*Cellular parameters predict favourable response treatment with vedolizumab?*

Disruption of intestinal homeostasis lies at the heart of a variety of
intestinal disorders in man including Crohn*s disease, a chronic inflammatory
disorder of the gastrointestinal tract that can affect both the distal ileum
and colon. While the actual disease trigger is unknown, there is strong
evidence that a combination of genetic and environmental factors lead to
impaired barrier function in the intestine, allowing translocation of bacterial
products and/or commensals into the mucosal tissue which leads to immune
activation and inflammation. In the absence of appropriate regulatory
mechanisms this can lead to chronic inflammation affecting the intestinal
mucosa. Consequently, many studies have focussed on the role of the mucosal
immune system in CD. The prominent role of the immune system is evidenced by
the strong involvement of a variety of cytokines, including macrophage derived
IL12, IL6, IL23 and TNF* which impacts on adaptive Th1 and Th17 T cell subsets,
leading to the secretion of the pro-inflammatory cytokines IFN*, IL17 and IL21.
Despite significant improvements in the management of CD, remission is often
difficult to maintain. Consequently, there is significant morbidity in many
patients. In addition, due to unknown causes the incidence of childhood CD is
rising. Novel therapeutic approaches are therefore highly desirable and need to
be explored.
Fistulisation in Crohn*s disease (CD) is a major problem which can result in
considerable morbidity. Around 35% of the CD patients have at least one fistula
episode. Of these, 54% is perianal and 9% is rectovaginal [1]. Patients with
perianal fistulas can present with symptoms such as constant anal pain or pain
after defecation, (painful) swelling around the anus, continuous (malodourous)
discharge of pus and/or blood from the external opening with skin irritation
around the anus, fever and even incontinence [2]. Patients with colonic and
active rectal disease have more frequently perianal fistulas compared to
patients with isolated ileal or ileocolonic disease [1,3-8]. Male gender, age
at diagnosis of CD and smoking are risk factors for the development of perianal
fistulas although data are conflicting [1,3,5,7-12]. While a range of potent
drugs and advanced surgical techniques are available nowadays, the treatment of
perianal fistulising CD remains challenging.
The Accent II trial and subgroup analysis of the CHARM study have demonstrated
the efficacy of anti-TNF* with varying degrees of success in achieving closure
of the fistula benefitting up to one half of patients [13,14]. Previous studies
have suggested that combined surgical drainage and anti-TNF* can result in
closure of the fistula in 40*70% of patients [15-18]. Recently Sandborn et al.
[19] reported the results of the first induction and maintenance trials of
vedolizumab, an *4*7-integrin specific antibody. This antibody inhibits the
interaction between immune cells and the intestinal vasculature resulting in a
decreased migration of systemic leukocytes to the inflamed intestinal tissues.
Although the number of patients with fistulas at baseline in this trial was
quite low (165/1115; 14.8%) and the number of patients available for evaluation
at week 52 even lower (n = 57), vedolizumab every 8 weeks resulted in a
significant higher closure rate (41.2%) compared to placebo (11.1%) (p = 0.03)
[19]. Due to the highly variable clinical outcome, however, there is a strong
need to determine biomarkers that correlate with response to treatment.
Next to a prominent role for the adaptive immune system, recent studies have
indicated a crucial role for innate lymphoid cells (ILCs) in inflammatory bowel
diseases as well. ILC have been found in mucosal tissues and are functionally
specialized cells characterized by the expression of lineage-defining
transcription factors: cytotoxic Eomes+ cells (cNK), IFN*-producing T-bet+
cells (ILC1), IL-5-producing GATA-3+ cells (ILC2) and IL-22-producing ROR*t+
cells (ILC3) [20]. Each subset displays highly distinct functions that appear
to parallel those of T-helper cells subsets with pro- (ILC1) or
anti-inflammatory properties (ILC3). Strikingly, these cells display plasticity
as evidence has been presented that in response to cytokines ILC3 cells can
differentiate into ILC1 cells and vice versa, implying that they will modulate
immune responses depending on the local cytokine milieu. Moreover, in our
recent 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) [21]. 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 lymphocytes and their functions, we have most
recently applied high-parameter mass cytometry (cytometry by time-of-flight;
CyTOF). To this end, we designed a CyTOF panel with 32 monoclonal antibodies
recognizing lineage markers, activation markers, and chemokine and cytokine
receptors. 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 and fistulizing Crohn*s disease). By combining
bio-informatics tools that allow unsupervised hierarchical clustering (SPADE)
[22] and nonlinear dimensionality reduction (viSNE) [23], 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.
In our initial analysis we observed highly distinctive tissue and
disease-specific expression profiles both within the adaptive and innate
lymphocyte compartments. For example, a distinct central memory T cell subset
was found to be exclusively present in small intestinal biopsy material of
patients with celiac disease while an ILC3 population was exclusively
identified in rectum biopsy material of CD patients in remission. Moreover, in
a pilot study (7 patients), we analyzed material from the fistula (obtained
from the surgeon during examination under anesthesia). Results of the immune
system composition demonstrate that there is a dominant myeloid cell population
(average 66.7%) present in the fistulas of five patients while in the fistulas
of the two other patients lower percentages of myeloid cells were found (30%)
while a higher percentage of T cells was observed, indicative of inter-patient
variability that could correlate with response to treatment with vedolizumab as
this would block the migration of pro-inflammatory T cells to the fistula. More
general, the analysis revealed heterogeneity within the mucosal immune system
which is far greater than previously appreciated. Therefore, CyTOF technology
offers unprecedented depth in the analysis of cellular heterogeneity of both
the innate and adaptive lymphocyte compartment present in intestinal biopsies.
This offers a unique opportunity to determine if cellular parameters can be
identified that correlate with and predict response to treatment, an important
step towards personalized and (cost-) effective treatment strategies.

Next to the involvement of the mucosal immune system, there is significant
evidence for an important role of the stromal cell compartments, including the
epithelium, in the maintenance of intestinal homeostasis. The epithelium not
only forms a barrier between the intestinal lumen and the underlying submucosa,
but also has important functions in the specific defence against invading
pathogens as evidenced by the secretion of antimicrobial peptides, the
expression of pattern recognition receptors and the ability to produce and
respond to a variety of cytokines. Next to the epithelium, stromal cell
populations of mesenchymal origin are found throughout the intestinal mucosa
and their phenotype is location dependent. In the small intestinal villi, for
example, stromal myofibroblasts form a subepithelial layer just below the basal
membrane in the lower part of the villi while towards the tip of the villi
fibroblasts occupy this niche, indicating specialized functions. In this niche
both myofibroblasts and fibroblasts can establish close contacts with all
immune components in the lamina propria and epithelium as well as with the
epithelial cells themselves. There is emerging evidence that these CD45-CD90+
myofibroblasts can play a crucial role in the maintenance and breaking of
intestinal homeostasis as they express a variety of pattern recognition
receptors, can act as non-professional antigen presenting cells and produce and
respond to a variety of cytokines. In fact, the immune modulatory properties of
mesenchymal stromal cells are well established. Exploration of the phenotypical
and functional heterogeneity in patients with CD is thus highly warranted as
this may reveal additional biomarkers relevant to response to treatment. A
special CyTOF antibody panel designed for this purpose is currently available.

To exploit CyTOF technology and carry out an in depth investigation of the
phenotypical and functional heterogeneity of the innate and adaptive immune
cell compartments and the stromal cell compartments present in rectum biopsies
of patients with CD with perianal fistulas before (week 0) and after treatment
with vedolizumab (week 24) and to correlate these data with the clinical
outcome of the patients at week 24.

Pilot study.
Patients with CD with peri-anal fistulas, who will start with vedolizumab
treatment, age group 18-75 years old, who undergo endoscopy, will be included
for taking biopsies for the current protocol. After treatment of 24 weeks this
prcedure is repeated.
The biopsies will be evaluated by CyTOF.

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.