried out at approximately Day 2-4 and Day 7 to 14. 
The total duration of this study will be up to 10 weeks, including the screening 
period.

A data look will be carried out after the recruitment of a cohort of up to 5 HVs to 
determine if the quality and quantity of cells derived from either fine needle aspirate 
or core biopsy of the inguinal lymph node and from peripheral blood are likely to be 
sufficient to continue the study to meet its primary objective. Full details on this 
Day 1: lymph node biopsy 
& blood collection
Screening
Period
Study 
Visit
Follow Up
Period 2
Day ~ - 56 to Day - 7
Day ~7 to Day ~14
Day ~2 to Day ~4
Follow Up 
Period 1
T1D 
Diagnosis(N.B. “T1D diagnosis” is not applicable to healthy volunteers)

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feasibility analysis will be provided in the Study Reference Manual (SRM) and 
Reporting and Analysis Plan (RAP).

An interim analysis will be carried out after the recruitment of 5 evaluable HVs and 
5 evaluable NOT1D subjects. The primary purpose of this interim analysis will be to 
facilitate decision making and study design for a potential follow-up interventional 
study.
Type and Number of Subjects
Up to 15 evaluable NOT1D subjects and up to 15 evaluable HVs will be enrolled. If 
subjects prematurely discontinue the study, additional replacement subjects (up to 5 
subjects) may be enrolled at the discretion of the sponsor in consultation with the 
investigator. Evaluable subjects are defined as subjects that provide both iLN biopsy (at 
least sufficient amounts of cells by one of the two biopsy methods employed to enable 
the conduct of assays related to at least the primary endpoint) AND at least sufficient 
amounts of peripheral blood to enable the conduct of assays related to at least the primary 
endpoint). 
Analysis
Descriptive statistics will be calculated for each of the primary flow cytometry cell types 
by group and sample type (blood / biopsy). Generalized linear models with appropriate 
link functions will be used to analyze data separately for each flow cytometry cell type to 
provide estimates for comparisons.

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2.
INTRODUCTION
Lymph nodes (LNs) are important sites in the body where either an immune response to 
pathogenic antigens or tolerance is initiated [Sainte-Marie, 2010]. In T1D there is a break 
down in tolerance against self antigens resulting in immune attack of the beta cells of the 
pancreas.
In the non-obese mouse model of T1D (NOD mouse) islet specific T-cells can be 
detected in the pancreatic LN before the onset of disease [Höglund, 1999] and removal of 
these LNs rescues NOD mice from developing T1D [Gagnerault, 2002]. In addition, in 
subjects with T1D, auto-reactive T cells isolated from the blood are capable of destroying 
islet cells when co-cultured in vitro, suggesting that immune activation is not only 
occurring at the pancreas but also in other lymphoid tissues [Unger, 2012]. However, 
identification of clonal signatures unique to T1D subjects has proven to be difficult due to 
the low frequency of antigen specific cells in the blood of these subjects. To date, 
researchers have studied pathogenic T cells in response to insulin-specific antigens in the 
peripheral blood but the results have shown no significant differences between HVs and 
T1D subjects [Herold, 2009], most likely due to lack of response to antigen by low 
affinity T-cell Receptors (TCRs). In addition, emerging data suggest that post-
translational modification of insulin-related antigens is playing a role in break of 
tolerance and destruction of beta cells [van Lummel, 2014]. 
2.1.
Study Rationale
This study will assess and compare the molecular immune profile of cells derived from 
LNs in HVs and NOT1D subjects, to better understand the processes that may lead to 
beta cell destruction. Since retrieving peri-pancreatic LN cells is not feasible in a research 
setting and immune cells circulate quickly throughout the body, we will address our 
questions using cells from the LNs in the inguinal region. This method has been utilised 
in subjects with rheumatoid arthritis, subjects at risk of rheumatoid arthritis and HVs [van 
Baarsen, 2013].
It is hypothesised that early changes in the immune system in subjects with NOT1D can 
be detected in LNs that will be distinct from changes observed in cells derived from  
peripheral blood, such as PBMCs. As a result, novel biomarkers of disease activity may 
be identified, potentially supporting the early readout of therapeutic efficacy in future 
T1D trials.
2.2.
Brief Background
2.2.1.
Type 1 Diabetes Mellitus
T1D represents 5-10% of all subjects with Diabetes Mellitus [Maahs, 2010] and is 
primarily a result of the T cell-mediated destruction of beta cells in the pancreas 
[Atkinson, 2014]. The disease occurs most often in children, adolescents, and young 
adults, but can present at any age. T1D has strong Human leukocyte antigen (HLA) 
associations, particularly with linkage to DR and DQ genes. Autoreactive antibodies 
precede the onset of disease and have been used for risk stratification in susceptible 

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subjects [Wasserfall, 2006], however, they cannot predict disease progression. Due to the 
varying length of time between autoantibody seroconversion and clinical symptoms in 
subjects, it is postulated that the environment also plays a role in the development of the 
disease. Environmental factors such as viruses [Yeung, 2011], lack of vitamin D 
[Hyppönen, 2001], weaning and improved hygiene [Knip, 2010] have all been implicated 
as triggers for T1D, but their roles remain controversial. Major steps to address this are 
being undertaken through the longitudinal ‘TEDDY’ study (The environmental 
determinants of diabetes in the young), which is monitoring genetically susceptible 
children from birth (https://teddy.epi.usf.edu/). 
During the preclinical period between seroconversion and disease onset there is 
progressive loss of beta cells in the pancreatic islets, which impairs the ability of an 
individual to maintain blood glucose levels within a physiologic range [Rowe, 2011]. A 
complex interplay between the innate and adaptive arms of the immune system, in 
response to islet-specific antigens, is responsible for the pathogenesis of T1D. The 
precise events that trigger the development of an autoimmune attack on and destruction 
of the beta cells are unclear, but may be driven primarily by cytotoxic T cells. Indeed, 
islet-derived CD8+ T cells expressed T-cell receptors bound to MHC class 1 tetramers 
with the beta-cell autoantigen Islet-specific glucose-6-phosphatase catalytic subunit-
related protein (IGRP) and other target peptides in NOT1D subjects [Skowera, 2015]. In 
addition, autoreactive CD8+ and CD4+ T lymphocytes can be detected in peripheral 
circulation and are directed to many of the same epitopes in recent-onset diabetes 
subjects [Velthuis, 2010; Unger, 2011] Interestingly, analyses of pancreata from subjects 
diagnosed with diabetes within 1 year suggest that subjects under the age of 14 have 
higher incidence of insulitis (68%) compared to those between 15 and 39 (39%) In't Veld, 
2014]. This suggests that a more vigorous autoimmune attack occurs in children. As a 
consequence of this autoimmune attack, autoantibodies against: glutamic acid 
decarboxylase (GAD), protein tyrosine phosphatase-related islet antigen-2 protein (IA 2), 
IA 2, islet-cell antigen (ICA), zinc transporter 8 (ZnT8) and/or insulin (IAA) are 
detectable in approximately 85% to 90% of subjects with T1D [Vermeulen, 2011]. But as 
discussed earlier, these autoantibodies can neither predict absolute disease progression 
nor be used to monitor therapeutic efficacy.
The net effect of autoimmune destruction of beta cells coupled to little or no regeneration 
marks a progressive deterioration of glucose homeostasis, culminating in an absolute 
dependence on exogenous insulin replacement therapy. It is estimated that only 10-20% 
of beta-cells are still functioning at the time of clinical diagnosis [Knip, 2002]. The rate 
of progression from normo-glycaemia, through pre-clinical impairment of glucose 
handling to insulin dependence is variable. Indeed, even 1-5 years after diagnosis, about 
50% of T1D subjects show clinically meaningful beta-cell function [Palmer, 2009].
The destruction process of beta cells is not a continuous phenomenon and it has been 
argued that T1D is a relapsing-remitting disease [Von Herrath, 2007]. Therefore in order 
to prevent further deterioration of the pancreas it is vital that subjects are treated as close 
to or prior to diagnosis if possible.
Clinical trials in T1D/NOT1D usually rely on metabolic measurements as endpoints, 
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[Aronson, 2014]. Reliable biomarkers of immune dysfunction and beta cell destruction 
are not available at the moment and it is hypothesized that analysis of lymphocyte 
phenotypes and additionally the deep phenotyping of T and B cells in the earliest stages 
of the autoimmune process in T1D / NOT1D may provide a window on the immune 
system, which will eventually enable the characterisation of biomarkers and allow an 
early evaluation of the efficacy of immunomodulatory regimes in future studies.
2.2.2.
Lymph node biopsy immunophenotyping
iLN biopsies will be carried out by two related methods in this study: fine-needle aspirate 
biopsy (referred to as “fine needle aspirate” in this protocol) and core needle biopsy 
(referred to as “core biopsy” in this protocol). 
Immunophenotyping of cells obtained by iLN core biopsies in autoimmune disease was 
described by van Baarsen [van Baarsen, 2013]. The authors examined the cellular 
composition of iLNs in HVs compared to subjects with early rheumatoid arthritis (RA) 
and subjects at risk of developing RA and showed significant changes in B and T cell 
composition by flow cytometry analysis between groups. This difference was not 
detected in PBMCs, indicating that studying the composition of immune cells of the iLN 
has additional value in understanding molecular events in the early phases of an 
autoimmune disease. Furthermore, biopsy by fine needle aspirate of lymph nodes has 
been described as a method to immunophenotype cells derived from axillary lymph nodes 
following skin antigen delivery [Tatovic, 2015].

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3.
OBJECTIVE(S) AND ENDPOINT(S)
Objectives
Endpoints
Primary
1)To assess the frequency and phenotype 
of leukocyte subsets in iLN and 
peripheral blood in HVs and NOT1D 
subjects

Absolute number and/or proportion 
of leukocyte subsets in iLN and 
peripheral blood
Secondary
1) To assess the frequency and phenotype 
of leukocyte subsets in iLN core biopsies 
and iLN fine needle aspirates in HVs and 
NOT1D subjects

Absolute number and/or proportion 
of leukocyte subsets in iLN core 
biopsies and iLN fine needle 
aspirates.
2) To assess the safety and tolerability of 
iLN biopsy as well as expectations and 
experience of the biopsy procedure.

Number of AEs and SAEs 
following lymph node biopsy 
procedure

Descriptive data obtained by a 
questionnaire on the acceptability 
of iLN biopsy in research setting.  
Exploratory*
1) To assess suppression activity of T 
regulatory lymphocytes from the iLN and 
peripheral blood in HVs and NOT1D 
subjects

Relative levels of T lymphocyte 
suppressive activity of cells from 
iLN and peripheral blood
respectively
2) To assess the frequency of cytokine-
producing lymphocytes in HVs and 
NOT1D subjects

Proportion of lymphocyte 
populations producing pro-
inflammatory and/or anti-
inflammatory cytokines in iLN and 
peripheral blood
3) To assess clonal expansion of T and B 
lymphocyte populations in HVs and 
NOT1D subjects

Comparison of TCR and BCR 
usage in lymphocytes in iLN and 
peripheral blood
4) To assess the gene expression 
fingerprint of auto-antigen specific 
lymphocytes in HVs and NOT1D subjects

Gene expression levels of sorted 
auto antigen stimulated T 
lymphocyte populations in iLN and 
peripheral blood 

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Objectives
Endpoints
5)To assess the phenotype and 
transcriptomic fingerprint of stromal cells 
from iLN core biopsy samples in HVs 
and NOT1D subjects

Absolute number and/or proportion 
of stromal cell subsets and gene 
expression levels of stromal cell 
subsets from iLN core biopsy 
samples 
* The Exploratory endpoints are listed in order of priority and the conduct of any experimental assays will 
be dependent on the material available (i.e. number of cells obtained) from iLN biopsies and peripheral 
blood.
See Section 8.1 for a description of the intended use of cells from each biopsy procedure. 
4.
STUDY DESIGN

A multi-centre, non-drug treatment study to compare differences in immune cells 
derived from iLN and peripheral blood of NOT1D subjects and HVs.

After informed consent, each subject will attend a screening visit followed by a study 
visit for iLN biopsy and peripheral blood collection (Day 1). Follow up phone calls 
after the study visit will be carried out at approximately 2 to 4 days and 7 to 14 days 
after the study visit. The total duration of this study will be up to 10 weeks, including 
the screening period.

A data look will be carried out after the recruitment of a cohort of up to 5 HVs to 
determine if the quality and quantity of cells derived from either fine needle aspirate 
or core biopsy of the inguinal lymph node and from peripheral blood are likely to be 
sufficient to continue the study to meet its primary objective. Full details on this 
feasibility analysis will be provided in the SRM and Reporting and Analysis Plan 
(RAP).

Following the data look, for the remainder of the study the team will seek to match 
NOT1D subjects to HVs at a group level according to the number of subjects for 
each gender and the distribution of age within gender. Full details on group matching 
will be provided in the SRM. 

An interim analysis will be carried out after the recruitment of 5 evaluable HVs and 
5 evaluable NOT1D subjects. The primary purpose of this interim analysis will be to 
facilitate decision making and study design for a potential follow-up interventional 
study.
4.1.
Overall Design
Day 1: lymph node biopsy 
& blood collection
Screening
Period
Study 
Visit
Follow Up
Period 2
Day ~ -56 to Day -7
Day ~7 to Day ~14
Day ~2 to Day ~4
Follow Up 
Period 1
T1D 
Diagnosis(N.B. “T1D diagnosis” is not applicable to healthy volunteers)

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4.2.
Type and Number of Subjects
Up to 15 evaluable NOT1D subjects and up to 15 evaluable HVs will be enrolled. If 
subjects prematurely discontinue the study, additional replacement subjects (up to 5 
subjects) may be enrolled at the discretion of the sponsor in consultation with the 
investigator. Evaluable subjects are defined as subjects that provide both iLN biopsy (at 
least sufficient amounts of cells by one of the two biopsy methods employed to enable 
the conduct of assays related to at least the primary endpoint) AND at least sufficient 
amounts of peripheral blood to enable the conduct of assays related to at least the primary 
endpoint). 
4.3.
Design Justification
Immunophenotyping of cells obtained by iLN biopsies with a similar study design was 
described by van Baarsen [van Baarsen, 2013]. The authors of that report examined the 
cellular composition of inguinal lymph nodes in HVs and subjects with early 
inflammatory arthritis or individuals at risk of developing the disease. A similar study 
design is applied in this study.
4.4.
Benefit:Risk Assessment
The following section outlines the risk assessment and mitigation strategy for this 
protocol:

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4.4.1.
Risk Assessment
Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
Study Procedures
Short and/or persistent pain following the 
lymph node biopsy procedure 
Short period of pain in the groin region has 
been reported in up to 7% of subjects and 
persistent pain for up to 2 months has been 
reported in up to 0.9% of subjects after the 
lymph node biopsy procedure previously 
[de Hair, 2012].

Subjects will be followed up as 
described in the Time & Events 
Table (see Section 6.1) and asked to 
promptly report any pain. Subjects 
will be asked to contact the 
investigator for an examination if 
they report any symptoms related to 
persistent pain in the biopsy area. 

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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
Bruising / Bleeding at biopsy site
Core biopsy and fine needle aspirate have 
the risk of moderate bleeding, especially if a 
large 
blood 
vessel 
is 
punctured. 
A 
haematoma incidence of up to 80 % has 
been reported in the literature and post 
procedure haematoma may cause further 
discomfort for the subject [de Hair, 2012].

Biopsy procedure will be performed 
under ultrasound guidance by an 
experienced radiologist. Adequate 
manual pressure haemostasis will be 
performed and the puncture site will 
be reviewed by a physician prior to 
completion of the study visit.

Subjects with bleeding disorders or 
those who take anticoagulant / 
antiplatelet agents will be excluded 
from the study as described in 
Section 5.2.

Subjects in whom the procedure may 
be technically difficult will be 
excluded on a case by case basis 
subject to investigator’s discretion.

The number of core biopsies taken in 
this study will be limited to a 
maximum of five core biopsies in 
addition to a maximum of two fine 
needle aspirate passages in each 
subject.

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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
Infection risk
All biopsy procedures carry a risk of 
infection. This risk may be higher in 
subjects 
with 
(uncontrolled) 
Diabetes 
Mellitus.

Subjects in whom the procedure may 
place them at increased risk of 
infection (e.g., large pannus, poor 
glycemic control) will be excluded 
on a case by case basis subject to 
investigator’s discretion.

Subjects with groin infection will be 
excluded.

Strict aseptic technique during the 
procedure will be followed.

Subjects will be followed up as 
described in the Time and Events 
Table (see Section 6.1) and will be 
asked to contact the investigator for 
an examination if they report any 
signs or symptoms of infection. 
Inadvertent trauma to femoral nerve
The biopsy procedure carries a hypothetical 
risk of trauma to the femoral nerve which 
may 
cause 
persisting 
paraesthesia 
(hypothetical because the site of biopsy is 
distant to the femoral nerve).

Ultrasound guided biopsy procedure 
will avert the risk of inadvertent 
damage to large nerves.
Post-procedure lymphoedema
Damage to the lymphatic system carries a 
hypothetical risk of lymphoedema in the 

The number of core biopsies taken in 
this study will be limited to a 
maximum of five core biopsies in 

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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
ipsilateral leg. 
addition to a maximum of two fine 
needle aspirate passages in each 
subject. Subjects will be followed up 
as described in the Time and Events 
Table (see Section 6.1) and will be 
asked to contact the investigator for 
an examination if they report any 
signs or symptoms of lymphoedema.
Other 
Toxicity from Local Anaesthetic agents
Administration of these agents may cause 
systemic e.g., hypersensitivity, cardiac or 
neurological 
toxicity 
in 
susceptible 
individuals.

Subjects 
with 
allergy 
to 
local 
anaesthetic agents will be excluded.

The administration and use will be in 
keeping with the relevant SmPC.

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4.4.2.
Benefit Assessment
This is an exploratory study to identify peripheral immune system biomarkers associated 
with T1D activity and there is no anticipated potential for direct benefit to subjects. The 
results from this study may contribute to understanding of disease pathogenesis, and 
identifying potential new biomarkers for future early phase clinical studies, in which the 
pharmacology of new treatments of NOT1D subjects will be tested.
4.4.3.
Overall Benefit:Risk Conclusion
No medical benefit is anticipated by NOT1D subject’s participation in this study. Taking 
into account the measures taken to minimise risk to subjects, the potential study 
procedure-associated risks are justified by the anticipated general benefits in the future 
that may be afforded to subjects affected by T1D or other diseases.
5.
SELECTION OF STUDY POPULATION AND 
WITHDRAWAL CRITERIA
Specific information regarding warnings, precautions, contraindications, adverse events, 
and other pertinent information that may impact subject eligibility is provided in the 
SRM.
Deviations from inclusion and exclusion criteria are not allowed because they can 
potentially jeopardize the scientific integrity of the study, regulatory acceptability or 
subject safety. Therefore, adherence to the criteria as specified in the protocol is essential.
Subjects may be re-screened for the study, provided that re-screening can be 
accomplished to meet the timeframe of the inclusion criteria of a subject entering the 
study, i.e. with an interval of up to 8 weeks between the initial diagnosis and day 1 of the 
study (see Section 5.1).
5.1.
Inclusion Criteria
A subject will be eligible for inclusion in this study only if all of the following criteria 
apply:
AGE (all subjects)
1. Between 18 and 40 years of age inclusive, at the time of signing the informed 
consent.
TYPE OF SUBJECT AND DIAGNOSIS INCLUDING DISEASE SEVERITY 
Healthy volunteers
2. Healthy as determined by the investigator or medically qualified designee based on a 
medical evaluation including medical history, physical examination and laboratory 
tests.
3. Subject values for the following parameters: fasted glucose, HbA1c, INR, APTT, 

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platelet count, red blood cells and total lymphocyte count within the normal range at 
screening.
NOT1D subjects
4. Documented diagnosis of diabetes mellitus according to ADA and WHO criteria and 
consistent with Type 1a (autoimmune) Diabetes Mellitus, with an interval of up to 8
weeks between the initial diagnosis and day 1 of the study (day 1 =  “iLN biopsy” 
day, see Section 6.1, Time and Events Table).
5. Currently requires insulin treatment for T1D and has received insulin therapy for at 
least 7 days prior to screening.
6. Positive, at screening, for at least one autoantibody associated with T1D: anti-GAD, 
anti-IA 2, anti-ICA, anti-IAA, anti-ZnT8.
7. Evidence, at screening, of residual functioning β cells as measured by fasted C-
peptide levels ≥0.15 nmol/L.
8. Subject values for the following parameters: INR, APTT, platelet count, red blood 
cells and total lymphocyte count within the normal range at screening.
SEX (all subjects)
9. Both, male or female subjects are eligible to participate in this study.
A female subject is only eligible to participate if she is not pregnant (as confirmed by a 
negative urine) human chorionic gonadotrophin (hCG) test), not lactating at screening 
AND study visit(s) or has documented evidence to not be of child bearing potential.
INFORMED CONSENT (all subjects)
10. Capable of giving signed informed consent which includes compliance with the 
requirements and restrictions listed in the consent form and in this protocol.
OTHER (all subjects)
11. A subject with a clinical abnormality or laboratory parameter(s) which is/are not 
specifically listed in the inclusion or exclusion criteria, outside the reference range for 
the population being studied may be included only if the investigator, in consultation 
with the Medical Monitor if required, agree and document that the finding is unlikely 
to introduce additional risk factors and will not interfere with the study procedures.

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5.2.
Exclusion Criteria
A subject will not be eligible for inclusion in this study if any of the following criteria 
apply:
HEALTHY VOLUNTEERS
1. Family history of T1D (i.e. 1st degree relative has been diagnosed with T1D)
2. Presence of one or more of serum autoantibodies, such as anti-GAD, anti-IA2, anti-
ICA, 
anti-IAA, 
anti-ZnT8, 
anti-thyroid 
peroxidase 
antibodies, 
anti-tissue 
transglutaminase antibodies, anti-nuclear antibodies.
NOT1D SUBJECTS
3. History of autoimmune disease other than T1D
4. Presence of one or more of serum autoantibodies of the following: anti-thyroid 
peroxidase antibodies, anti-tissue transglutaminase antibodies or anti-nuclear 
antibodies 
ALL SUBJECTS: 
CONCURRENT CONDITIONS/MEDICAL HISTORY (INCLUDES LIVER 
FUNCTION
5. Allergy or intolerance to local anaesthetic agents
6. Any localised groin condition which would contraindicate biopsy procedure including 
but not limited to: Active infection/inflammation at the intended puncture site, 
previous surgery/scarring or any other anatomical abnormality as deemed relevant to 
the procedure by the investigator, in consultation with the Medical Monitor if 
required.
7. History of bleeding disorders, current or anticipated continuous use of anticoagulant 
(including but not limited to warfarin, rivaroxaban) and antiplatelet agents (including 
but not limited to NSAIDS, clopidogrel, etc.)
8. Active or unresolved bacterial infection, viral infection, fungal infection within 4 
weeks prior to day 1.
9. Known febrile episode over 38 degrees Celsius within 4 weeks prior to day 1. 
10. Active organ dysfunction or previous organ allograft.
11. History of malignancy (with the exception of resected basal carcinoma of the skin or 
cervical carcinoma in situ).

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12. Has undergone any major surgical procedure within 30 days before screening, and/or 
is planning to undergo any such surgery during the period of the study (i.e. from 
screening until the last follow-up telephone call, see Section 6.1).
CONCOMITANT MEDICATIONS
13. Present or previous treatment with any cell depleting therapies or immune-modulating 
or suppressive agents (e.g., oral steroids), including investigational agents such as the 
following but not limited to e.g., IL-2, CAMPATH, anti-CD4, anti-CD5, anti-CD3, 
anti-CD19, anti-CD-20.
14. Vaccination ≤ 28 days before day 1 of the study or planned during the study period.
15. Current participation in an interventional clinical trial. Subjects, who participated in 
an interventional clinical trial previously, must wait for 3 months after completing the 
previous interventional clinical trial before participating in this study.
CONTRAINDICATIONS 
16. Any medical history or clinically relevant abnormality that is deemed by the 
investigator and/or medical monitor to make the subject ineligible for inclusion 
because of a safety concern or any situation that, in the investigator’s judgment, is 
likely to cause the subject to be unable or unwilling to participate in study procedures 
or to complete all scheduled assessments.
17. A positive pre-study drug/alcohol screen (unless positive due to prescription 
medication). A minimum list of drugs that will be screened for include
amphetamines, barbiturates, cocaine, opiates, cannabinoids and benzodiazepines.
18. Inability to access the groin area to perform the biopsy procedure as judged by the 
investigator.
5.3.
Screening/Baseline/Run-in Failures
Screen failures are defined as subjects who consent to participate in the clinical trial but 
did not undergo the study procedures (iLN biopsy and peripheral blood collection). In 
order to ensure transparent reporting of screen failure subjects, meet the Consolidated 
Standards of Reporting Trials (CONSORT) publishing requirements, and respond to 
queries from Regulatory authorities, a minimal set of screen failure information is 
required including Demography, Screen Failure details, Eligibility Criteria, and Serious 
Adverse Events (see Section 6.7.1.5).

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5.4.
Withdrawal/Stopping Criteria
The following actions must be taken in relation to a subject who fails to attend the clinic 
for a required visit or does not complete telephone calls:

The site must attempt to contact the subject and re-schedule the missed 
visit/telephone call as soon as possible. 

The site must counsel the subject on the importance of maintaining the assigned 
visit/telephone calls schedule and ascertain whether or not the subject wishes to 
and/or should continue in the study. 

In cases where the subject is deemed ‘lost to follow up’, the investigator or designee 
must make every effort to regain contact with the subject (where possible, 3 
telephone calls and if necessary a certified letter to the subject’s last known mailing 
address or local equivalent methods). These contact attempts should be documented 
in the subject’s medical record. 

Should the subject continue to be unreachable, only then will he/she be considered to 
have withdrawn from the study with a primary reason of “Lost to Follow-up”. 
A subject may withdraw from the study at any time at his/her own request, or may be 
withdrawn at the discretion of the investigator at any time if the subject undergoes a 
change, which would render her/him unsuitable to take part in the study according to the 
stated inclusion / exclusion criteria. If a subject withdraws from the study, he/she may 
request destruction of any samples taken, and the investigator must document this in the 
site study records.
Replacement of withdrawn subjects for inclusion in the protocol will be at the discretion 
of the PI in consultation with the medical monitor.
5.5.
Subject and Study Completion
A completed subject is one who has completed all phases of the study including the 
follow-up call(s).The end of the study is defined as the last subject’s last telephone call.
5.6.
Treatment after the End of the Study
Subjects will not receive any treatment from GSK after completion of the study because 
no medicinal products are being investigated in this study, the indication being studied is 
not life threatening or seriously debilitating and other treatment options are available.
The investigator is responsible for ensuring that consideration has been given to the post-
study care of the subject’s medical condition.

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5.7.
Lifestyle and/or Dietary Restrictions
5.7.1.
Caffeine, Alcohol, and Tobacco

Subjects should maintain their usual caffeine- or xanthine-containing products 
consumption levels (e.g., coffee, tea, cola drinks and chocolate). 

During each study session, subjects will abstain from alcohol for 24 hours prior to 
the screening visit, each assessment day and whilst present in the hospital / clinical 
unit. No more than 21 units (for men) and 14 units (for women) of alcohol per week 
should be consumed from the time of screening until the end of the clinical phase of 
the study.
5.7.2.
Activity
Subjects must abstain from strenuous exercise for 24 hours post biopsy, including weight 
lifting, repetitive leg movements, riding a non-motorized bicycle or sexual intercourse.
Subjects must refrain from operating any motorised vehicle for at least 24h following the 
procedure.
5.8.
Concomitant Medications and Non-Drug Therapies
5.8.1.
Permitted Medications and Non-Drug Therapies
All concomitant medications for co-morbid conditions taken during the study will be 
recorded in the CRF. The minimum requirement is that drug name, total daily dose and 
the frequency of administration are to be recorded. See Section 5.8.2 for subjects’ 
reporting obligations.
All participants

Medication use by eligible subjects may be considered on a case by case basis by the 
Investigator (in consultation with the Medical Monitor if required).

Eligible subjects who have well controlled concurrent medical conditions must be 
stable on their current treatment prior to study enrolment, and for the duration of the 
study, but may be excluded from the study at the investigators discretion.
NOT1D subjects

Regular medication can be continued throughout the study but see Section 5.8.2 for 
reporting obligations.
5.8.2.
Prohibited Medications and Non-Drug Therapies
Subjects must report the use of any prescription and non-prescription medications during 
the study period to the investigator.

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6.
STUDY ASSESSMENTS AND PROCEDURES
Protocol waivers or exemptions are not allowed with the exception of immediate safety 
concerns. Therefore, adherence to the study design requirements, including those 
specified in the Time and Events Table, are essential and required for study conduct.
This section lists the procedures and parameters of each planned study assessment. The 
timing of each assessment is listed in the Time and Events Table Section 6.1

The change in timing or addition of time points for any planned study assessments 
must be documented in a Note to File which is approved by the relevant GSK study 
team member and then archived in the study sponsor and site study files, but this will 
not constitute a protocol amendment. 

The IRB/IEC will be informed of any safety issues that require alteration of the 
safety monitoring scheme or amendment of the Informed Consent Form. 

No more than 500 mL of blood will be collected over the duration of the study, 
including any extra assessments that may be required.

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6.1.
Time and Events Table

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Screening procedures in addition to those listed below are (outpatient visit): Informed consent; Inclusion / Exclusion criteria; demography.
Procedure
Screening (between 
-56 to 7 days prior 
to study session)
Study Session
Day 1
Telephone 
follow-up
Day ~2 to ~ 
Day 4
Telephone 
follow-up 
Day ~7 to 
Day ~14
Notes
Pre biopsy 
Biopsy
End of study 
session visit 
Full physical 
exam
X
To be completed if a 
post study visit is 
required. Attendance 
to the unit post study 
sessions only if 
necessary (at 
discretion of 
investigator)
Medical history / 
Past and current 
medical 
conditions
X
X
To be completed if a 
post study visit is 
required. Attendance 
to the unit post study 
sessions only if 
necessary (at 
discretion of 
investigator)

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Procedure
Screening (between 
-56 to 7 days prior 
to study session)
Study Session
Day 1
Telephone 
follow-up
Day ~2 to ~ 
Day 4
Telephone 
follow-up 
Day ~7 to 
Day ~14
Notes
Pre biopsy 
Biopsy
End of study 
session visit 
Laboratory 
assessments  (see 
Section  6.7.5)
X1
X2
To be completed if a 
post study visit is 
required. Attendance 
to the unit post study 
sessions only if 
necessary (at 
discretion of 
investigator)
1. Blood testing in 
fasted condition at 
screening
2. Haematology and 
clinical chemistry 
only  on Day 1 in a 
non fasted 
condition
Concomitant 
medication 
review
X
X
X
X
Alcohol Urine 
Test and Test for 
Drugs of Abuse
X
X
Urine Pregnancy 
test (in women of 
child bearing 
potential)
X
X

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Procedure
Screening (between 
-56 to 7 days prior 
to study session)
Study Session
Day 1
Telephone 
follow-up
Day ~2 to ~ 
Day 4
Telephone 
follow-up 
Day ~7 to 
Day ~14
Notes
Pre biopsy 
Biopsy
End of study 
session visit 
Vital signs
X
X
X
To be completed if a 
post study visit is 
required. Attendance 
to the unit post study 
sessions only if 
necessary (at 
discretion of 
investigator)
Proposed biopsy 
site review
X
Peripheral blood 
collection for cell 
isolation 
X
Peripheral blood 
collection for 
genetics
X
Optional in 
consenting subjects
Inguinal lymph 
node biopsy
(fine needle 
aspirate & core 
biopsy)
X
Wound 
assessment
X
X (telephone)
X 
(telephone)
To be completed if a 
post study visit is 
required. Attendance 
to the unit post study 
sessions only if 
necessary (at 
discretion of 
investigator)

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Procedure
Screening (between 
-56 to 7 days prior 
to study session)
Study Session
Day 1
Telephone 
follow-up
Day ~2 to ~ 
Day 4
Telephone 
follow-up 
Day ~7 to 
Day ~14
Notes
Pre biopsy 
Biopsy
End of study 
session visit 
Pre- & post-
biopsy 
questionnaire to 
assess subject 
experience of 
lymph node 
biopsy
X
X
(fill in 
questionnaire 
on the day 
after the 
procedure, 
communicate 
answers via 
telephone a 
few days 
after)
SAE review
----------------------------------------------------------------------------------------------------------------------------
AE review
----------------------------------- -------------------------------------------------------------

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6.2.
Screening and Critical Baseline Assessments
The following demographic parameters will be captured: year of birth, sex, race and 
ethnicity.
Medical/medication/family history will be assessed as related to the inclusion/exclusion 
criteria listed in Section 5.
Procedures conducted as part of the subject’s routine clinical management, for example 
but not limited to laboratory assessment, and obtained prior to signing of informed 
consent may be utilized for Screening or baseline purposes provided the procedure(s) 
meet(s) the protocol-defined criteria and has (have) been performed within the timeframe 
of the study.
6.3.
Pre-& post-biopsy Questionnaire
All 
subjects 
will 
be 
asked 
to 
complete 
a 
questionnaire 
about 
their 
expectations/experiences of undergoing the procedure of fine needle aspirate biopsy 
followed by core needle biopsy. The questionnaire focuses on the motivation of taking 
part in this study and aspects associated with experience before and after the procedure. 
Please see T&E table to study timings (see Section 6.1) and SRM for the questionnaire.
6.4.
T1D auto-antibodies and other auto-antibodies
Tests for one or more antibodies will be performed at screening. These can include anti-
GAD, anti-IA 2, anti-ICA, anti-IAA, anti-ZnT8, anti-nuclear antibodies, anti-thyroid 
peroxidise antibodies, anti-tissue glutaminase antibodies.
6.5.
Fine needle aspirate & core biopsy of the inguinal lymph 
node
All study participants will undergo needle biopsies in an outpatient clinical setting by a 
radiologist. Both, iLN fine needle aspirate and iLN core biopsy will be taken after 
obtaining local anaesthesia and by using ultrasonography. First a fine-needle aspirate of a 
lymph node will be obtained and following that procedure up to five core biopsies of a 
lymph node will be acquired.
Using a standard clean technique, and with the ultrasound probe covered, local 
anaesthetic will be administered into the skin and subcutaneous tissues adjacent to the 
identified lymph node(s). The iLN will be localised using the assistance of 
ultrasonography and marked by the radiologist. For the fine-needle aspirate, an iLN will 
be localised by ultrasonography and sampled using a 21-gauge needle and a 5-ml syringe, 
as described by Tatovic et al 2015 [Tatovic, 2015]. Briefly, the LN cortex will be 
sampled using small to and fro needle movements while applying 1 ml suction with the 
syringe. One to two passages will be obtained per subject using this method.
Following the fine-needle aspirate, an approximately 0.5 cm long incision will be made 
as described by De Hair et al 2012 [de Hair, 2012]. From the same or a separate inguinal 
lymph node to the one sampled by fine-needle aspirate, up to five core biopsies (14 or 16 

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or 18 gauge) will be obtained using the assistance of ultrasonography , according to local 
standard operating procedures. All samples will be processed and stored immediately 
according to standard procedures for the various analyses (see SRM).
Following the procedures, manual pressure will be applied to the area for approximately 
5 minutes to ensure haemostasis and to reduce the risk of haematoma formation. A sterile 
dressing will be applied to the small wound. Written information about after care will be 
provided to the subject, which will include advice about wound care. 
6.6.
Wound assessment
A visual assessment of the LN biopsy site will be conducted by the investigator before 
the end of the study visit to ensure adequate haemostasis and wound management. A 
sterile adhesive dressing will be applied to the wound. Subjects will be asked to contact 
the investigator for an examination if they report any signs or symptoms related to 
persistent pain in the biopsy area or signs or symptoms indicating infection.
6.7.
Safety
Planned time points for all safety assessments are listed in the Time and Events Table 
(Section 6.1). Additional time points for safety tests (such as vital signs, physical exams 
and laboratory safety tests) may be added during the course of the study based on newly 
available data to ensure appropriate safety monitoring, but this will not constitute a 
protocol amendment. The IRB/IEC will be informed of any safety issues that require 
alteration of the safety monitoring scheme.
6.7.1.
Adverse Events (AE) and Serious Adverse Events (SAEs)
The definitions of an AE or SAE can be found in Appendix 3.
The investigator and their designees are responsible for detecting, documenting and 
reporting events that meet the definition of an AE or SAE. 
6.7.1.1.
Time period and Frequency for collecting AE and SAE information

Any SAEs assessed as related to study participation (e.g., protocol-mandated 
procedures, invasive tests) will be recorded from the time a subject consents to 
participate in the study up to and including any follow-up contact. 

AEs will be collected from: the start of study procedures until the follow-up contact 
(see Section 6.7.1.3), at the time points specified in the Time and Events Table (see 
Section 6.1).

Medical occurrences that begin prior to the start of study procedure but after 
obtaining informed consent may be recorded on the Medical History/Current 
Medical Conditions section of the CRF.

All SAEs will be recorded and reported to GSK within 24 hours, as indicated in 
Appendix 3.

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
Investigators are not obligated to actively seek AEs or SAEs in former study 
subjects. However, if the investigator learns of any SAE, including a death, at any 
time after a subject has been discharged from the study, and he/she considers the 
event reasonably related to the study procedures or study participation, the 
investigator must promptly notify GSK.
NOTE: The method of recording, evaluating and assessing causality of AEs and SAEs 
plus procedures for completing and transmitting SAE reports to GSK are provided in 
Appendix 3.
6.7.1.2.
Method of Detecting AEs and SAEs
Care will be taken not to introduce bias when detecting AEs and/or SAEs. Open-ended 
and non-leading verbal questioning of the subject is the preferred method to inquire about 
AE occurrence. Appropriate questions include:

“How are you feeling?” 

“Have you had any (other) medical problems since your last visit/contact?” 

“Have you taken any new medicines, other than those provided in this study, since 
your last visit/contact?” 
6.7.1.3.
Follow-up of AEs and SAEs
After the initial AE/SAE report, the investigator is required to proactively follow each 
subject at subsequent visits/contacts.  All SAEs, and non-serious AEs of special interest 
(as defined in Section 11.3) will be followed until resolution, until the condition 
stabilizes, until the event is otherwise explained, or until the subject is lost to follow-up 
(as defined in Section 5.4). Further information on follow-up procedures is given in 
Section 11.3.
6.7.1.4.
Death Events
The Death sections of the CRF will be required to be completed for all deaths. 
The Death CRF is provided immediately after the occurrence or outcome of death is 
reported. Initial and follow-up reports regarding death must be completed within one 
week of when the death is reported.
6.7.1.5.
Regulatory Reporting Requirements for SAEs
Prompt notification by the investigator to GSK or designee of SAEs related to study 
procedures (even for non- interventional post-marketing studies) is essential so that legal 
obligations and ethical responsibilities towards the safety of subjects (and the safety of a 
product under clinical investigation are met). 
GSK will comply with country specific regulatory requirements relating to safety 
reporting to the regulatory authority, Institutional Review Board (IRB)/Independent 
Ethics Committee (IEC) and investigators.

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Investigator safety reports are prepared for suspected unexpected serious adverse 
reactions according to local regulatory requirements and GSK policy and are forwarded 
to investigators as necessary.
An investigator who receives an investigator safety report describing a SAE(s) or other 
specific safety information (e.g., summary or listing of SAEs) from GSK will file it and 
will notify the IRB/IEC, if appropriate according to local requirements.
6.7.2.
Pregnancy

Details of all pregnancies in female subjects will be collected after the start of the 
study until completion of the study.

If a pregnancy is reported then the investigator should inform GSK within 2 weeks 
of learning of the pregnancy and should follow the procedures outlined in Section 
11.4.
6.7.3.
Physical Exams

A complete physical examination will include, at a minimum, assessment of the 
Cardiovascular, Respiratory, Gastrointestinal and Neurological systems, as well as 
palpation of lymph nodes and proposed puncture sites. Height and weight will also 
be measured and recorded. 

A proposed biopsy site review will involve palpation of lymph nodes and proposed 
puncture sites as well as an assessment of the skin around the area of the proposed 
puncture site.

Investigators should pay special attention to clinical signs related to previous serious 
illnesses
6.7.4.
Vital Signs
Vital signs will be measured in semi-supine position after approximately 5 minutes rest 
and will include temperature, systolic and diastolic blood pressure and pulse rate. These 
will be recorded in the CRF.
6.7.5.
Clinical Safety & Other Laboratory Assessments
All protocol required laboratory assessments, as defined in Table 1, must be conducted in 
accordance with the SRM, and Protocol Time and Events Schedule (see Section 6.1). 
Laboratory requisition forms must be completed and samples must be clearly labelled 
with the subject number, protocol number, site/centre number, and visit date. Details for 
the preparation and shipment of samples will be provided by the laboratory and will be 
detailed in the SRM. Reference ranges for all safety &other parameters will be provided 
to the site by the laboratory responsible for the assessments.
If additional non-protocol specified laboratory assessments are performed at the 
institution’s local laboratory and result in a change in subject management or are 
considered clinically significant by the investigator (e.g., SAE or AE) the results must be 
recorded in the CRF. 

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Information for appropriate processing and handling of samples to avoid duplicate and/or 
additional blood draws will be described in the SRM. 
All study-required laboratory assessments will be performed by a central laboratory, apart 
from:

Haematology, Clinical Chemistry, Routine Urinalysis and “Other Screening Tests” 
as described in the appropriate row of Table 1 below, which will be conducted 
locally at each site (with the exception of auto-antibody screening which may be 
conducted by a central laboratory)
NOTE:  Local laboratory results are only required in the event that the central laboratory 
results are not available in time for either a treatment and/or response evaluation to be 
performed. If a local sample is required it is important that the sample for central analysis 
is obtained at the same time. Additionally if the local laboratory results are used to make 
either a treatment or response evaluation, the results must be entered into the CRF.
Haematology, clinical chemistry, urinalysis and additional parameters to be tested are 
listed in Table 1. 

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Table 1
Protocol Required Safety & Other Laboratory Assessments
Laboratory 
Assessments
Parameters
Haematology
Platelet Count
RBC Indices:
WBC count with Differential:
RBC Count
MCV
Neutrophils
Hemoglobin
MCH
Lymphocytes
Hematocrit
Monocytes
INR (derived from 
Prothrombin time) and 
APTT
Eosinophils
Basophils
Clinical 
Chemistry 
Urea
Potassium
AST (SGOT)
Total and direct 
bilirubin
Creatinine
Sodium
ALT (SGPT)
Total Protein
Glucose1
Calcium
Alkaline phosphatise
Albumin
Routine 
Urinalysis

Specific gravity

pH, glucose, protein, blood and ketones by dipstick

Microscopic examination (if blood or protein is abnormal)
Other 
Screening 
Tests

HIV

Hepatitis B (HBsAg)

Hepatitis C (Hep C antibody)

Alcohol and drug screen 

Urine hCG Pregnancy test (as needed for women of child bearing 
potential)2

Fasted C-peptide 

HbA1c

anti-GAD, anti-IA2, anti-ICA, anti-IAA, anti-ZnT8, anti-thyroid 
peroxidase, anti-tissue transglutaminase and anti-nuclear antibody
NOTES :
1. Fasted glucose at screening, non fasted glucose on Day 1.
2. Local urine testing will be standard for the protocol unless serum testing is required 
by local regulation or ethics committee.
                                                                                                                                                                             
6.8.
Biomarker(s)
With the subject’s consent, iLN and peripheral blood samples will be collected during 
this study and used for the purposes of measuring biomarkers to aid the understanding of 
the pathogenesis of NOT1D and/or medically related conditions. If relevant, this 
approach will be extended to include the identification of biomarkers associated with 
adverse events. A peripheral blood sample may also be used for genotyping. Further 
details of experimental procedures will be provided in the SRM. 

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Blood samples (volume up to 30 ml) and up to two fine needle aspirate passages and up 
to five lymph node core biopsy samples will be collected at the time point indicated in 
Section 6.1.
Primary endpoint: Candidate biomarkers (= absolute numbers and/or proportion of 
leukocyte subsets) associated with either location of cells (i.e. iLNs derived leukocytes 
versus peripheral blood derived cells) and/or disease-status (i.e. HV versus NOT1D 
subjects) may be identified by application of flow cytometry.

Objective: To assess the frequency and phenotype of leukocyte subsets in iLN and 
peripheral blood in HVs and T1D subjects
o
Leukocyte subset phenotyping will be carried out on peripheral blood and 
iLN-derived cells by assessing expression of but not restricted to the 
following antigens; CD3, CD4, CD8, CD11c, CD14, CD16, CD19, CD24, 
CD25,  CD38, CD45RA, CD56, HLA-DR and FOXP3.
Secondary endpoint: Candidate biomarkers (=absolute numbers and/or proportion of 
leukocyte subsets) associated with type of biopsy (core biopsy versus fine needle 
aspirate) may be identified by application of flow cytometry.

Objective: To assess the frequency and phenotype of leukocyte subsets from iLN 
core biopsy versus iLN fine needle aspirate in HVs and T1D subjects:
o
Leukocyte subset phenotyping will be carried out on iLN-derived cells by 
assessing expression of but not restricted to the following antigens; CD3, 
CD4, CD8, CD11c, CD14, CD16, CD19,  CD25, CD24, CD38, CD45RA, 
CD56, HLA-DR and FOXP3
Exploratory endpoints: Novel candidate biomarkers and functional analysis of iLN-
derived cells and cells from peripheral blood will be assessed depending on the material 
available.

Objective: To assess suppression activity of T regulatory lymphocytes from the 
iLN and peripheral blood in HVs and NOT1D subjects
o
T regulatory suppression analysis may be carried out by a micro 
suppression assay or alternative equivalent. 

Objective: To assess the frequency of cytokine-producing lymphocytes in HVs 
and NOT1D subjects.
o
Percentages of lymphocytes (and subsets) producing pro- and or anti-
inflammatory cytokines may be identified by flow cytometry (for 
example, but not restricted to, Th1 and Th17 cells). 

Objective: To assess clonal expansion of T and B lymphocyte populations in HVs 
and NOT1D subjects
o
T and B lymphocyte clones may be assessed by TCR and BCR deep 
sequencing. 

Objective: To assess the gene expression fingerprint of auto-antigen specific 
lymphocytes in HVs and NOT1D subjects

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o
For analysis of autoreactive T lymphocyte function, iLN-derived 
lymphocytes and PBMCs may be stimulated with antigens such as GAD65 
or proinsulin. Gene expression of autoreactive T lymphocytes may be 
analysed by RNA (Ribonucleic Acid) sequencing (and/or equivalent 
methodologies) of sorted T cell populations. The RNAs assayed/identified 
may be those involved with the pathogenesis of T1D. 

Objective: To assess the phenotype and transcriptomic fingerprint of stromal cells 
from iLN core biopsy samples in HVs and NOT1D subjects
o
Phenotyping of stromal cells, including but not limited to fibroblastic reticular 
cells, from iLN core biopsy samples may be carried out by assessing 
expression of, but not limited to, the following antigens: CD45, CD31, gp38, 
MHC II. 
o
Gene expression of stromal cells, including but not limited to fibroblastic 
reticular cells, may be analysed by RNA sequencing (and/or equivalent 
methodologies)
All samples will be retained for a maximum of 15 years after the last subject completes 
the trial. 
6.9.
Genetics
Information regarding genetic research is included in Appendix 2.
7.
DATA MANAGEMENT

For this study, subject data will be entered into GSK defined CRFs, transmitted 
electronically to GSK or designee and combined with data provided from other 
sources in a validated data system.  

Management of clinical data will be performed in accordance with applicable GSK 
standards and data cleaning procedures to ensure the integrity of the data, e.g., 
removing errors and inconsistencies in the data.

Adverse events and concomitant medications terms will be coded using MedDRA 
(Medical Dictionary for Regulatory Activities) and an internal validated medication 
dictionary, GSKDrug.  

CRFs (including queries and audit trails) will be retained by GSK, and copies will be 
sent to the investigator to maintain as the investigator copy.  Subject initials or other 
Personally Identifiable Information (PII) will not be collected or transmitted to GSK 
according to GSK policy.

Screen failure information will be entered in to the clinical database (see also Section 
5.3)

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8.
STATISTICAL CONSIDERATIONS AND DATA 
ANALYSES
This is a non-randomized study, without an investigational drug.
All data required for the analysis of primary, secondary and exploratory objectives will 
be captured in the clinical database and included in the clinical report. Further details will 
be provided in the RAP.
8.1.
Hypotheses
This study is designed to explore the phenotype of immune cells in the iLN and 
peripheral blood (such as, but not restricted to PBMCs) of NOT1D subjects compared to 
HVs.
There are no formal hypotheses being tested due to the exploratory nature of the study. 
Primary comparisons of NOT1D subjects to HVs will be made using an estimation 
approach, providing point estimates and confidence intervals.
Secondary comparisons of biopsy methods will be made within NOT1D subjects and 
HVs using an estimation approach:

iLN biopsies will be carried out by two related methods, fine-needle aspirate and 
core biopsy sampling: 

If sufficient numbers of cells to meet the cell number requirements of the primary 
endpoint are obtained by both biopsy methods, the primary endpoint analysis will be 
carried out independently for each biopsy method, otherwise only the biopsy method 
for which sufficient numbers of cells were obtained will be used for primary 
analysis.

If no meaningful differences between biopsy methods are observed, material from 
both biopsy methods may be pooled to meet exploratory objectives 1, 2, 3 and 4. 
Material from fine-needle aspirate and core biopsies will not be pooled unless 
supported by the comparison described above.

If meaningful differences in the proportion of leukocyte subsets between cells 
derived from each biopsy method are observed, then :
o
If sufficient number of core biopsy derived cells are available, these will 
be analysed for exploratory objectives 1, 2 and 5.
o
Fine needle aspirate derived cells will be used to meet exploratory 
objectives 3 and 4. If insufficient number of core biopsy derived cells are 
available to meet exploratory objectives 1 and 2, then fine needle aspirate 
derived cells may be used to meet either or both of those exploratory 
objectives

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8.2.
Sample Size Considerations
This is an exploratory study and there are no formal sample size calculations. The upper 
limit for sample size (n=15 per group) has been based on feasibility.
8.2.1.
Sample Size Assumptions
For PBMCs, literature data was available for lymphocytes CD4+, CD8+ and regulatory 
T-cells in HVs using a similar gating strategy to that planned here. Computer simulation 
were performed to assess the expected precision of estimation of odds ratios and relative 
risks for a cross sectional comparison of PBMC regulatory T-cells for a range of 
differences between NOT1D subjects and HVs. The average precision for both PBMC 
estimates is expected to be within 1% of the point estimate using n=15 per group
8.2.2.
Sample Size Sensitivity
No formal sample size sensitivity analysis has been performed.
The precision for iLN estimates is expected to be lower than the precision for PBMC 
estimates, since it is anticipated that fewer cells will be available from iLN biopsies for 
flow cytometry analysis. No published data was found for the phenotypes of interest in 
iLN biopsies.
8.2.3.
Sample Size Re-estimation or Adjustment
Data for primary endpoints may be reviewed and/or analyzed on an ongoing basis. Based 
on this information the study team may make a recommendation to the investigator that 
sufficient data has been obtained prior to the maximum of n=15 subjects per group, such 
that further recruitment of subjects is not needed.
8.3.
Data Analysis Considerations
8.3.1.
Analysis Populations
Population
Definition / Criteria
Safety

Comprised of all subjects who complete any study assessment.
Core Biopsy

Subjects in the ‘Safety’ population for whom at least one lymph node core 
biopsy was taken
Fine needle 
aspirate 
Biopsy

Subjects in the ‘Safety’ population for whom at least one lymph node fine 
needle aspirate biopsy was taken
Biopsy

Subjects in both ‘Core Biopsy’ and ‘fine needle aspirate biopsy’ populations
Blood

Subjects in the ‘Safety’ population for whom one blood sample was taken for 
analysis of cells derived from peripheral blood.

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8.3.2.
Interim Analysis
A data look will be carried after the recruitment of a cohort of up to 5 HVs to determine 
if the quality and quantity of cells derived from either fine needle aspirate or core biopsy 
of the inguinal lymph node and from peripheral blood are likely to be sufficient to 
continue the study to meet its primary objective.
An interim analysis will be carried out after the recruitment of 5 evaluable HVs and 5 
evaluable NOT1D subjects. The primary purpose of this interim analysis will be to 
facilitate decision making and study design for a potential follow-up interventional study. 
In addition the interim analysis results would also help to enable prioritization of the 
exploratory assays and to facilitate the final RAP. Due to uncertainty around variability
and precision, the primary analyses described in Section 8.4.1 will be performed in order 
to re-assess the precision of estimates for the comparisons of interest.
8.4.
Key Elements of Analysis Plan
8.4.1.
Primary Analyses
Descriptive statistics will be calculated for each of the primary flow cytometry cell types 
by group and sample type (blood / core biopsy / fine needle aspirate biopsy). The flow 
cytometry data for each cell type will be displayed graphically using scatter plots of cell 
count and % cell count versus group and/or sample type.
Generalized linear models with appropriate link functions will be used to analyze data 
separately for each flow cytometry cell type. Models will include terms for group, sample 
type, and the interaction of group with sample type. Subject will be included as a random 
effect term.

For absolute cell counts, these models will estimate the mean cell count for each 
sample type in each group, comparing means as outlined in the primary hypothesis 
section. 

For proportional cell counts, these models will estimate the average odds (or relative 
risk) of having a particular cell type for each sample type in each group, comparing 
these odds (or relative risks) by estimating the ratios for the primary comparisons of 
interest.
All estimates will be presented with 95% confidence intervals.
Model assumptions will be checked, and modifications to the model or alternative 
approaches may be considered in the event of significant deviations from model 
assumptions.
Further details will be provided in the Reporting and Analysis Plan (RAP).

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8.4.2.
Secondary Analyses
Generalized linear models as described in Primary Analyses will be used to compare 
absolute and proportion cell counts between core biopsies and fine needle aspirate 
biopsies.
Details of this and further secondary analyses will be provided in the RAP.
8.4.3.
Other Analyses
Details of other analyses will be provided in the RAP.
9.
STUDY GOVERNANCE CONSIDERATIONS
9.1.
Posting of Information on Publicly Available Clinical Trial 
Registers
Study information from this protocol will be posted on publicly available clinical trial 
registers before enrolment of subjects begins.
9.2.
Regulatory and Ethical Considerations, Including the 
Informed Consent Process
The study will be conducted in accordance with all applicable regulatory requirements, 
and with GSK policy.
The study will also be conducted in accordance with ICH Good Clinical Practice (GCP), 
all applicable subject privacy requirements, and the guiding principles of the current 
version of the Declaration of Helsinki.  This includes, but is not limited to, the following:

IRB/IEC review and favourable opinion/approval of the study protocol and 
amendments as applicable

Obtaining signed informed consent

Investigator reporting requirements (e.g., reporting of AEs/SAEs/protocol deviations 
to IRB/IEC)

GSK will provide full details of the above procedures, either verbally, in writing, or 
both.

Signed informed consent must be obtained for each subject prior to participation in 
the study

The IEC/IRB, and where applicable the regulatory authority, approve the clinical 
protocol and all optional assessments, including genetic research.  

Optional assessments (including those in a separate protocol and/or under separate 
informed consent) and the clinical protocol should be concurrently submitted for 
approval unless regulation requires separate submission.  

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
Approval of the optional assessments may occur after approval is granted for the 
clinical protocol where required by regulatory authorities.  In this situation, written 
approval of the clinical protocol should state that approval of optional assessments is 
being deferred and the study, with the exception of the optional assessments, can be 
initiated.
9.3.
Quality Control (Study Monitoring)

In accordance with applicable regulations including GCP, and GSK procedures, 
GSK monitors will contact the site prior to the start of the study to review with the 
site staff the protocol, study requirements, and their responsibilities to satisfy 
regulatory, ethical, and GSK requirements.  

When reviewing data collection procedures, the discussion will also include 
identification, agreement and documentation of data items for which the “CRF” will 
serve as the source document.
GSK will monitor the study and site activity to verify that the:

Data are authentic, accurate, and complete.

Safety and rights of subjects are being protected.

Study is conducted in accordance with the currently approved protocol and any other 
study agreements, GCP, and all applicable regulatory requirements.
The investigator and the head of the medical institution (where applicable) agrees to 
allow the monitor direct access to all relevant documents
9.4.
Quality Assurance

To ensure compliance with GCP and all applicable regulatory requirements, GSK 
may conduct a quality assurance assessment and/or audit of the site records, and the 
regulatory agencies may conduct a regulatory inspection at any time during or after 
completion of the study. 

In the event of an assessment, audit or inspection, the investigator (and institution) 
must agree to grant the advisor(s), auditor(s) and inspector(s) direct access to all 
relevant documents and to allocate their time and the time of their staff to discuss the 
conduct of the study, any findings/relevant issues and to implement any corrective 
and/or preventative actions to address any findings/issues identified.
9.5.
Study and Site Closure

Upon completion or premature discontinuation of the study, the GSK monitor will 
conduct site closure activities with the investigator or site staff, as appropriate, in 
accordance with applicable regulations including GCP, and GSK Standard Operating 
Procedures.

GSK reserves the right to temporarily suspend or prematurely discontinue this study 
at any time for reasons including, but not limited to, safety or ethical issues or severe 
non-compliance. For multicenter studies, this can occur at one or more or at all sites.  

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
If GSK determines such action is needed, GSK will discuss the reasons for taking 
such action with the investigator or the head of the medical institution (where 
applicable). When feasible, GSK will provide advance notification to the investigator 
or the head of the medical institution, where applicable, of the impending action.

If the study is suspended or prematurely discontinued for safety reasons, GSK will 
promptly inform all investigators, heads of the medical institutions (where 
applicable) and/or institution(s) conducting the study. GSK will also promptly 
inform the relevant regulatory authorities (where applicable) of the suspension or 
premature discontinuation of the study and the reason(s) for the action.  

If required by applicable regulations, the investigator or the head of the medical 
institution (where applicable) must inform the IRB/IEC promptly and provide the 
reason for the suspension or premature discontinuation.
9.6.
Records Retention

Following closure of the study, the investigator or the head of the medical institution 
(where applicable) must maintain all site study records (except for those required by 
local regulations to be maintained elsewhere), in a safe and secure location.  

The records must be maintained to allow easy and timely retrieval, when needed 
(e.g., for a GSK audit or regulatory inspection) and must be available for review in 
conjunction with assessment of the facility, supporting systems, and relevant site 
staff.  

Where permitted by local laws/regulations or institutional policy, some or all of these 
records can be maintained in a format other than hard copy (e.g., microfiche, 
scanned, electronic); however, caution needs to be exercised before such action is 
taken.  

The investigator must ensure that all reproductions are legible and are a true and 
accurate copy of the original and meet accessibility and retrieval standards, including 
re-generating a hard copy, if required.  Furthermore, the investigator must ensure 
there is an acceptable back-up of these reproductions and that an acceptable quality 
control process exists for making these reproductions.

GSK will inform the investigator of the time period for retaining these records to 
comply with all applicable regulatory requirements.  The minimum retention time 
will meet the strictest standard applicable to that site for the study, as dictated by any 
institutional requirements or local laws or regulations, GSK standards/procedures, 
and/or institutional requirements.  

The investigator must notify GSK of any changes in the archival arrangements, 
including, but not limited to, archival at an off-site facility or transfer of ownership 
of the records in the event the investigator is no longer associated with the site.

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9.7.
Provision of Study Results to Investigators, Posting of 
Information on Publically Available Clinical Trials Registers 
and Publication
Where required by applicable regulatory requirements, an investigator signatory will be 
identified for the approval of the clinical study report. The investigator will be provided 
reasonable access to statistical tables, figures, and relevant reports and will have the 
opportunity to review the complete study results at a GSK site or other mutually-
agreeable location.
GSK will also provide the investigator with the full summary of the study results.  The 
investigator is encouraged to share the summary results with the study subjects, as 
appropriate.
GSK will provide the investigator with the randomization codes for their site only after 
completion of the full statistical analysis, if applicable.
The procedures and timing for public disclosure of the results summary and for 
development of a manuscript for publication will be in accordance with GSK Policy.
A manuscript will be progressed for publication in the scientific literature if the results 
provide important scientific or medical knowledge.

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10.
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11.
APPENDICES
11.1.
Appendix 1: Abbreviations and Trademarks
Abbreviations
AE
Adverse Event
ADA
American Diabetes Association
CD
Cluster of Differentiation (glycoprotein)
CRF
Case Report Form
DNA
Deoxyribonucleic acid
GAD
Glutamic acid decarboxylase
GSK
GlaxoSmithKline
HLA
Human Leukocyte Antigen
hCG
Human chorionic gonadotrophin
HV
Healthy Volunteers
IAA
Insulin autoantibodies
IA-2
Protein tyrosine phosphatase-related islet antigen-2
ICA
Islet-cell antigen
IGRP
Islet-specific glucose-6-phosphatase catalytic subunit-related 
protein
iLN
inguinal lymph node
IRB/IEC
Institutional Review Board / Institutional Ethics Committee
LN
Lymph node
MHC
Major Histocompatibility Complex
mRNA
messenger RNA
NMR
Nuclear Magnetic Resonance spectroscopy
NOD
Non-Obese Diabetic
NOT1D
New Onset Type 1 Diabetes Mellitus
PBMC
Peripheral Blood Mononuclear Cells
RA
Rheumatoid Arthritis
RAP
Reporting and Analysis Plan
RNA
Ribonucleic Acid
SAE
Serious Adverse Event
SmPC
Summary of Product Characteristics
SRM
Study Reference Manual
T1D
Type 1 Diabetes
TCR / BCR
T-cell Receptor / B-cell Receptor
WHO
World Health Organisation
ZnT8
Zinc transporter 8
Trademark Information
Trademarks owned by the 
GlaxoSmithKline group of companies
Trademarks not owned by the 
GlaxoSmithKline group of companies
NONE
None

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11.2.
Appendix 2: Genetic Research
Genetics – Background
Naturally occurring genetic variation may contribute to inter-individual variability in 
response to medicines, as well as an individual's risk of developing specific diseases. 
Genetic factors associated with disease characteristics may also be associated with 
response to therapy, and could help to explain some clinical study outcomes. For 
example, genetic variants associated with age-related macular degeneration (AMD) are 
reported to account for much of the risk for the condition [Gorin, 2012] with certain 
variants reported to influence treatment response [Chen, 2012]. Thus, knowledge of the 
genetic aetiology of disease may better inform understanding of disease and the 
development of medicines. Additionally, genetic variability may impact the 
pharmacokinetics (absorption, distribution, metabolism, and elimination), or 
pharmacodynamics (relationship between concentration and pharmacologic effects or the 
time course of pharmacologic effects) of a specific medicine and/or clinical outcomes 
(efficacy and/or safety) observed in a clinical study.
Genetic Research Objectives and Analyses
The objectives of the genetic research are to investigate the relationship between genetic 
variants and: 

antigen-specific T-cells in T1D and T1D susceptibility, severity, and progression 
Genetic data may be generated while the study is underway or following completion of 
the study.  Genetic evaluations may include focused candidate gene approaches and/or 
examination of a large number of genetic variants throughout the genome (whole genome 
analyses). Genetic analyses will utilize data collected in the study and will be limited to 
understanding the objectives highlighted above.  Analyses may be performed using data 
from multiple clinical studies to investigate these research objectives.
Appropriate descriptive and/or statistical analysis methods will be used. A detailed 
description of any planned analyses will be documented in a Reporting and Analysis Plan 
(RAP) prior to initiation of the analysis. Planned analyses and results of genetic 
investigations will be reported either as part of the clinical RAP and study report, or in a 
separate genetics RAP and report, as appropriate.
Study Population
Any subject who is enrolled in the study can participate in genetic research. Any subject 
who has received an allogeneic bone marrow transplant must be excluded from the 
genetic research.
Study Assessments and Procedures
A key component of successful genetic research is the collection of samples during 
clinical studies. Collection of samples, even when no a priori hypothesis has been 

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identified, may enable future genetic analyses to be conducted to help understand 
variability in disease and medicine response.  

A 6 ml blood sample will be taken for Deoxyribonucleic acid (DNA) extraction. A 
blood sample is collected at the study visit (Day 1), after the subject has been 
randomized and provided informed consent for genetic research. Instructions for 
collection and shipping of the genetic sample are described in the laboratory manual. 
The DNA from the [blood/saliva] sample may undergo quality control analyses to 
confirm the integrity of the sample. If there are concerns regarding the quality of the 
sample, then the sample may be destroyed. The blood sample is taken on a single 
occasion unless a duplicate sample is required due to an inability to utilize the 
original sample.  
The genetic sample is labelled (or “coded”) with the same study specific number used to 
label other samples and data in the study.   This number can be traced or linked back to 
the subject by the investigator or site staff. Coded samples do not carry personal 
identifiers (such as name or social security number). 
Samples will be stored securely and may be kept for up to 15 years after the last subject 
completes the study, or GSK may destroy the samples sooner.  GSK or those working 
with GSK (for example, other researchers) will only use samples collected from the study 
for the purpose stated in this protocol and in the informed consent form.  Samples may be 
used as part of the development of a companion diagnostic to support the GSK medicinal 
product.
Subjects can request their sample to be destroyed at any time.
Informed Consent
Subjects who do not wish to participate in the genetic research may still participate in the 
study.  Genetic informed consent must be obtained prior to any blood being taken.
Subject Withdrawal from Study
If a subject who has consented to participate in genetic research withdraws from the 
clinical study for any reason other than being lost to follow-up, the subject will be given a 
choice of one of the following options concerning the genetic sample, if already 
collected:

Continue to participate in the genetic research in which case the genetic DNA sample 
is retained

Discontinue participation in the genetic research and destroy the genetic DNA 
sample 
If a subject withdraws consent for genetic research or requests sample destruction for any 
reason, the investigator must complete the appropriate documentation to request sample 
destruction within the timeframe specified by GSK and maintain the documentation in 
the site study records. 

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Genotype data may be generated during the study or after completion of the study and 
may be analyzed during the study or stored for future analysis. 

If a subject withdraws consent for genetic research and genotype data has not been 
analyzed, it will not be analyzed or used for future research.  

Genetic data that has been analyzed at the time of withdrawn consent will continue 
to be stored and used, as appropriate. 
Screen and Baseline Failures
If a sample for genetic research has been collected and it is determined that the subject 
does not meet the entry criteria for participation in the study, then the investigator should 
instruct the subject that their genetic sample will be destroyed. No forms are required to 
complete this process as it will be completed as part of the consent and sample 
reconciliation process.  In this instance a sample destruction form will not be available to 
include in the site files.
Provision of Study Results and Confidentiality of Subject’s Genetic Data
GSK may summarize the genetic research results in the clinical study report, or 
separately and may publish the results in scientific journals. 
GSK may share genetic research data with other scientists to further scientific 
understanding in alignment with the informed consent. GSK does not inform the subject, 
family members, insurers, or employers of individual genotyping results that are not 
known to be relevant to the subject’s medical care at the time of the study, unless 
required by law. This is due to the fact that the information generated from genetic 
studies is generally preliminary in nature, and therefore the significance and scientific 
validity of the results are undetermined.  Further, data generated in a research laboratory 
may not meet regulatory requirements for inclusion in clinical care.

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11.3.
Appendix 3: Definition of and Procedures for Recording, 
Evaluating, Follow-Up and Reporting of Adverse Events
11.3.1.
Definition of Adverse Events
Adverse Event Definition:

In this study an AE is any untoward medical occurrence in a patient or clinical 
investigation subject, temporally associated with study procedures

NOTE: An AE can therefore be any unfavourable and unintended sign (including an 
abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally 
associated with the study procedures.
Events meeting AE definition include:

Any abnormal laboratory test results (haematology, clinical chemistry, or urinalysis) 
or other safety assessments (e.g., ECGs, radiological scans, vital signs 
measurements), including those that worsen from baseline, and felt to be clinically 
significant in the medical and scientific judgement of the investigator.

Exacerbation of a chronic or intermittent pre-existing condition including either an 
increase in frequency and/or intensity of the condition.

New conditions detected or diagnosed after study procedure even though it may have 
been present prior to the start of the study.
Events NOT meeting definition of an AE include:

Any clinically significant abnormal laboratory findings or other abnormal safety 
assessments which are associated with the underlying disease, unless judged by the 
investigator to be more severe than expected for the subject’s condition.

The disease/disorder being studied or expected progression, signs, or symptoms of 
the disease/disorder being studied, unless more severe than expected for the subject’s
condition.

Medical or surgical procedure (e.g., endoscopy, appendectomy): the condition that 
leads to the procedure is an AE.

Situations where an untoward medical occurrence did not occur (social and/or 
convenience admission to a hospital).

Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present 
or detected at the start of the study that do not worsen.

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11.3.2.
Definition of Serious Adverse Events
If an event is not an AE per definition above, then it cannot be an SAE even if serious 
conditions are met (e.g., hospitalization for signs/symptoms of the disease under study, 
death due to progression of disease, etc).
Serious Adverse Event (SAE) is defined as any untoward medical occurrence that, 
at any dose:
a.
Results in death
b.
Is life-threatening
NOTE:
The term 'life-threatening' in the definition of 'serious' refers to an event in which the 
subject was at risk of death at the time of the event.  It does not refer to an event, which 
hypothetically might have caused death, if it were more severe.
c.
Requires hospitalization or prolongation of existing hospitalization
NOTE:

In general, hospitalization signifies that the subject has been detained (usually 
involving at least an overnight stay) at the hospital or emergency ward for 
observation and/or treatment that would not have been appropriate in the physician’s 
office or out-patient setting.  Complications that occur during hospitalization are 
AEs.  If a complication prolongs hospitalization or fulfils any other serious criteria,
the event is serious.  When in doubt as to whether “hospitalization” occurred or was 
necessary, the AE should be considered serious.

Hospitalization for elective treatment of a pre-existing condition that did not worsen 
from baseline is not considered an AE.
d.
Results in disability/incapacity
NOTE:

The term disability means a substantial disruption of a person’s ability to conduct 
normal life functions.

This definition is not intended to include experiences of relatively minor medical 
significance such as uncomplicated headache, nausea, vomiting, diarrhoea, influenza, 
and accidental trauma (e.g., sprained ankle) which may interfere or prevent everyday 
life functions but do not constitute a substantial disruption.
e.
Is a congenital anomaly/birth defect
f.
Other situations:

Medical or scientific judgment should be exercised in deciding whether reporting is 
appropriate in other situations, such as important medical events that may not be 
immediately life-threatening or result in death or hospitalization but may jeopardize 
the subject or may require medical or surgical intervention to prevent one of the 

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other outcomes listed in the above definition.  These should also be considered 
serious.

Examples of such events are invasive or malignant cancers, intensive treatment in an 
emergency room or at home for allergic bronchospasm, blood dyscrasias or 
convulsions that do not result in hospitalization, or development of drug dependency 
or drug abuse.
11.3.3.
Recording of AEs and SAEs
AEs and SAE Recording:

When an AE/SAE occurs, it is the responsibility of the investigator to review all 
documentation (e.g., hospital progress notes, laboratory, and diagnostics reports) 
relative to the event.

The investigator will then record all relevant information regarding an AE/SAE in 
the CRF

It is not acceptable for the investigator to send photocopies of the subject’s medical 
records to GSK in lieu of completion of the GSK, AE/SAE CRF page.

There may be instances when copies of medical records for certain cases are 
requested by GSK.  In this instance, all subject identifiers, with the exception of the 
subject number, will be blinded on the copies of the medical records prior to 
submission of to GSK.

The investigator will attempt to establish a diagnosis of the event based on signs, 
symptoms, and/or other clinical information.  In such cases, the diagnosis will be 
documented as the AE/SAE and not the individual signs/symptoms.

Subject-completed questionnaires and the collection of AE data are independent 
components of the study.

Responses to each question in the questionnaire will be treated in accordance with 
standard scoring and statistical procedures detailed by the scale’s developer.
11.3.4.
Evaluating AEs and SAEs
Assessment of Intensity
The investigator will make an assessment of intensity for each AE and SAE reported 
during the study and will assign it to one of the following categories:

Mild:  An event that is easily tolerated by the subject, causing minimal discomfort 
and not interfering with everyday activities.

Moderate:  An event that is sufficiently discomforting to interfere with normal 
everyday activities

Severe:  An event that prevents normal everyday activities. - an AE that is assessed 

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as severe will not be confused with an SAE. Severity is a category utilized for rating 
the intensity of an event; and both AEs and SAEs can be assessed as severe.

An event is defined as ‘serious’ when it meets at least one of the pre-defined 
outcomes as described in the definition of an SAE.
Assessment of Causality

The investigator is obligated to assess the relationship between study procedure and 
the occurrence of each AE/SAE.

A "reasonable possibility" is meant to convey that there are facts/evidence or 
arguments to suggest a causal relationship, rather than a relationship cannot be ruled 
out.

The investigator will use clinical judgment to determine the relationship.

Alternative causes, such as natural history of the underlying diseases, concomitant 
therapy, other risk factors, and the temporal relationship of the event to the study 
procedure will be considered and investigated.

The investigator will also consult the Investigator Brochure (IB) and/or Product 
Information (where applicable), for marketed products, in the determination of 
his/her assessment.

For each AE/SAE the investigator must document in the medical notes that he/she 
has reviewed the AE/SAE and has provided an assessment of causality.

There may be situations when an SAE has occurred and the investigator has minimal 
information to include in the initial report to GSK.  However, it is very important 
that the investigator always make an assessment of causality for every event 
prior to the initial transmission of the SAE data to GSK.

The investigator may change his/her opinion of causality in light of follow-up 
information, amending the SAE data collection tool accordingly.

The causality assessment is one of the criteria used when determining regulatory 
reporting requirements.
Follow-up of AEs and SAEs

The investigator is obligated to perform or arrange for the conduct of supplemental 
measurements and/or evaluations as may be indicated or as requested by GSK to 
elucidate as fully as possible the nature and/or causality of the AE or SAE.

The investigator is obligated to assist.  This may include additional laboratory tests 
or investigations, histopathological examinations or consultation with other health 
care professionals.

If a subject dies during participation in the study or during a recognized follow-up 
period, the investigator will provide GSK with a copy of any post-mortem findings, 

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including histopathology.

New or updated information will be recorded in the originally completed CRF.

The investigator will submit any updated SAE data to GSK within the designated 
reporting time frames.
11.3.5.
Reporting of SAEs to GSK
SAE reporting to GSK via electronic data collection tool

Primary mechanism for reporting SAEs to GSK will be the electronic data collection 
tool

If the electronic system is unavailable for greater than 24 hours, the site will use the 
paper SAE data collection tool and fax it to the Medical Monitor.

Site will enter the serious adverse event data into the electronic system as soon as it 
becomes available.

The investigator will be required to confirm review of the SAE causality by ticking 
the ‘reviewed’ box at the bottom of the eCRF page within 72 hours of submission of 
the SAE.

After the study is completed at a given site, the electronic data collection tool (e.g., 
InForm system) will be taken off-line to prevent the entry of new data or changes to 
existing data

If a site receives a report of a new SAE from a study subject or receives updated data 
on a previously reported SAE after the electronic data collection tool has been taken 
off-line, the site can report this information on a paper SAE form or to the [Medical 
Monitor or the SAE coordinator] by telephone.

Contacts for SAE receipt can be found at the beginning of this protocol on the 
Sponsor/Medical Monitor Contact Information page.

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11.4.
Appendix 4: Collection of Pregnancy Information
The investigator will collect pregnancy information on any female subject who becomes 
pregnant while participating in this study. The investigator will record pregnancy 
information on the appropriate form and submit it to GSK within 2 weeks of learning of a 
subject's pregnancy. The subject will also be followed to determine the outcome of the 
pregnancy. Information on the status of the mother and child will be forwarded to GSK. 
Generally, follow-up will be no longer than 6 to 8 weeks following the estimated delivery 
date. Any premature termination of the pregnancy will be reported.
While pregnancy itself is not considered to be an AE or SAE, any pregnancy 
complication or elective termination of a pregnancy for medical reasons will be recorded 
as an AE or SAE.
A spontaneous abortion is always considered to be an SAE and will be reported as such.
Furthermore, any SAE occurring as a result of a post-study pregnancy and is considered 
reasonably related to the study treatment/procedure by the investigator, will be reported 
to GSK as described in Appendix 3. While the investigator is not obligated to actively 
seek this information in former study participants, he or she may learn of an SAE through 
spontaneous reporting.

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11.5.
Appendix 5: Country Specific Requirements
No country-specific requirements exist.

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11.6.
Appendix 6: Protocol changes
This amendment applies to all sites.
11.6.1.
Amendement No. 3
General summary of changes:

Extended the interval between the initial diagnosis and Day 1, from 6 weeks to 8 
weeks following recruitment challenges. 8 weeks is the upper limit for the 
NOTID population and does not affect the scientific rationale for the study.  The 8 
week change was updated throughout the protocol.

Clarified exclusion criteria, vaccination ≤ 28 days before day 1.

Interim Analysis: Added additional information for the  Interim Analysis after the 
recruitment of 5 evaluable HVs and 5 evaluable NOTID subjects. This will 
facilitate the decision making of the study and prioritization of the exploratory 
assays.

Clarified fasting at screening in the Time and Event table and added footnote to 
Table 1 to clarify fasted glucose at screening and non fasted glucose on Day 1
List of specific changes: 
Section 1: Overall Design. Updated schematic to reflect extended screening window 
from Day~ -42 to Day~ -56. The change was also applied to Section 4: Study Design 
and Section 6.1: Time and Events Table.
Original:

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Amended: 
Section 1: Overall Design. Last sentence in second bullet point, changed the total 
duration of the study from 8 weeks to 10 weeks. This change was also applied to
Section 4. Study Design.
Original:

After informed consent, each subject will attend a screening visit followed by a study 
visit for iLN biopsy and peripheral blood collection (Day 1). Follow up phone calls 
after the study visit will be carried out at approximately Day 2-4 and Day 7 to 14. 
The total duration of this study will be up to 8 weeks, including the screening period.
Amended:

After informed consent, each subject will attend a screening visit followed by a study 
visit for iLN biopsy and peripheral blood collection (Day 1). Follow up phone calls 
after the study visit will be carried out at approximately Day 2-4 and Day 7 to 14. 
The total duration of this study will be up to 8 10 weeks, including the screening 
period.
Section 1: Overall Design, added Interim Analysis (4th bullet point). This was also 
added to Section 4 (5th bullet point).
Added text:

An interim analysis will be carried out after the recruitment of 5 evaluable HVs and 
5 evaluable NOT1D subjects. The primary purpose of this interim analysis will be to 
facilitate decision making and study design for a potential follow-up interventional 
study.

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Section 5: Selection of study population and withdrawal criteria. Changed 
timeframe of the inclusion criteria to 8 weeks. This change also applies to section 5.1 
NOTID subjects bullet point number 4.
Original:
Subjects may be re-screened for the study, provided that re-screening can be 
accomplished to meet the timeframe of the inclusion criteria of a subject entering the 
study, i.e. with an interval of up to 6 weeks between the initial diagnosis and day 1 of the 
study (see Section 5.1).
Amended:
Subjects may be re-screened for the study, provided that re-screening can be 
accomplished to meet the timeframe of the inclusion criteria of a subject entering the 
study, i.e. with an interval of up to 86 weeks between the initial diagnosis and day 1 of 
the study (see Section 5.1).
Section 5.1.
Original:
Amended:
Section 5.2. Exclusion Criteria: Concomitant medications: Bullet point number 14. 
Deleted i.e. from screening visit to last telephone call to make clearer exclusion 
period applied to before Day 1.
Original:
Amended:

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Section 6.1. Time and Events Table. Screening days updated in header and clarified 
fasting at screening and non fasting on Day 1 in the notes associated to laboratory 
assessments.
Original:
Amended:

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Section 6. Table 1: Clinical Chemistry added footnote to clarify fasted glucose at 
screening, non fasted glucose on Day 1.
Original:
Amended:
Section 8.3.2. Interim Analysis.  Added additional text for interim analysis.
Original:

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Amended:
11.6.2.
Amendement No. 2
General summary of changes:

Time and Event table: Duplicate assesment removed and updated (medical 
history/past current medical conditions and laboratory assessments). Clarified when 
telephone call for Follow Up Period 1 would be collected. Clarified genetics testing 
was optional in consenting subjects. Added SAE/AE review.

Added HbA1c to Clinical Chemistry in Table 1.
List of specific changes: 
Section 6.1. Time and Events table

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Original:
Amended:

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Orignal:
Amended:
Original:
Amended:

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Section 6.7.5, Table 1:
Added HbA1c test to other screening test to support inclusion criteria and changed Fasted 
C-peptide levels to Fasted C-peptide.
11.6.3.
Amendment No. 1
General summary of changes:

Objective "To assess the biochemical fingerprint derived from metabonomic 
analyses of peripheral blood in HVs and NOT1D subjects" & associated endpoint 
removed in Section 3. References to this objective and endpoint in Section 6.1; and 
Section 6.8 were removed accordingly. This objective and associated endpoint were 
removed because they should be addressed in separate studies in the future.

New exploratory objective (“To assess the phenotype and transcriptomic fingerprint 
of stromal cells from iLN core biopsy samples in HVs and NOT1D subjects”) and 
associated endpoint added in Section 3, Section 6.8 and Section 8.1 were updated 
accordingly. This objective and associated endpoint were added to provide additional 
insights into the exploration of the peripheral immune system in subjects with New 
Onset T1 Diabetes, which were not envisaged during the finalisation of the original 
protocol.

The Questionnaire in Appendix 1 was modified to avoid any duplication with 
Informed Consent procedures and duplication of data collection in eCRF. The entire 
questionnaire has been moved from the Study protocol to the Study Reference 
Manual (SRM) and is therefore not present in the amended protocol anymore. 

All appendix numberings and in-text references to appendices have been updated to 
reflect that the Lymph Node Questionnaire is no longer located in Appendix 1 of the 
Study Protocol.

Clarifying wording was added and typos were corrected in Section 6.2; Section 6.7.5 
and Section 6.8.

Appendix 1 - Abbreviations and Trademarks lists were updated.

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List of specific changes: 
Original:
Amended & Rationale for amendment
Section 3, Objectives & Endpoint:
5) To assess 
the 
biochemical 
fingerprint 
derived from 
metabonomic 
analyses of 
peripheral 
blood in HVs 
and NOT1D 
subjects

List of 
bioche
mical 
markers 
/ 
biomark
ers 
(immun
e &
non-
immune 
function 
related) 
derived 
from 
analysis 
of 
peripher
al blood 
Section 6.1 Time and Events Table, 
Procedure 10:

Peripheral blood collection for 
cell isolation and metabonomics 
analyses
5) To assess 
the phenotype 
and 
transcriptomic 
fingerprint of 
stromal cells 
from iLN core 
biopsy 
samples in 
HVs and 
NOT1D 
subjects

Absolute 
number 
and/or 
proportion 
of stromal 
cell subsets
and gene 
expression 
levels of 
stromal cell 
subsets from 
iLN core 
biopsy 
samples 
Section 6.1 Time and Events Table, Procedure 
10:

Peripheral blood collection for cell 
isolation and metabonomics analyses
The original exploratory objective & endpoint 
were removed because they should be 
addressed in separate studies in the future. 
A new exploratory objective & endpoint were 
added to provide additional insights into the 
exploration of the peripheral immune system 
in subjects with New Onset T1 Diabetes, 
which were not envisaged during the 
finalisation of the original protocol.
Section 11.1, Appendix 1: Lymph Node 
Questionnaire: Core-needle Biopsy and 
Fine Needle Aspirate of inguinal lymph 

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nodes
Part 1:

Please check the appropriate box 
to identify which study group you 
belong to:
□
Newly Diagnosed Diabetes 
Mellitus subject 
□
Healthy volunteer
1. Gender:
□
Male
□
Female
2. Age:..............……………… years
3. Did 
you 
ever 
undergo 
a 
procedure 
under 
local 
anaesthetics?
□
No Go to question 5

6. Please circle the appropriate box 
below to indicate how well you are 
prepared for the lymph node biopsy 
procedure, with respect to:
a.
understanding the aim of the 
procedure and the background 
information
b. what 
to 
expect 
during 
the 
procedure
c.
after-care
d. possible complications
Part 2:
1. With the benefit of hindsight, 
what aspects do you feel could 
have been better explained about 
the procedure:
□ None, I am content with 
the given information and 
how it was discussed with 
me
Or:
More 
detailed 

Please check the appropriate box 
to identify which study group you 
belong to:
□
Newly 
Diagnosed 
Diabetes 
Mellitus subject 
□
Healthy volunteer
1. Gender:
□
Male
□
Female
2. Age:..............……………… years
3. Did you ever undergo a procedure 
under local anaesthetics?
□
No Go to question 3

6. Please circle the appropriate box below 
to indicate how well you are prepared for 
the lymph node biopsy procedure, with 
respect to:
a.
understanding 
the 
aim 
of 
the 
procedure 
and 
the 
background 
information
b. what 
to 
expect 
during 
the 
procedure
c.
after-care
d. possible complications
1. With 
the 
benefit 
of 
hindsight, 
what aspects do you feel could 
have been better explained about 
the procedure:
□ None, I am content with 
the given information and 
how it was discussed with 
me
Or:
More 
detailed 

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explanations about:
□ The procedure itself
□ Anaesthetic procedure 
□ After-care
□ Complications to be 
expected
explanations about:
□ The procedure itself
□ Anaesthetic procedure 
□ After-care
□ 
Complications 
to 
be 
expected
Part 1 study group and age information, Part 
1 question 6 and Part 2, Question 1 (“After 
care” option) were deleted to avoid any 
duplication of data collection in eCRF and 
duplication with Informed Consent procedures 
respectively. A cross-reference in Part 1 
question 3 was updated to reflect updated 
numbering 
of 
questions. 
The 
entire 
questionnaire has been removed from the 
protocol and added to the SRM.
Section 6.2, Screening and Critical 
Baseline Assessments; Paragraph 3, 
sentence 1:

Procedures conducted as part of the 
subject’s routine clinical 
management, for example but not 
limited to blood count, and obtained 
prior to signing of informed consent 
may be utilized for Screening or 
baseline purposes provided the 
procedure(s) meet(s) the protocol-
defined criteria and has (have) been 
performed within the timeframe of 
the study

Procedures conducted as part of the 
subject’s routine clinical management, for 
example but not limited to blood count
laboratory assessment, and obtained prior 
to signing of informed consent may be 
utilized for Screening or baseline purposes 
provided the procedure(s) meet(s) the 
protocol-defined criteria and has (have) 
been performed within the timeframe of 
the study.
Clarified wording by using a more general 
example of subject’s routine “clinical 
management”.
Section 6.7.5, Clinical Safety Laboratory 
Assessments
Paragraph 1, sentence 1:

All protocol required laboratory 
assessments, as defined in Table 
1, must be conducted in 
accordance with the Laboratory 
Section 6.7.5, Clinical Safety & Other 
Laboratory Assessments
Clarified wording by adding “other” to both 
Section Title; in paragraph 1, sentence 4 and 
Table 1 Title.

All protocol required laboratory 
assessments, as defined in Table 1, 
must be conducted in accordance with 
the Laboratory Manual (and/or SRM)

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Manual (and/or SRM), and 
Protocol Time and Events 
Schedule (see Section 6.1).
and Protocol Time and Events 
Schedule (see Section 6.1).
Clarified wording to reflect that the 
Laboratory manual will be included in the 
SRM.
Section 6.8, Biomarkers
Paragraphs 4 and 6:

[...]by assessing expression of but 
not restricted to the following
antigens; CD3, CD4, CD8, 
CD11c, CD14, CD16, CD19, 
CD24, CD25, CD24, CD38, 
CD45RA, CD56, HLA-DR and 
FOXP3

[...]by assessing expression of but 
not restricted to the following 
antigens; CD3, CD4, CD8, 
CD11c, CD14, CD16, CD19, 
CD24, CD25, CD24, CD38, 
CD45RA, CD56, HLA-DR and 
FOXP3
Deleted duplication of antigen “CD24” from 
list of antigens.