Post on 11-Aug-2020
transcript
EVERY STEP OF THE WAY
DISCOVERY
Complex Biology In Vitro Assays: Immunology T Cell Transfer Model of Inflammatory Bowel Disease (IBD)The inflammatory bowel diseases (IBDs) of Crohn’s disease & ulcerative colitis are idiopathic chronic
inflammatory disorders of the intestine and/or colon.
No single animal model completely recapitulates the clinical and histopathological characteristics of
human IBD, but the following components are required to model human disease:
1. Chronic gut inflammation largely mediated by T lymphocytes (T cells)
2. Presence of commensal enteric bacteria, required for the initiation and perpetuation of intestinal and/
or colonic inflammation
3. The genetic background of the animal represents an important modulator/modifier of disease onset
and severity1
To identify and test novel therapies targeted at regulating the immunological mechanisms responsible
for the induction, perpetuation, and/or regulation of IBD as well as the role of T-regulatory cells (Treg),
T cell-dependent models of IBD are significantly more relevant to human disease than are the erosive,
chemically-induced self-limiting models of acute colitis.
Adoptive transfer of CD4+CD45RBhigh T cells (naive T cells, depleted of the Treg population) from healthy
wild-type (WT) mice into syngeneic recipients that lack T and B cells induces a pancolitis and small
bowel inflammation at 5-8 weeks following T cell transfer2-7.
SummaryAdoptive transfer of a subset of
naive CD4+ T cells to syngeneic
SCID or Rag-knock-out mice
results in the development of
the chronic, progressive colitis
and wasting seen in patients
with IBD. This model is used
extensively for studying the
immunologic background for the
disease and testing new drug
candidates.
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• Isolate CD4+CD45RBhigh and CD4+CD45RBlow cells (Control)
• Transfer cells to recipient Rag2-/- mice
Clinical readouts:• Body weight• Clinical scores• Stool consistency
Readouts post termination:• Colon length• Histopathology• Flow cytometry (LP, MLN)• Cytokines (Luminex)• Gene expression (Nanostring, QPCR)
Day 0 Day 47
Table 1. Assay Workflow
Figure 1. Purity of sorted T cells pre-transfer to Rag2-/- recipients. Cells were stained with CD4, CD45RB and CD62L and sorted into CD4+CD45RB low and
CD4+CD45RB high populations. (A) Purity of pre-sorted (red histogram) and pre-transferred cells (orange and blue histograms). (B) CD62L expression within the
pre-sorted cells (red histogram), CD4+CD45RB low (Orange histogram) and CD4+CD45RB high (Blue histogram) sorted subsets.
100 101 102 103 104 105
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Pre-sort CD4+CD45RB low
CD4+CD45RB high
Assay Principle Naïve CD4+ T cells from C57Bl/6 are enriched by MACS® sorting (Miltenyi Biotec) and FACS sorted based on CD45RB using
FACSAria™ Fusion. These CD4+CD45RBhigh cells are then transferred into RAG KO mice on Day 0. CD4+CD45RBlow T cells
serve as experimental controls (Figure 1). Abatacept (α-CTLA4) is included as a drug control.
A B
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In Vivo Readouts - Clinical Observations and ScoresFrom Day 0, animals are monitored daily for non-specific clinical signs and weighed regularly. From 3 weeks post adoptive
transfer until the end of the experiment, animals are monitored daily for clinical signs (including bodyweight loss, loose stools
and clinical scores). A scoring system (max. score =12) is used, and both raw and analyzed data is provided (Fig 2).
Figure 2. Clinical read-outs. (A) Bodyweight as a percentage of start weight and (B) bodyweight in grams. Area highlighted in which clinical scoring and
Abatacept administration began. (C) Total clinical score our of twelve indicated per group over time. (D) Area-under-curve analysis of total clinical scores. Rag
KO untreated is significantly different to other groups. Statistical analysis unpaired t-test, P<0.05.
A
C D
CD4+CD45RB low control
CD4+CD45RB hi + vehicle
CD4+CD45RB hi + Abatacept *****
Daily Scoringand Treatments
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otal
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Figure 3. Phenotypic analysis of lamina propria cells after re-stimulation to determine the effect of a therapy (example shown here; Abatacept) on T cell
phenotype and function as determined by intracellular cytokine staining. Representative flow cytometry of each group of mice showing (A) IFNγ versus IL-17 or
(B) IFNγ versus CD44. Average expression of (C) total IL17+, (D) total IFNγ+ and (E) total CD44+ cells per group. (*p<0.05; Mann-Whitney).
CD
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xpre
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Ex Vivo Readouts : Flow Cytometry To determine the effect of a therapy on the cell populations within relevant tissues highly specific and sensitive, multiparameter
flow cytometric analysis of individual cells can be carried out on cells from colon lamina propria, spleen, and lymph node
tissue; single cell suspensions are prepared at end time point(s) and cell counts and percentages for each population of
interest from each are determined (Fig 3, 4). Analysis of lineage markers determines the cell types present within the tissue
and analysis of cytokine expression, effector molecule and transcription factor expression indicate whether a therapy has
modulated immune cell function.
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B
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1.84 1.14
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ControlCD4+CD45RBlow
IL-1
7C
D44
IFNγ IFNγ IFNγ
UntreatedCD4+CD45RBhi
AbataceptCD4+CD45RBhi
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Exp
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IFNγ
(%)
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Figure 4. Phenotype analysis of draining lymph node and spleen cells after restimulation. Expression of the effector cytokines IFNg and IL-17 indicates the level
of pathology causing Th1 and Th17 T cells within the tissue, CD44 is expressed on activated T cells. Average expression of (A) total IL17+, (B) total IFNγ+
cells and (C) total CD44+ cells per group. (* p<0.05; **P<0.005; ***p<0.0005; Mann-Whitney).
Figure 5: Histopathological data shown as mean score ± SEM. Statistical analysis by 2-way ANOVA followed by Sidak’s multiple comparisons test. *
indicates p<0.05 between the two groups for the criterion.
Figure 6: Illustrative histopathological data (A) An untreated animal showing mucosal erosion, epithelial hyperplasia and submucosal and transmural inflammation.
Scale bar: 100 µm (B) An untreated animal showing crypt abscesses and lamina propria infiltrates of neutrophils and mononuclear cells. Scale bar: 50 µm (C) An
Abatacept treated animal showing intact mucosa, normal goblet cell numbers and minimal inflammatory cell infiltrates in the lamina propria. Scale bar: 100 µm
HistopathologyHistopathology can provide information on how a therapeutic limits immune driven pathology and damage of the gut
barrier epithelial layer; At termination, ‘Swiss rolls’ of the colon can be processed for paraffin-embedding, sectioning
and haematoxylin and eosin (H&E) staining. Sections will be scored by a qualified histopathologist according to a semi-
quantitative scoring system. Representative images will be provided. Figure 5 shows histology scores for mice receiving
CD4+CD45RBhi cells (untreated) and those receiving CD4+CD45RBhi cells and Abatacept. Figure 6 shows representative
H&E staining from each group and illustrates the changes in tissue morphology and immune infiltration in the untreated
versus Abatacept group. Overall treatment with Abatacept inhibits immune infiltration and damage to the gut tissue.
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Figure5:Histopathologicaldatashownasmeanscore±SEM.Statisticalanalysisby2-wayANOVAfollowedbySidak’smultiplecomparisonstest.*indicatesp<0.05betweenthetwogroupsforthecriterion.
Figure 6: Illustrative histopathological data (A) An untreated animal showing mucosal erosion, epithelial hyperplasia and submucosal and transmural inflammation. Scale bar: 100 µm (B) An untreated animal showing crypt abscesses and lamina propria infiltrates of neutrophils and mononuclear cells. Scale bar: 50 µm (C) An Abatacept treated animal showing intact mucosa, normal goblet cell numbers and minimal inflammatory cell infiltrates in the lamina propria. Scale bar: 100 µm.
Luminex® Bio-Plex Multiplex Immunoassays can be performed from serum obtained from terminal bleeds or colon homogenates (for use with Bio-Plex Pro™ Mouse Cytokine Th17 panel, Th1/Th2 panel or custom panel).
NanoString Analysis A piece of distal colon can be taken prior to histological sampling for NanoString analysis allowing measurement of gene expression within the tissue (up to 770 genes on either a prebuilt or custom gene panel such as the nCounter® Autoimmune Profiling Panel) and how this is modulated by a therapy allowing identification of potential biomarkers and identification of potential MOA.
Summary The T cell transfer model of colitis recapitulates the clinical pathology (colitis and small bowel inflammation) observed in human intestinal inflammatory diseases such as Crohn’s
7
Figure5:Histopathologicaldatashownasmeanscore±SEM.Statisticalanalysisby2-wayANOVAfollowedbySidak’smultiplecomparisonstest.*indicatesp<0.05betweenthetwogroupsforthecriterion.
Figure 6: Illustrative histopathological data (A) An untreated animal showing mucosal erosion, epithelial hyperplasia and submucosal and transmural inflammation. Scale bar: 100 µm (B) An untreated animal showing crypt abscesses and lamina propria infiltrates of neutrophils and mononuclear cells. Scale bar: 50 µm (C) An Abatacept treated animal showing intact mucosa, normal goblet cell numbers and minimal inflammatory cell infiltrates in the lamina propria. Scale bar: 100 µm.
Luminex® Bio-Plex Multiplex Immunoassays can be performed from serum obtained from terminal bleeds or colon homogenates (for use with Bio-Plex Pro™ Mouse Cytokine Th17 panel, Th1/Th2 panel or custom panel).
NanoString Analysis A piece of distal colon can be taken prior to histological sampling for NanoString analysis allowing measurement of gene expression within the tissue (up to 770 genes on either a prebuilt or custom gene panel such as the nCounter® Autoimmune Profiling Panel) and how this is modulated by a therapy allowing identification of potential biomarkers and identification of potential MOA.
Summary The T cell transfer model of colitis recapitulates the clinical pathology (colitis and small bowel inflammation) observed in human intestinal inflammatory diseases such as Crohn’s
7
Figure5:Histopathologicaldatashownasmeanscore±SEM.Statisticalanalysisby2-wayANOVAfollowedbySidak’smultiplecomparisonstest.*indicatesp<0.05betweenthetwogroupsforthecriterion.
Figure 6: Illustrative histopathological data (A) An untreated animal showing mucosal erosion, epithelial hyperplasia and submucosal and transmural inflammation. Scale bar: 100 µm (B) An untreated animal showing crypt abscesses and lamina propria infiltrates of neutrophils and mononuclear cells. Scale bar: 50 µm (C) An Abatacept treated animal showing intact mucosa, normal goblet cell numbers and minimal inflammatory cell infiltrates in the lamina propria. Scale bar: 100 µm.
Luminex® Bio-Plex Multiplex Immunoassays can be performed from serum obtained from terminal bleeds or colon homogenates (for use with Bio-Plex Pro™ Mouse Cytokine Th17 panel, Th1/Th2 panel or custom panel).
NanoString Analysis A piece of distal colon can be taken prior to histological sampling for NanoString analysis allowing measurement of gene expression within the tissue (up to 770 genes on either a prebuilt or custom gene panel such as the nCounter® Autoimmune Profiling Panel) and how this is modulated by a therapy allowing identification of potential biomarkers and identification of potential MOA.
Summary The T cell transfer model of colitis recapitulates the clinical pathology (colitis and small bowel inflammation) observed in human intestinal inflammatory diseases such as Crohn’s
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Lamina propria mononuclear infiltrates
Lamina propria neutrophil infiltrates
Goblet cell loss
Transmural inflammation
Submucosal inflammation
Mucosal erosion
Crypt abscess
Epithelial hyperplasia
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© 2020, Charles River Laboratories International, Inc.
Luminex®
Bio-Plex Multiplex Immunoassays can be performed from serum obtained from terminal bleeds or colon homogenates (for
use with Bio-Plex Pro™ Mouse Cytokine Th17 panel, Th1/Th2 panel or custom panel).
NanoString AnalysisA piece of distal colon can be taken prior to histological sampling for NanoString analysis allowing measurement of
gene expression within the tissue (up to 770 genes on either a prebuilt or custom gene panel such as the nCounter®
Autoimmune Profiling Panel) and how this is modulated by a therapy allowing identification of potential biomarkers and
identification of potential MOA.
Summary The T cell transfer model of colitis recapitulates the clinical pathology (colitis and small bowel inflammation) observed
in human intestinal inflammatory diseases such as Crohn’s disease and ulcerative colitis1-3. Adoptive transfer of naïve
CD4 T cells depleted of Treg (CD4+CD45RBhi) leads to colitis and the presence of activated (CD44+) T cells expressing
the effector cytokines IL-17 and IFNγ in the colonic lamina propria and draining mesenteric lymph nodes. In contrast, in
the control group transferred with CD4+CD45RBlow T cells which contain Treg cells, animals remain healthy and T cells
are not activated in the lamina propria. Administration of a T cell immunomodulator (Abatacept) following transfer of
the CD4+CD45RBhi cells acts as a positive control, demonstrating a decrease in T cell activation and effector cytokine
expression and a concomitant decrease in clinical scores.
Histopathology of the distal colon obtained from mice with active disease in this T cell transfer colitis model reveals
transmural inflammation, epithelial cell hyperplasia, polymorphonuclear leukocyte (PMN) and mononuclear leukocyte
infiltration, crypt abscesses, and epithelial cell erosions, as is observed in human disease1. Furthermore, mice exhibited
varying degrees of weight loss, loose stools, and diarrhea, like the human disease. However, the major advantages
of this in vivo model compared to the chemically-induced models of colitis are that one can examine the very earliest
immunological events associated with the induction of gut inflammation as well as the perpetuation of disease. In addition,
this model draws parallels to human disease, as it is T cell driven whereas the chemically-induced models of colitis are
largely innate cell-driven.
Therefore, it is important to consider target of a therapeutic when selecting a model. This model is responsive to a variety
of different immunological and antibiotic treatment protocols, as illustrated by the Abatacept control used in the data
provided and other published data2,6,7.
Assay Reference Code
References
1. Ostanin DV et al. Am J Physiol Gastrointest Liver Physiol. 2009, 296(2): G135-146.
2. Powrie F et al. Immunity. 1994, 1(7): 553-562.
3. Steinbach EC et al. J Vis Exp 2015, 98: 52533.
4. Powrie F. Immunity. 1995, 3(2): 171-174.
5. Mottet C et al. J Immunol. 2003 170(8): 3939-3943.
6. Powri F. Ann N Y Acad Sci. 2004 1029: 132-141.
7. Liu Z et al. J Immunol. 2000, 164(11): 6005-6014.
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