Complex Biology In Vitro Assays: Immunology...EVERY STEP OF THE WAY DISCOVERY Complex Biology In Vitro Assays: Immunology T Cell Transfer Model of Inflammatory Bowel Disease (IBD)

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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FITC-A :: CD45RB

Nor

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PE-A :: CD62L

Nor

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To

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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








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

0 5 10 15 20 25 30 35 40 45 50

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[email protected] • www.criver.com https://www.criver.com/consult-pi-ds-how-can-we-support-your-program-immunology•



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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

44 e

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)

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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.

A

B

0 104 105

1.84 1.14

6.2290.8

105

104

103

0

-103

0 104 105

31.8 4.67

0.9062.7

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8.34 7.21

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40.4 43.8

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24.6 19.7

2.6053.0

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-103

ControlCD4+CD45RBlow

IL-1

7C

D44

IFNγ IFNγ IFNγ

UntreatedCD4+CD45RBhi

AbataceptCD4+CD45RBhi








Exp

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IFNγ

(%)

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Lymph nodes Spleen

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IL17

(%)

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4030

20101086420

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A

A

B

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C

C

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.

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










© 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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Complex Biology In Vitro Assays: Immunology...EVERY STEP OF THE WAY DISCOVERY Complex Biology In Vitro Assays: Immunology T Cell Transfer Model of Inflammatory Bowel Disease (IBD)