of November 16, 2018.This information is current as Trichuris murisNematode Parasite

Intestine of Mice Infected with the Intestinal and Isolated Lymphoid Follicles in the LargeLymphocytes, Lamina Propria Leukocytes, The Characterization of Intraepithelial

Kathryn J. ElseMatthew C. Little, Louise V. Bell, Laura J. Cliffe and

http://www.jimmunol.org/content/175/10/6713doi: 10.4049/jimmunol.175.10.6713

2005; 175:6713-6722; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/175/10/6713.full#ref-list-1

, 21 of which you can access for free at: cites 41 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on Novem

ber 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

by guest on N

ovember 16, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

The Characterization of Intraepithelial Lymphocytes, LaminaPropria Leukocytes, and Isolated Lymphoid Follicles in theLarge Intestine of Mice Infected with the Intestinal NematodeParasite Trichuris muris1

Matthew C. Little,2 Louise V. Bell, Laura J. Cliffe, and Kathryn J. Else

Despite a growing understanding of the role of cytokines in immunity to the parasitic helminth Trichuris muris, the local effectormechanism culminating in the expulsion of worms from the large intestine is not known. We used flow cytometry and immunohisto-chemistry to characterize the phenotype of large intestinal intraepithelial lymphocytes (IEL) and lamina propria leukocytes (LPL) fromresistant and susceptible strains of mouse infected with T. muris. Leukocytes accumulated in the epithelium and lamina propria afterinfection, revealing marked differences between the different strains of mouse. In resistant mice, which mount a Th2 response, thenumber of infiltrating CD4�, CD8�, B220�, and F4/80� IEL and LPL was generally highest around the time of worm expulsion fromthe gut, at which point the inflammation was dominated by CD4� IEL and F4/80� LPL. In contrast, in susceptible mice, which mounta Th1 response, the number of IEL and LPL increased more gradually and was highest after a chronic infection had developed. At thispoint, CD8� IEL and F4/80� LPL were predominant. Therefore, this study reveals the local immune responses underlying the expulsionof worms or the persistence of a chronic infection in resistant and susceptible strains of mouse, respectively. In addition, for the first time,we illustrate isolated lymphoid follicles in the large intestine, consisting of B cells interspersed with CD4� T cells and having a centralzone of rapidly proliferating cells. Furthermore, we demonstrate the organogenesis of these structures in response to T. murisinfection. The Journal of Immunology, 2005, 175: 6713–6722.

T richuris muris is a natural mouse model of the nematodeparasite, Trichuris trichiura, one of the most prevalent hu-man helminth infections worldwide. The range of protec-

tive immunity mounted against T. muris in the mouse infectionmodel varies depending upon the background genetics of the in-bred strain of mouse (1, 2) and parallels the range of responsesobserved within an outbred human population exposed to T. tri-chiura. The majority of mouse strains, such as BALB/c, are resis-tant to T. muris and quickly expel the parasite, whereas a fewstrains, such as AKR, are susceptible, allowing the development offecund adult parasites, culminating in a chronic infection of thececum and proximal colon.

It is now well established that a Th2-dominated response, char-acterized by the production of IL-4, IL-5, IL-9, and IL-13, is anabsolute requirement for the expulsion of worms by resistantstrains of mouse (3–6). Susceptible strains, rather than failing torespond to T. muris, instead mount an inappropriate Th1 responseassociated with high levels of IFN-� and IL-12 (7, 8). Despite thisknowledge, the effector mechanism ultimately responsible for theexpulsion of T. muris by the host is not understood. Many of theTh2 responses typically associated with helminth infection, such asmastocytosis, eosinophilia, and strong parasite-specific Ab re-sponses, are not essential (9, 10). Resistance can be conferred to

immunodeficient SCID mice by the transfer of CD4� donor cells(9, 11). However, in this model, protective immunity can be ab-rogated (using a combination of Ab against the gut-homing adhe-sion molecules mucosal addressin cell adhesion molecule-1, �7

integrin, and CD103 (11)) by blocking T cell migration to the gut(12). This supports the theory that locally acting T cell-dependanteffector mechanisms are responsible for the expulsion of T. murisfrom the large intestine.

Lamina propria leukocytes (LPL)3 and intraepithelial lympho-cytes (IEL) are the effector compartments of the gut mucosal im-mune system (13). By virtue of their anatomical location, IEL havethe closest direct contact with foreign Ags derived from the gutlumen and are thought to play a key role in the immune responsesto these Ags and in the pathogenesis of a variety of disease states.

Small intestinal IEL have been extensively studied in the mouse.Most are T cells, but compared with peripheral T cells found insecondary lymphoid organs, a high proportion of IEL are CD8�,express TCR��, and develop independently of the thymus. Thy-mus-independent IEL, which can be either TCR��� or TCR���,are relatively abundant and express CD8 in its �� homodimericform. Contrastingly, the thymus-dependent population expressesTCR�� and bears either CD4 or CD8 in its more familiar ��heterodimeric form (14–19).

However, IEL from the large intestine are seldom studied de-spite the marked differences in both function and luminal environ-ment between the different regions of the intestine and the devel-opment of diseases specific to the large intestine, such as ulcerativecolitis and colon cancer. Accordingly, a few studies have shown

Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom

Received for publication January 10, 2005. Accepted for publication July 22, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Wellcome Trust Grant 044494/Z (to M.C.L., L.V.B.,and K.J.E.).2 Address correspondence and reprint requests to Dr. Matthew C. Little, Faculty ofLife Sciences, University of Manchester, Michael Smith Building, Oxford Road,Manchester, U.K. M13 9PT. E-mail address: matthew.c.little@manchester.ac.uk

3 Abbreviations used in this paper: LPL, lamina propria leukocyte; E/S, excretory/secretory; IEL, intraepithelial lymphocyte; ILF, isolated lymphoid follicle; MLN,mesenteric lymph node; p.i., postinfection.

The Journal of Immunology

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

by guest on Novem

ber 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

that IEL of the large intestine have a different phenotype and func-tion than those of the small intestine (20–22). Within the largeintestine, the proportion of CD8� cells is lower; however, they stillconstitute a major subset of the T cell pool, with the ratio of CD4�

to CD8� being approximately equal. Principally, although thy-mus-independent T cells, characterized by their expression ofCD8�� and TCR��, predominate in the small intestine, they aremuch less abundant in the large intestine (20–22). IEL from thelarge intestine have much less cytolytic activity in vitro than IELfrom the small intestine (20). Furthermore, although similar pat-terns of IFN-� production are seen, more of the type 2 cytokines,IL-4 and IL-5, are produced by IEL of the large intestine (21, 23).Therefore, a pronounced regional specialization of epithelial Tcells is found in the gut.

Given that T. muris forms syncitial tunnels within the epitheliumof the cecum and proximal colon (24), IEL are especially close tothe parasites and their Ag. Therefore, IEL may play a major role inthe immune response to and, ultimately, the elimination of T.muris. Because Th cells are essential for the expulsion of T. muris,we hypothesize that these cells migrate to the large intestine inresistant strains of mouse in temporal association with the expul-sion of worms. In susceptible strains of mouse, the accumulationof inappropriate subsets of leukocytes in the large intestine mightunderlie their inability to expel the parasite, leading to the devel-opment of a chronic infection. This study characterizes large in-testinal IEL throughout the infection of resistant and susceptiblemice with T. muris.

Materials and MethodsMice

Specific pathogen-free AKR and BALB/c mice were purchased from Har-lan U.K. and were maintained in individually ventilated cages. In all ex-periments, male mice were infected with T. muris when they were 6–8 wkold. SCID mice were used to investigate the influx of macrophages into thelarge intestine during T. muris infection in the absence of an adaptiveimmune system and to examine whether isolated lymphoid follicles (ILF)-like structures could develop in the absence of lymphocytes. SCID micewere bred in isolators at the University of Manchester, and male mice usedwhen they were 6–8 wk old. The animal studies were reviewed and ap-proved by the Home Office and were performed under the legal require-ments of the Animal (Scientific Procedures) Act (1986).

Parasite

T. muris was maintained as described previously (24). Mice were infectedorally with �150 infective eggs. Mice were killed at various time pointspostinfection (p.i.), and the worm burdens in the large intestine were as-sessed as described previously (1, 2). T. muris excretory/secretory (E/S) Agwas prepared from adult worms after a 4-h in vitro culture as describedpreviously (24).

Cell culture

Mesenteric lymph nodes (MLN) were removed, and single-cell suspen-sions were prepared. Total MLN cells were suspended in RPMI 1640 me-dium supplemented with 5% FCS, 2 mmol/l L-glutamine, 100 U/ml peni-cillin, 100 �g/ml streptomycin (all from Invitrogen Life Technologies),and 60 �mol/l monothioglycerol (Sigma-Aldrich). The cells were stimu-lated with 50 �g/ml T. muris E/S Ag in 48-well plates (5 � 106 cells/well)at 37°C for 24 h. The cell supernatants were harvested and stored at �20°Cuntil they were assayed for cytokines.

Cytokine ELISA

Cytokines were analyzed by sandwich ELISA as described previously (25).The following mAb pairs were used: IFN-�, R4-6A2, and XMG1.2; IL-4,11B11, and BVD-24G.2; IL-5, TRFK5, and TRFK4 (all from BD Bio-sciences); IL-9, 249.2 (E. Schmitt, University of Mainz, Mainz, Germany),and DC9302C12 (BD Biosciences); and IL-12 p40, C15.6 (G. Trinchieri,Schering-Plough, Dardilly, France), and C17.8 (BD Biosciences). The de-tection Ab were biotinylated, and a streptavidin-peroxidase (Roche) systemwas used in conjunction with the substrate ABTS (Sigma-Aldrich). The

samples were quantified using recombinant murine cytokine standards(R&D Systems). The plates were read at 405 nm.

Parasite-specific Ab ELISA

Serum was assayed by capture ELISA for T. muris-specific IgG1 andIgG2a as described previously (26). Briefly, Immulon 96-well plates(Thermo Electron) were coated with 5 �g/ml T. muris E/S Ag and incu-bated with serum diluted through eight serial 2-fold dilutions from 1/20 to1/2560. Parasite-specific Ig was detected using either biotinylated anti-murine IgG1 (Serotec) or IgG2a (BD Biosciences).

Isolation of IEL

IEL were isolated by an accepted modification of the method described byDavies and Parrott (27). Briefly, large intestines (cecum and �6 cm ofproximal colon) were removed, and macroscopically visible lymphoid ag-gregates on the cecum were cut off and dispensed with. Fat and connectivetissues were removed, and the large intestines were opened longitudinally,then washed twice, to remove the feces, in calcium- and magnesium-freeHBSS containing 2% FCS (at 4°C). The intestinal tissue from 10 mice waspooled and then cut into 1-cm pieces. This tissue was placed in 50-ml tubesand washed three times in HBSS containing 2% FCS at 4°C. The tissue wastransferred to 25-cm3 tissue culture flasks and incubated at 37°C in HBSScontaining 10% FCS, 0.2 mmol/l EDTA, 1 mmol/l DTT, 100 U/ml peni-cillin, and 100 �g/ml streptomycin. After 20 min, the flasks were shakenvigorously for 30 s, and the supernatant containing the IEL was separatedfrom the tissue fragments using a stainless steel sieve. The supernatant wascollected and put on ice, the tissue fragments were retuned to the flasks,and the process was repeated. After this process, the tissue pieces wereexamined microscopically to ensure that the epithelium had been removedand that the characteristic folds and ridges of the lamina propria were stillintact. The epithelial cell suspensions from both incubations were pooled,washed, and suspended in RPMI 1640 at 4°C, then passed through nylonwool columns. The cell suspension was collected and suspended in 44%Percoll, which was layered on top of 67.5% Percoll and centrifuged at600 � g for 20 min at 4°C. The IEL were collected from the interfacebetween the Percoll gradients and prepared for phenotypic analysis by flowcytometry.

Flow cytometry

IEL were washed in PBS containing Dulbecco’s A and B salts, 0.1% so-dium azide, and 2% FCS. Triple staining was performed on samples of 1 �106 cells using a combination of the following Abs: anti-CD3-PE, biotin-ylated anti-CD25 used in conjunction with streptavidin-TriColor (BD Bio-sciences), and one of the following FITC-conjugated Abs: anti-CD4, anti-B220, anti-CD30 (Serotec), anti-CD69, or anti-CD103. Alternatively,triple staining was conducted using anti-CD8�-PE or anti-TCR�-PE, anti-CD8�-FITC or anti-TCR��-FITC, and biotinylated anti-CD25 used in con-junction with streptavidin-TriColor (BD Biosciences). Appropriate isotypecontrols of irrelevant specificity (rat IgG2a-PE, rat IgG2b-PE, rat IgG2a-FITC, rat IgG2b-FITC, hamster IgG-FITC, and biotinylated rat IgG2a)were included. All Abs were obtained from BD Biosciences unless other-wise stated. All cells were stained for 30 min in the dark on ice and thenfixed by the addition of 2% formaldehyde in PBS, 0.1% sodium azide, and2% FCS. The data were acquired on a FACSCalibur flow cytometer andanalyzed using CellQuest Pro software (both from BD Biosciences).

Immunohistochemistry

Mice were killed at various time points p.i. with T. muris. Age-matched,uninfected control mice were killed on day 21 p.i. Approximately 6 mm ofthe proximal colon (juxtaposed to the distal cecum) was removed, trisected,and carefully positioned in OCT embedding medium (R. A. Lamb). Thetissue was snap-frozen in liquid nitrogen-chilled isopentane (BDH-Merck),and 6-�m sections were cut using a cryomicrotome. The tissue was air-dried for 1 h to maximize its adhesion to gelatin-coated microscope slides,then fixed using 4% paraformaldehyde (Sigma-Aldrich) in PBS for 10 minat 4°C. Slides were washed in PBS, and endogenous peroxidase activitywas quenched using 0.064 mg/ml sodium azide, 1.5 U/ml glucose oxidase,and 1.8 mg/ml D-glucose (Sigma-Aldrich) in PBS for 20 min at 37°C. Afteranother wash in PBS, nonspecific binding sites in the sections were blockedusing 10% normal rat serum (Sigma-Aldrich) in PBS for 1 h at roomtemperature. Endogenous avidin and biotin binding sites were blocked us-ing a commercial kit according to the manufacturer’s instructions (VectorLaboratories). The sections were incubated at room temperature for 1 hwith one of the following rat anti-mouse biotinylated mAb: anti-CD4(5 �g/ml; BD Biosciences), anti-CD8� (10 �g/ml; BD Biosciences),

6714 LARGE INTESTINAL LEUKOCYTES AND T. muris INFECTION

by guest on Novem

ber 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

anti-B220 (10 �g/ml; BD Biosciences), anti-CD11b (5 �g/ml; BD Bio-sciences), anti-F4/80 (5 �g/ml; Caltag Laboratories), or anti-�7 integrin(10 �g/ml; BD Biosciences). Alternatively, a number of sections wereincubated in parallel with the appropriate biotinylated isotype control Abs(BD Biosciences). A Vectastain Elite avidin-biotin-peroxidase complexkit, followed by a 3,3�-diaminobenzidine chromagen kit, were then usedaccording to the manufacturer’s instructions (Vector Laboratories). Thesections were counterstained in Harris’s hematoxylin solution and mountedin Aquamount aqueous mounting medium (BDH-Merck). The number ofpositively stained cells per 20 crypt units was assessed in triplicate by lightmicroscopy after randomization and blinding.

In vivo labeling and in situ immunohistochemical visualizationof proliferating lymphocytes

Mice were injected i.p. with 10 mg of BrdU, which is taken up by prolif-erating cells during the S phase of the cell cycle. After 40 min, the micewere killed, and the detection of nuclei that had incorporated BrdU wasperformed by immunohistochemistry using an anti-BrdU mAb (Mas 250b;Harlan Sera Laboratories) as described previously (28).

Statistical analysis

Statistical analysis was performed by ANOVA and Tukey’s post-test (us-ing the statistical package GraphPad PRISM 3.0).

ResultsBALB/c mice are resistant to T. muris and mount a Th2response, whereas AKR mice are susceptible to infection andmount a Th1 response

After infection with T. muris, BALB/c mice expelled the majorityof the worms from the large intestine before day 21 p.i. and werefree of worms by day 35 p.i. In contrast, AKR mice failed to expelthe worms and were chronically infected with T. muris (Fig. 1A).MLN cells from uninfected mice and mice infected with T. muriswere stimulated in vitro with T. muris E/S Ag. The cells frominfected BALB/c mice produced the Th2 cytokines IL-4, IL-5, andIL-9, whereas AKR mice displayed a Th1-skewed, Ag-specificcytokine response, characterized by higher levels of IFN-� andIL-12 p40 (Fig. 1B). Furthermore, the Ag-specific Ab produced byBALB/c mice in response to infection were predominantly IgG1,in contrast to AKR mice, in which high levels of IgG2a weredetected (Fig. 1C). Taken together, these data confirmed that theimmune response to T. muris was Th2 and Th1 dominated in re-sistant BALB/c mice and susceptible AKR mice, respectively.

Reduction in yield of IEL during the infection

Using accepted standard methods, the number of IEL extractedfrom the large intestine of one uninfected mouse was typically inthe range of 1 � 106 to 1.5 � 106 (Fig. 1D). However, p.i., theyield of IEL decreased (Fig. 1D) in temporal association with theexpulsion of worms from the large intestine (Fig. 1A). The lowestyield of IEL from BALB/c mice occurred on days 14 and 21 p.i.,when the worms were being actively expelled. The IEL yield grad-ually returned to normal as the mice became free of infection. InAKR mice, a progressive reduction in the yield of IEL was notedas the chronic infection developed. The expulsion of worms or thedevelopment of a chronic infection is associated with enteropathyin the large intestine, including crypt hyperplasia, goblet cell hy-perplasia, and the hypersecretion of mucus (24). This appears tointerfere with our method of IEL extraction from the large intes-tine, leading to an artificially low yield p.i. The percentage of IELexpressing CD103 (�E�7 integrin) was �85%, and this was un-affected by infection (data not shown).

The number of CD4� IEL increased p.i.

Using flow cytometry, the percentage of IEL exhibiting a Th cellphenotype (CD3�CD4�) was �5% in both BALB/c and AKRmice (Fig. 2A). However, p.i., dynamic changes in the percentage

of CD3�CD4� IEL occurred that differed between the two strainsof mouse. In BALB/c mice, the percentage of CD3�CD4� IELincreased gradually, and at its peak (21 days p.i.) had risen by�2-fold (Fig. 2A). There followed a decline in the percentage ofCD3�CD4� IEL in BALB/c mice, approaching preinfection levelsby 35 days p.i. (Fig. 2A). In contrast, the percentage ofCD3�CD4� IEL in AKR mice continued to rise throughout theinfection, reaching a 3-fold increase by 35 days p.i. (Fig. 2A).

CD4 detection by immunohistochemistry allowed the numericalquantification of CD4� IEL and LPL within the large intestine.Dynamic changes in the number of CD4� IEL (Fig. 2, B and C)and LPL (Fig. 2, B and D) occurred p.i. with T. muris, revealing

FIGURE 1. The immune response to infection with T. muris and theisolation of IEL from the large intestine. Susceptible (AKR) and resistant(BALB/c) strains of mouse were infected with �150 T. muris ova. Micewere killed at several time points p.i. (three mice per group), and the num-ber of T. muris worms inhabiting the large intestine was counted and isexpressed as the mean � SEM (A). MLN cells, isolated from uninfectedmice and from infected mice on day 21 p.i., were stimulated in vitro for24 h with T. muris E/S Ag. The supernatant was analyzed for cytokines byELISA. The data are expressed as the mean � SEM of five mice (B).Ag-specific IgG was analyzed in the serum of uninfected (naive) mice andinfected mice 35 days p.i. The serum was serially diluted, and only data forthe 1/80 dilution (within the linear range) are expressed as the mean �SEM of five mice (C). IEL were isolated from the large intestine of unin-fected (naive) mice and from mice infected with T. muris at various timepoints p.i., as indicated. A representative example of the yield of IELduring the infection is shown in D.

6715The Journal of Immunology

by guest on Novem

ber 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

differences between the strains of mouse. Initially, in BALB/cmice, the number of CD4� IEL increased, reaching a peak 21 daysp.i., then declined at later time points. In contrast, the number ofCD4� IEL in AKR mice continued to increase throughout theinfection (Fig. 2, B and C). These strain-specific patterns of changein CD4� IEL during infection are essentially similar to those dem-onstrated by flow cytometry (Fig. 2A). The number of CD4� LPLincreased in both strains of mouse p.i. (Fig. 2, B and D). Therewere roughly 15 times more CD4� cells in the lamina propria thanin the epithelium regardless of infection with T. muris (Fig. 2, Cand D).

The number of CD8� IEL increased p.i.

The expression by IEL of both isoforms of CD8 was investigatedusing flow cytometry. This revealed major differences between thestrains of mouse in the relative abundance of both CD8��� andCD8��� IEL in the large intestine. Twice the percentage ofCD8��� IEL were detected in AKR mice compared with BALB/cmice before infection (Fig. 3A). Conversely, although a significantproportion of IEL expressed CD8�� in BALB/c mice, the per-centage of these cells was negligible in AKR mice (Fig. 3A). Afterinfection, CD8�� IEL were more abundant in AKR mice than inBALB/c mice (Fig. 3B).

Immunohistochemical detection of the CD8 �-chain (which isexpressed by all CD8� cells) enabled all CD8� IEL and LPL

within the large intestine to be quantified. Paradoxically, althoughCD8� IEL were found in BALB/c mice by flow cytometry (Fig. 3,A and B), virtually no CD8� IEL or LPL were detected by immu-nohistochemistry in uninfected BALB/c mice (Fig. 3D). After in-fection, there was a limited influx of CD8� IEL and LPL into thelarge intestine in BALB/c mice (Fig. 3, C–E). Compared withBALB/c mice, CD8� IEL and LPL were relatively abundant inuninfected AKR mice, and the number of these cells was consid-erably greater p.i. (Fig. 3, C–E). Approximately 20 times moreCD8� leukocytes were found in the lamina propria than in theepithelium in uninfected AKR mice (Fig. 3, D and E).

FIGURE 2. Analysis of Th cells in the large intestine of susceptible(AKR) and resistant (BALB/c) strains of mouse infected with T. muris. IELwere isolated from the large intestine of uninfected (naive) mice and frommice infected with T. muris at various time points p.i., as indicated.CD3�CD4� IEL were analyzed by flow cytometry. The data are presentedgraphically in A as the mean � SD of four separate experiments. Immu-nohistochemical staining of CD4� cells in the proximal colon was con-ducted at all time points p.i. A representative photographic example isshown for BALB/c mice 21 days p.i., where positively stained cells arebrown. Arrows show examples of IEL and LPL. Scale bar � 50 �m (B).Quantitative analysis of the immunohistochemistry is illustrated in C(CD4� IEL) and D (CD4� LPL). The values represent the mean � SEMof five mice at each time point, and the results are representative of threeseparate experiments. �, p 0.05; ��, p 0.01; ���, p 0.001.

FIGURE 3. Analysis of CTLs in the large intestine of susceptible(AKR) and resistant (BALB/c) strains of mouse infected with T. muris. IELwere isolated from the large intestine of uninfected (naive) mice and frommice infected with T. muris at several time points p.i., as indicated. CD8�-and CD8�-expressing IEL were analyzed by flow cytometry. A represen-tative example for naive mice is shown in A, where the percentage of gatedcells in each quadrant of the scatter plots is denoted. The flow cytometrydata are presented graphically (as the mean � SD of three separate exper-iments) in B only for CD8�� IEL. Immunohistochemical staining ofCD8�� cells in the proximal colon was conducted at all time points p.i. Arepresentative photographic example is shown for BALB/c mice 21 daysp.i., where positively stained cells are brown. Arrows show examples ofIEL and LPL. Scale bar � 50 �m (C). Quantitative analysis of the immu-nohistochemistry is illustrated in D (CD8�� IEL) and E (CD8�� LPL).The values represent the mean � SEM of five mice at each time point, andthe results are representative of two separate experiments. ��, p 0.01;���, p 0.001.

6716 LARGE INTESTINAL LEUKOCYTES AND T. muris INFECTION

by guest on Novem

ber 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Analysis of TCR subunits

There were �4 times more TCR��� IEL than TCR��� IEL inuninfected mice, as determined by flow cytometry (data notshown). In BALB/c mice, the percentages of TCR��� andTCR��� IEL were 59 and 15%, respectively. No clear pattern ofchange over time p.i. to TCR��� or TCR��� IEL was evident ineither strain of mouse (data not shown).

The number of B220� IEL increased p.i.

The percentages of T cells (CD3�B220�) and B cells(B220�CD3�) in the IEL compartment were evaluated by flowcytometry. T cells were always more abundant than B cells (datanot shown). In BALB/c mice, the percentage of B220�CD3� IELincreased 21 days p.i. (Fig. 4A). An increase in the percentage ofB220�CD3� IEL was associated with a decrease in the percentageof CD3�B220� IEL (data not shown).

B cells in the large intestine were also examined by immuno-histochemistry. There were �4 times more B220� IEL than LPLin uninfected mice (Fig. 4, C and D). After infection, there weregreater numbers of B220� IEL and LPL (Fig. 4, B–D). The pat-terns of this change over time broadly mirrored those found byflow cytometry (Fig. 4A). That is to say, the number of B cellsinitially increased p.i. in BALB/c mice and subsequently returned

to normal levels, whereas in AKR mice the highest levels of Bcells occurred at later time points (Fig. 4, C and D). However,there was also some disparity in the data between the two methodsof B cell analysis. Most strikingly, by immunohistochemical anal-ysis, there were significantly more B220� IEL in AKR mice thanin BALB/c mice 21 days p.i. (Fig. 4, B and C), although by flowcytometry the converse was found (Fig. 4A).

Large influx of macrophages into the large intestine p.i.

Because no macrophage markers are entirely specific, two suchmarkers (F4/80 and CD11b) were used to investigate more clearlythe influx of macrophages into the large intestine by immunohis-tochemistry. In practice, there was little difference between the twomethods of analysis. In uninfected mice, F4/80� and CD11b� IELwere scarce (Fig. 5, A, B, D, and E), whereas F4/80� and CD11b�

LPL were relatively abundant (Fig. 5, A, C, D, and F). After in-fection, there was a significant increase in the number of F4/80�

and CD11b� IEL (Fig. 5, A, D, B, and E). There was a morestriking increase in the number of F4/80� and CD11b� cells in thelamina propria p.i., uncovering major differences between the twostrains of mouse. In BALB/c mice, the number of F4/80� andCD11b� LPL reached a peak 21 days p.i., declining at later timepoints. In contrast, the numbers of F4/80� and CD11b� LPL inAKR mice continued to increase throughout the infection (Fig. 5,C and F). Interestingly, there were twice as many F4/80� andCD11b� cells in the lamina propria of BALB/c mice than in AKRmice 21 days p.i. (Fig. 5, A, C, D, and F). In uninfected SCID micethere were 52 � 11 F4/80� LPL/20 crypts (data not shown). Afterinfection, the number of F4/80� LPL continued to increase inSCID mice (139 � 12 F4/80� LPL/20 crypts after 21 days and192 � 17 F4/80� LPL/20 crypts after 35 days; data not shown),resembling that found in AKR mice (Fig. 5C).

Expression of activation markers by IEL does not change p.i.

There were more CD25� IEL in AKR mice than in BALB/c mice(Fig. 6, A–C). Only a small proportion of CD3� IEL expressedCD25 in either strain of mouse (Fig. 6A); accordingly, few CD4�

IEL expressed CD25 (Fig. 6B). The majority of CD25� IEL inAKR mice were B cells (Fig. 6C). In contrast to BALB/c mice, inwhich few B220� IEL expressed CD25, in AKR mice mostB220� IEL expressed CD25 (Fig. 6C). A high proportion of CD3�

IEL expressed the activation marker CD69 (Fig. 6D). The infec-tion of mice by T. muris caused no discernable difference in thepercentage of CD25� or CD69� IEL (Fig. 6). Less than 0.5% ofthe IEL expressed CD30 (an early marker of activation) in unin-fected mice or at any time point p.i. (data not shown).

Lymphoid follicles filled mainly with B cells are present in largeintestine

Numerous pronounced follicular structures were discovered in thelarge intestine of both AKR and BALB/c mice. These follicleswere comprised primarily of closely packed B cells interspersed bysmall clusters of CD4� T cells. CD8� T cells were much lesscommon, but could occasionally be found at the edge of the fol-licles. Macrophages were found at the marginal zone of the folli-cles, and occasionally, individual macrophages could also be de-tected more centrally. Some cells around the outside of the folliclesexpressed �4 integrin (Fig. 7A). BrdU was incorporated by leuko-cytes in the core of the follicles, suggesting a central zone of pro-liferating B cells (Fig. 7B). Infrequently, structures resembling fol-licles were also found in the large intestine of infected SCID mice,although they consisted of neither B cells nor macrophages (Fig.7C). In some mice, multiple follicles were found in the gut sections(Fig. 7D). In AKR mice, but not in BALB/c mice, there was a

FIGURE 4. Analysis of B cells in the large intestine of susceptible(AKR) and resistant (BALB/c) strains of mouse infected with T. muris. IELwere isolated from the large intestine of uninfected (naive) mice and frommice infected with T. muris at several time points p.i., as indicated.B220�CD3� IEL were analyzed by flow cytometry, and the data are pre-sented in A as the mean � SD of three separate experiments. Immunohis-tochemical staining of B220� cells in the proximal colon was conducted atall time points p.i. A representative photographic example is shown forBALB/c mice 21 days p.i., where positively stained cells are brown. Ar-rows show examples of IEL and LPL. Scale bar � 50 �m (B). Quantitativeanalysis of the immunohistochemistry is illustrated in C (B220� IEL) andD (B220� LPL). The values represent the mean � SEM of five mice ateach time point, and the results are representative of two separate experi-ments. �, p 0.05; ��, p 0.01; ���, p 0.001.

6717The Journal of Immunology

by guest on Novem

ber 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

significant increase, per mouse in the number of follicles at latertime points p.i. (Fig. 7E). However, the follicles tended to be largerin BALB/c mice than in AKR mice, particularly p.i. (Fig. 7F).

DiscussionTypical of previous investigations, only �1 � 106 IEL were ob-tained from the large intestine of an individual uninfected mouse.Consequently, to analyze their phenotype comprehensively, it iscommon practice to pool IEL from several individuals (20–22). Aconsiderable percentage of IEL extracted from the large intestineexpressed the classical IEL marker CD103 (�E�7 integrin), con-firming the reliability of our preparation technique. Some differ-ences in the phenotype of IEL were apparent between uninfected

BALB/c and AKR strains of mouse. CD8� IEL were more abun-dant in AKR mice than in BALB/c mice. Furthermore, althoughCD8��� IEL were found in both strains of mouse, CD8��� cellswere found only in BALB/c mice. Indeed, there are numerousexamples in the literature of phenotypic differences betweenstrains (20–22).

Regardless of interstrain differences, we were able to comparethe present study with previous investigations because BALB/cmice are routinely used. Consistent with previous descriptions ofIEL isolated from the large intestine (20, 21), �76% were CD3�

T cells, of which 80% were TCR��� and 20% were TCR���.However, a discrepancy with previous reports was evident whenthe T cell subsets were subjected to a more detailed analysis. In thepresent study the proportions of CD3� IEL expressing CD4 andCD8 were 7 and 51%, respectively, and by deduction, the remain-ing T cells (�42%) were double negative (CD4�CD8�). Othersestimate a higher proportion of CD4� T cells (between 32 and72%), with the ratio of CD4� to CD8� being approximately equal(20–22). Indeed, the relative abundance of CD4� T cells from thelarge intestine is thought to distinguish them from T cells of thesmall intestine, where CD8� cells predominate (20–22). Further-more, contrary to previous reports, CD8��� cells were found to bemore plentiful than CD8���, again resembling the phenotypecommonly associated with the small intestine (17, 21, 22). Al-though double-negative (CD4�CD8�) cells constituted a major Tcell subset in the present study, previous reports suggest they areless prevalent (from 1 to 27%) (20–22). Therefore, in this study wereproducibly define a large intestinal T cell phenotype that con-trasts with previous descriptions. It was vital in the present studyto use only mice that were free of gastrointestinal infections beforeinfection with the cecum-dwelling nematode T. muris. However,laboratory mice are often chronically infected with the gut-dwell-ing nematodes Aspiculuris tetraptera and Syphacia obvelata (29),and as we discuss later, infection does alter the balance of differentIEL subsets. Thus, our results may differ from those of previousreports in part due to the use of specific pathogen-free laboratorymice housed in individually ventilated cages. Hence, this studychallenges previous descriptions of IEL isolated from the largeintestine (suggesting that they are phenotypically similar to IELfrom the small intestine), and therefore, fundamentally, the poten-tial for CD8-mediated cytotoxicity in the large intestine is greaterthan described previously.

The number of IEL extracted from the large intestine p.i. ap-peared to be affected by infection-associated gut enteropathy. It istherefore misleading and does not reflect the actual number of IELin the large intestine p.i. However, a reliable account of IEL num-bers is given by histological examination, demonstrating themarked accumulation of IEL in the large intestine p.i., uncoveringdifferences between the contrasting stains of mouse and reinforc-ing the value of using both flow cytometry and immunohistochem-istry. In BALB/c mice, the number of IEL increased (peaking at�21 days p.i.), then reverted toward normal levels, correspondingwith the kinetics of worm expulsion. In contrast, the number ofIEL in AKR mice increased and remained high as the infectionprogressed.

Th cells are known to play a pivotal role in the mechanism of T.muris expulsion, because the depletion of CD4� cells confers asusceptible phenotype to resistant strains of mouse (30). As weconfirm, the generation of a Th2 response is essential for the ex-pulsion of worms (3–6). A locally acting mechanism for Th2 cellshas been postulated, yet no previous studies have shown the mi-gration of CD4� cells into the large intestine. Importantly, thepresent study demonstrates for the first time that in resistant miceexhibiting a Th2 response, CD4� Th cells do indeed accumulate in

FIGURE 5. Analysis of macrophages in the large intestine of suscepti-ble (AKR) and resistant (BALB/c) strains of mouse infected with T. muris.Staining for either F4/80 or CD11b, macrophages were detected by immu-nohistochemistry in the proximal colon of uninfected (naive) mice andfrom mice infected with T. muris. Representative photographic examplesof F4/80 (A) or CD11b (D) staining are shown for BALB/c mice 21 daysp.i. Positively stained cells are brown. Arrows show examples of IEL andLPL. Scale bars � 50 �m (A and D). Quantitative analysis of the immu-nohistochemistry is illustrated in B (F4/80� IEL), C (F4/80� LPL), E(CD11b� IEL), and F (CD11b� LPL). The values represent the mean �SEM of five mice at each time point. The results are representative of threeseparate experiments. �, p 0.05; ��, p 0.01; ���, p 0.001.

6718 LARGE INTESTINAL LEUKOCYTES AND T. muris INFECTION

by guest on Novem

ber 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

the epithelium of the large intestine around the time of worm ex-pulsion. In susceptible mice that mount a Th1 response, the num-ber of CD4� Th cells increases more gradually and is greatestduring the chronic phase of infection. Recently, several studieshave suggested various potential effector mechanisms by which T.muris may be expelled from the gut. These theories include anincreased rate of epithelial cell turnover (31) and the release offactors by goblet cells that may impair chemotaxis of the parasite(32). Nevertheless, both these potential mechanisms depend uponthe secretion of Th2 cytokines in the large intestine. Because thepresent study suggests that Th2 cells migrate to the large intestineat the time of worm expulsion, this bridges the gap in our knowl-edge between the well-characterized afferent immune responses tothe putative efferent immune effector mechanisms of wormexpulsion.

In contrast to resistant mice, a large population of CD8� cellsinfiltrated the mucosa of the large intestine in susceptible mice p.i.However, a recent study in our laboratory shows that the depletionof CD8� cells in susceptible mice fails to influence the develop-ment of a chronic infection (33). Therefore, although they are notessential for the development or maintenance of a chronic in-fection, the sheer magnitude of CD8� cell migration into thegut underlines the inability of susceptible mice to mount anappropriate protective immune response to the parasite. A muchlower number of CD8� IEL was found in resistant BALB/cmice p.i. These cells were detected less frequently by immu-nohistochemistry than by flow cytometric analysis of isolatedIEL, perhaps indicating a difference in the sensitivity of thecontrasting methods.

The accumulation of B cells in the gut was also noted p.i. B cellswere shown by immunohistochemistry to be more numerous insusceptible mice. However, in resistant mice, especially 21 daysp.i., the percentage of B cells in the isolated IEL (as shown by flowcytometry) was considerable, somewhat contradicting the immu-nohistochemical findings. During their extraction from the large

intestine, the contamination of IEL by B cells from ILF is inevi-table, because these follicular structures are intimately associatedwith the epithelium. Furthermore, we suggest (with relevance toall previous studies of isolated IEL) that although LPL can beexcluded from the preparations, a degree of contamination fromILF is to be expected. Because ILF tended to be larger inBALB/c mice than in AKR mice (particularly p.i.), this is themost likely explanation for the abundance of B cells in IELpreparations from resistant mice. Recently, other authors haveconsidered it inevitable that B cells from germinal centers con-taminate IEL suspensions (13). Therefore, the present studyhighlights the importance of immunohistochemistry as a vitaltool to address this problem.

It is difficult to describe the activation state of IEL, becausesome markers of activation were widely expressed by IEL, andothers were expressed by only a small minority of cells; the ma-jority of CD3� IEL expressed CD69, whereas CD25, in accor-dance with other studies (20), was expressed by a very small per-centage of T cells. Because CD3� IEL isolated from the largeintestine have been shown to express CD25 after TCR stimulationin vitro (20), it is perhaps surprising that there was no change in thefrequency of these cells p.i. with T. muris. Interestingly, CD25 wasexpressed most notably by B cells in AKR mice. A distinct pop-ulation of CD25�CD4� cells, namely, regulatory T cells, isthought to play a role in the persistence of infection to the parasiteLeishmania major (34). However, a role for regulatory T cells inthe immune response to T. muris seems unlikely, because 1% ofthe IEL were CD25�CD4�.

There was a sizeable influx of leukocytes into the lamina propriaof the large intestine p.i. The phenotype of LPL differed markedlyfrom that of IEL. CD4� cells were up to 10 times more abundantin the lamina propria than in the epithelium. Conversely, CD8�

and B220� LPL were less numerous than CD8� and B220� IEL.Macrophages made up a large fraction of the LPL p.i., whereasthey were rarely encountered in the IEL population. This work

FIGURE 6. The activation state of IEL in the largeintestine of susceptible (AKR) and resistant (BALB/c)strains of mouse infected with T. muris. The expressionof the lymphocyte activation markers CD25 and CD69was analyzed by flow cytometry in uninfected (naive)mice and in infected mice 21 days p.i. A, CD25 ex-pression by T cells. B, CD25 expression by Th cells. C,CD25 expression by B cells. D, CD69 expression by Tcells. The results are representative of two separateexperiments.

6719The Journal of Immunology

by guest on Novem

ber 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

confirms that IEL and LPL are distinct components of the GALT.Intriguingly, the migration of macrophages into the lamina propriareached a peak around the time of worm expulsion, at which pointthere were approximately twice as many macrophages in resistantmice than in susceptible mice. Greater numbers of macrophageswere observed p.i. in the lamina propria of SCID mice, suggestingthat the accumulation of macrophages in the large intestine is inpart an innate immune response to the parasite. A recent study inour laboratory shows that mice devoid of the macrophage chemo-kine CCL2 fail to expel T. muris, and this is associated with fewermacrophages in the lamina propria and an altered Th1/Th2 cyto-

kine balance (35). Taken together, this suggests a potential role formacrophages in the mechanism of worm expulsion from the gut.Future work will investigate the phenotype and role of laminapropria macrophages in this context.

Lymphoid structures known as ILF have been identified in bothsmall and large intestines of humans (36, 37); more recently, theyhave been discovered and characterized, in some detail, in thesmall intestine of mice (38, 39). They are composed of a large Bcell area, including a germinal center, and like Peyer’s patches, theepithelium overlying the follicles contains M cells, suggesting thatthey are inductive sites for local IgA responses. Although Hamada

FIGURE 7. Identification and char-acterization of ILF in the proximal colonof mice. Staining for a range of leuko-cyte markers, ILF were characterized byimmunohistochemistry in the proximalcolon of uninfected mice and from miceinfected with T. muris. Representativephotographic examples are shown foruninfected BALB/c mice (except forF4/80 staining, which is from an unin-fected AKR mouse), where positivelystained cells are brown (A). Immuno-staining revealed the incorporation ofBrdU into proliferative cells in the prox-imal colon. A representative photo-graphic example of BrdU staining(where BrdU-containing cells arebrown) is shown for a particularly largeILF from an AKR mouse 21 days p.i.(B). ILF-like structures were character-ized by immunohistochemistry in theproximal colon of SCID mice 35 daysp.i. (C). A typical example of immuno-histochemical staining of B220� cells inAKR mice 28 days p.i., showing multi-ple ILF (D). The number of ILF per6-mm section of proximal colon was de-termined histologically. A representativeexample is shown in E, where five miceper group were used (except for AKRmice 35 days p.i., where six mice wereused). Each dot on the graph denotes thenumber of ILF found per 6-mm sectionof proximal colon in an individualmouse. To represent the relative size ofILF between AKR and BALB/c mice 21days p.i., examples (ILF of average sizestained for B220) are shown in F. Allscale bars � 100 �m. The results arerepresentative of three separate experi-ments. �, p 0.05.

6720 LARGE INTESTINAL LEUKOCYTES AND T. muris INFECTION

by guest on Novem

ber 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

et al. (38) and Dohi et al. (40) discovered �50 ILF in the largeintestine of normal mice in addition to �10 colonic patches, theyneither described in detail nor showed any photographic examplesof these structures (38, 40). We found numerous pronounced fol-licular structures, invisible from the serosal or mucosal surface, inthe large intestine of both AKR and BALB/c mice. Consisting ofB cells interspersed with CD4� T cells and having a central zoneof rapidly proliferating cells, these lymphoid aggregations areanalogous to ILF. Therefore, the present study extends our knowl-edge of the GALT, illustrating ILF in the large intestine of mice forthe first time. While this manuscript was in revision, it was shownthat although colonic patches and ILF of the large intestine bothcontain M cells, they have a distinct structure and organogenesis(41). Lymphoid aggregations, equivalent in size to ILF, but devoidof B cells, were found in the large intestine of SCID mice. Thissuggests that the organogenesis of ILF depends on neither B cellsnor T cells. Indeed, similar structures have been identified in thesmall intestine of other immunodeficient mouse models, such asathymic nude mice (nu/nu) and RAG-2 knockout mice (38). It hasbeen shown that ILF are formed in the small intestine in responseto normal gut flora in the cecum (39). In the present study therewas a significant increase p.i. with T. muris in the number of largeintestinal ILF in AKR mice. These findings provide evidence forthe organogenesis of ILF in response to luminal stimuli. In ourmodel of T. muris infection, it is intriguing that ILF formationoccurs specifically in susceptible AKR mice. It may arise due tothe chronic infection of these mice, where the epithelium is ex-posed for a longer duration to T. muris Ag in the gut. This mayrepresent a diversion of the local immune response away from aneffective Th cell-dominated mechanism toward an inappropriateresponse dominated by B cells. The role of ILF in the pathogenesisof gastrointestinal infections and diseases should be investigated inthe future.

In conclusion, in this study we characterize for the first time theaccumulation of cells into the epithelium and lamina propria of thelarge intestine during infection of mice with T. muris. There weremarked differences in this respect between resistant and suscepti-ble strains of mouse. In resistant BALB/c mice, the local inflam-mation was dominated by CD4� IEL and F4/80� LPL at the timeof worm expulsion, in contrast to susceptible AKR mice, whereCD8� IEL and F4/80� LPL were predominant during thechronic phase of infection. Therefore, this study reveals thelocal immune responses underlying the expulsion of worms orthe persistence of chronic infection, respectively. Furthermore,we describe and illustrate ILF in the large intestine of mice anddemonstrate the organogenesis of these structures in response toT. muris infection.

AcknowledgmentsWe are grateful to Neil E. Humphreys for helpful discussions and advice.

DisclosuresThe authors have no financial conflict of interest.

References1. Else, K. J., D. Wakelin, D. L. Wassom, and K. M. Hauda. 1990. MHC-restricted

antibody responses to Trichuris muris excretory/secretory (E/S) antigen. ParasiteImmunol. 12: 509–527.

2. Else, K. J., D. Wakelin, D. L. Wassom, and K. M. Hauda. 1990. The influence ofgenes mapping within the major histocompatibility complex on resistance to Tri-churis muris infections in mice. Parasitology 101: 61–67.

3. Else, K. J., F. D. Finkleman, C. R. Maliszewski, and R. K. Grencis. 1994. Cy-tokine-mediated regulation of chronic intestinal helminth infection. J. Exp. Med.179: 347–351.

4. Bancroft, A. J., A. N. McKenzie, and R. K. Grencis. 1998. A critical role forIL-13 in resistance to intestinal nematode infection. J. Immunol. 160: 3453–3461.

5. Faulkner, H., J. C. Renauld, J. Van Snick, and R. K. Grencis. 1998. Interleukin-9enhances resistance to the intestinal nematode Trichuris muris. Infect. Immun. 66:3832–3840.

6. Richard, M., R. K. Grencis, N. E. Humphreys, J. C. Renauld, and J. Van Snick.2000. Anti-IL-9 vaccination prevents worm expulsion and blood eosinophilia inTrichuris muris-infected mice. Proc. Natl. Acad. Sci. USA 97: 767–772.

7. Else, K. J., L. Hultner, and R. K. Grencis. 1992. Cellular immune responses to themurine nematode parasite Trichuris muris. II. Differential induction of Th-cellsubsets in resistant versus susceptible mice. Immunology 75: 232–237.

8. Bancroft, A. J., K. J. Else, J. P. Sypek, and R. K. Grencis. 1997. Interleukin-12promotes a chronic intestinal nematode infection. Eur. J. Immunol. 27: 866–870.

9. Else, K. J., and R. K. Grencis. 1996. Antibody-independent effector mechanismsin resistance to the intestinal nematode parasite Trichuris muris. Infect. Immun.64: 2950–2954.

10. Betts, C. J., and K. J. Else. 1999. Mast cells, eosinophils and antibody-mediatedcellular cytotoxicity are not critical in resistance to Trichuris muris. ParasiteImmunol. 21: 45–52.

11. Betts, C. J., M. L. deSchoolmeester, and K. J. Else. 2000. Trichuris muris: CD4�

T cell-mediated protection in reconstituted SCID mice. Parasitology 121:631–637.

12. Picarella, D., P. Hurlbut, J. Rottman, X. Shi, E. Butcher, and D. J. Ringler. 1997.Monoclonal antibodies specific for �7 integrin and mucosal addressin cell adhe-sion molecule-1 (MadCAM-1) reduce inflammation in the colon of scid micereconstituted with CD45Rbhigh CD4� T cells. J. Immunol. 158: 2099–2106.

13. Brandtzaeg, P., and R. Pabst. 2004. Let’s go mucosal: communication on slipperyground. Trends Immunol. 25: 570–577.

14. Bonneville, M., C. A. Janeway, Jr., K. Ito, W. Haas, I. Ishida, N. Nakanishi, andS. Tonegawa. 1988. Intestinal intraepithelial lymphocytes are a distinct set of ��T cells. Nature 336: 479–481.

15. Goodman, T., and L. Lefrancois. 1988. Expression of the �-� T-cell receptor onintestinal CD8� intraepithelial lymphocytes. Nature 333: 855–858.

16. Bandeira, A., S. Itohara, M. Bonneville, D. O. Burlen, S. T. Mota, A. Coutinho,and S. Tonegawa. 1991. Extrathymic origin of intestinal epithelial lymphocytesbearing T-cell antigen receptor ��. Proc. Natl. Acad. Sci. USA 88: 43–47.

17. Guy-Grand, D., N. Cerf-Bensussan, B. Malissen, M. Malassis-Seris, C. Briottet,and P. Vassalli. 1991. Two gut intraepithelial CD8� lymphocyte populationswith different T cell receptors: a role for the gut epithelium in T cell differenti-ation. J. Exp. Med. 173: 471–481.

18. Lefrancois, L. 1991. Phenotypic complexity of intraepithelial lymphocytes of thesmall intestine. J. Immunol. 147: 1746–1751.

19. Rocha, B., P. Vassalli, and G. D. Guy. 1991. The V� repertoire of mouse guthomodimeric � CD8� intraepithelial T cell receptor �/�� lymphocytes reveals amajor extrathymic pathway of differentiation. J. Exp. Med. 173: 483–486.

20. Camerini, V., C. Panwala, and M. Kronenberg. 1993. Regional specialization ofthe mucosal immune system: intraepithelial lymphocytes of the large intestinehave a different phenotype and function than those of the small intestine. J. Im-munol. 151: 1765–1776.

21. Beagley, K. W., K. Fujihashi, A. S. Lagoo, S. Lagoo-Deenadaylan, C. A. Black,A. M. Murray, A. T. Sharmanov, M. Yamamoto, J. R. McGhee, C. O. Elson, etal. 1995. Differences in intraepithelial lymphocyte T cell subsets isolated frommurine small versus large intestine. J. Immunol. 154: 5611–5619.

22. Boll, G., A. Rudolphi, S. Spiess, and J. Reimann. 1995. Regional specializationof intraepithelial T cells in the murine small and large intestine. Scand. J. Im-munol. 41: 103–113.

23. Yamamoto, M., K. Fujihashi, K. W. Beagley, J. R. McGhee, and H. Kiyono.1993. Cytokine synthesis by intestinal epithelial lymphocytes. J. Immunol. 150:106–114.

24. Wakelin, D. 1967. Acquired immunity to Trichuris muris in the albino laboratorymouse. Parasitology 57: 515–524.

25. Else, K. J., and R. K. Grensis. 1991. Cellular immune responses to the murinenematode parasite Trichuris muris. I. Differential cytokine production duringacute or chronic infection. Immunology 72: 508–513.

26. Else, K. J., G. M. Entwistle, and R. K. Grensis. 1993. Correlations between wormburden and markers of Th1 and Th2 cell subset induction in an inbred strain ofmouse infected with Trichuris muris. Parasite Immunol. 15: 595–600.

27. Davies, M. D., and D. M. Parrott. 1981. Preparation and purification of lympho-cytes from the epithelium and lamina propria of murine small intestine. Gut 22:481–488.

28. Potten, C. S., D. Booth, N. J. Cragg, G. L. Tudor, J. A. O’Shea, C. Booth,F. A. Meineke, D. Barthel, and M. Loeffler. 2002. Cell kinetic studies in themurine ventral tongue epithelium: mucositis induced by radiation and its protec-tion by pre-treatment with keratinocyte growth factor (KGF). Cell Prolif.35(Suppl. 1): 32–47.

29. Sueta, T., I. Miyoshi, T. Okamura, and N. Kasai. 2002. Experimental eradicationof pinworms (Syphacia obvelata and Aspiculuris tetraptera) from mice coloniesusing ivermectin. Exp. Anim. 51: 367–373.

30. Koyama, K., H. Tamauchi, and Y. Ito. 1995. The role of CD4� and CD8� T cellsin protective immunity to the murine nematode parasite Trichuris muris. ParasiteImmunol. 17: 161–165.

31. Cliffe, L. J., N. E. Humphreys, T. E. Lane, C. S. Potten, C. Booth, andR. K. Grencis. 2005. Accelerated intestinal cell turnover: a new mechanism ofparasite expulsion. Science 308: 1463–1465.

32. Artis, D., M. L. Wang, S. A. Keilbaugh, W. He, M. Brenes, G. P. Swain,P. A. Knight, D. D. Donaldson, M. A. Lazar, H. R. P. Miller, et al. 2004. RELM�/FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinaltract. Proc. Natl. Acad. Sci. USA 101: 13596–13600.

6721The Journal of Immunology

by guest on Novem

ber 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

33. Humphreys, N. E., J. J. Worthington, M. C. Little, E. J. Rice, and R. K. Grensis.2004. The role of CD8� cells in the establishment and maintenance of Trichurismuris infection. Parasite Immunol. 26: 187–196.

34. Belkaid, Y., C. A. Piccrillo, S. Mendez, E. M. Shevach, and D. L. Sacks. 2002.CD4�CD25� regulatory T cells control Leishmania major persistence and im-munity. Nature 420: 502–507.

35. deSchoolmeester, M. L., M. C. Little, B. J. Rollins, and K. J. Else. 2003. Absenceof CC chemokine ligand 2 results in an altered Th1/Th2 cytokine balance andfailure to expel Trichuris muris infection. J. Immunol. 170: 4693–4700.

36. O’Leary, A. D., and E. C. Sweeney. 1986. Lymphoglandular complexes of thecolon: structure and distribution. Histopathology 10: 267–283.

37. Moghaddami, M., A. Cummins, and G. Mayrhofer. 1998. Lymphoid-filled villi:comparison with other lymphoid aggregations in the mucosa of the human smallintestine. Gastroenterology 115: 1414–1425.

38. Hamada, H., T. Hiroi, Y. Nishiyama, H. Takahashi, Y. Masunaga,S. Hachimura, S. Kaminogawa, H. Takahashi-Iwanaga, T. Iwanaga,

H. Kiyono, et al. 2002. Identification of multiple isolated lymphoid follicleson the antimesenteric wall of the mouse small intestine. J. Immunol. 168:57– 64.

39. Lorenz, R. G., D. D. Chaplin, K. G. McDonald, J. S. McDonough, andR. D. Newberry. 2003. Isolated lymphoid follicle formation is inducible anddependent upon lymphotoxin-sufficient B lymphocytes, lymphotoxin � receptor,and TNF receptor I function. J. Immunol. 170: 5475–5482.

40. Dohi, T., P. D. Rennert, K. Fujihashi, H. Kiyono, Y. Shirai, Y. I. Kawamura,J. L. Browning, and J. R. McGhee. 2001. Elimination of colonic patches withlymphotoxin � receptor-Ig prevents Th2 cell-type colitis. J. Immunol. 167:2781–2790.

41. Kweon, M.-N., M. Yamamoto, P. D. Rennert, E. J. Park, A.-Y. Lee, S.-Y. Chang,T. Hiroi, M. Nanno, and H. Kiyono. 2005. Prenatal blockade of lymphotoxin �receptor and TNF receptor p55 signaling cascade resulted in the acceleration oftissue genesis for isolated lymphoid follicles in the large intestine. J. Immunol.174: 4365–4372.

6722 LARGE INTESTINAL LEUKOCYTES AND T. muris INFECTION

by guest on Novem

ber 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from