CyTOF analysis of novel mucosal immune cells in Inflammatory bowel disease: relation with disease phenotype and location.

Inflammatory bowel disease (IBD) is a complex disease characterized by chronic,
idiopathic inflammation of the intestine. The etiology of IBD is
multifactorial, and depends upon the interaction of the host*s genetic
susceptibility, microbial antigens and adjuvants, environmental triggers that
initiate responses, and effector immune responses (1). Crohn*s disease (CD)
and ulcerative colitis (UC) represent the two main forms of IBD (Table 1).
These entities show partly pathognomonic differences, but in 10%-15% of cases a
clear assignment of the sub entity is not possible using established diagnostic
criteria resulting in an undeterminate colitis (2). To classify the disease, to
facilitate comparability in clinical trials and to identify disease subtypes we
use the Montreal Classification (Table 2) for adults and the Paris
classification for children (Table 3) (3,4). In comparison with adult onset
IBD, paediatric onset CD and CU tend to be more extensive and therefore more
often associated with severe exacerbations and worse prognosis (5).
The intestinal immune system is the most prominent defense mechamism of our
body and recent developments indicate proficient specialization within this
immune compartment. Changes in distribution of the innate, adaptive en
innate-like like immune cells at different sites of the intestine and their
contribution in the immune defense have become an important focus of research
(6).
In preliminary studies we defined four subsets of innate lymphocytes
(CD45+CD7+CD3-CD14-CD19-) in the human intestinal epithelium, distinguished by
the presence or absence of CD56 and IL-7R* (CD127) (7). Of these four subsets,
the CD56+CD127- subset bears strong resemblance to the recently described
innate-like lymphoid cells (ILC) type 1 in the epithelium. To investigate the
full diversity of intestinal innate lymphocytes and their relationship with the
ILC family, we applied high-parameter mass cytometry (cytometry by
time-of-flight; CyTOF). To this end, we designed a CyTOF panel with 36
monoclonal antibodies recognizing lineage markers, activation markers, and
chemokine and cytokine receptors, including several markers commonly used to
identify ILCs. This panel was used to define and compare the phenotypic
diversity of innate and adaptive lymphocytes in intestinal biopsies and blood
samples from non-diseased individuals and from patients with inflammatory
intestinal diseases (i.e. celiac disease, RCDII and fistulizing Crohn*s
disease). By combining bio-informatics tools that allow unsupervised
hierarchical clustering (SPADE) and nonlinear dimensionality reduction (viSNE),
we were able to visualize innate and adaptive lymphocytes at single-cell
resolution in a single map that took into account all phenotypic markers
concurrently. From our antibody panel, 20 markers were differentially expressed
by innate lymphocytes, including the cell-surface markers CD11c, CD45RA, CD8a,
c-Kit (CD117), CD161, NKp46 and several cytokine receptors. We observed two
distinct types of marker expression profiles within the innate lymphocyte
compartment: (1) Gradients of marker expression indicative of transitional
states between cell populations. (2) Highly restricted expression profiles
indicative of phenotypically distinct cell populations that were automatically
identified in an unbiased, data-driven manner with ACCENSE analysis. The latter
was indicative for the existence of at least five innate lymphocyte subsets in
the intestinal epithelium. In addition, we observed highly distinctive
disease-specific expression profiles both within the adaptive and innate
lymphocyte compartments (8). Therefore, CyTOF seems to offer unprecedented
depth in the analysis of cellular heterogeneity of both the innate and adaptive
lymphocyte compartment present in intestinal biopsies. We would like to perform
additional studies in order to obtain functional insight into the role of
innate and adaptive subpopulations in inflammatory bowel disease.

Until recently, high-dimensional cellular analysis implied the loss of tissue
context as they were mostly performed in single-cell suspensions. So also with
the technique of single cell CyTOF (mass cytometry) now used for these
analyses. Now the advent of imaging mass cytometry, next to single cell mass
cytometry, introduced the possibility to simultaneously detect a multitude of
cellular markers in tissue sections. Since we have a great expertise on both
techniques in our group we would like to apply this imaging technique also on
this cohort next to single cell analysis.
Besides that, we would like to strengthen our findings on protein level and
therefore we would like to also perform RNA-sequencing. This is a high-fidelity
way to look at the genes that are being actively expressed in cells and
complements really well the results on protein level.

1. Comprehensive phenotypic analysis and comparison of mucosal immune
compartment in patients and controls ex vivo (year 1-3);
2. Comprehensive phenotypic analysis and comparison of mucosal stromal cell
compartment in patients and controls ex vivo (year 1-3);
3. Comparison of immune and stromal cell compartments between pediatric and
adult patients (year 1-3);
4. Determination of correlates between changes in immune and stromal cell
compartments and response to therapy (year 2 and 3);
5. Comparison of the immune cell compartments between naïve patients and
patients with active disease already on treatment to determinate the response
on treatment.
6. Functional analysis of selected immune and stromal cell subsets based on
analysis in 1-4 above (year 3-4).

We will perform a case-control study in patients with both Crohn*s disease and
ulcerative colitis age 10-40 years old. *Healthy* controls, also age group
10-40 years, will be patients undergoing endoscopy for reasons unrelated to CD,
UC or microscopic colitis.
Patients will be recruited at the IBD outpatient clinic, both adults and
pediatric department.
The study duration will be 36 months.

Deliverables:
1. Inclusion of 25 CD patients / 25 UC patients / 15 controls at month 12.
2. Inclusion of additional 25 CD patients/ 25 UC patients / 15 controls and
interim-analysis at month 24.
3. Additional inclusion of patients with active disease or in remission (on
treatment): 35 CD patients / 35 UC patients / 20 controls at month 48.
4. Determination of correlates between cellular composition and response to
treatment at month 48.
5.Functional analysis and submission of manuscript(s) at month 60.


For population B a subject must meet all of the following criteria:
- Age 18-40 years old.
- A history of ileocoecal resection for Crohn*s disease, without any
immunosuppressive medication since operation.
- Endoscopic characteristics of an active Crohn*s disease at colonoscopy

For population C a subject must meet all of the following criteria:
- Age 18-40 years old.
- A history of ileoanal pouch reconstruction for ulcerative colitis without any
immunosuppressive medication or antibiotics during last 3 months
- A diagnosis of pouchitis during endoscopy

Minimal bleeding risk by taking 8 extra biopsies.
In adults there is a limited risk to taking biopsies like in children. Taking
biopsies during endoscopy can cause intra-intestinal or intramural haemorrhage,
or even perforation. The risk is estimated to be < 1:10000. There is no
additional risk in sampling an extra 10 ml of blood.