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Clinical Immunology 11

Commensal microbiota alter the abundance and TCR responsiveness

of splenic naıve CD4+ T lymphocytes

Tiffany Huang a, Bo Wei b, Peter Velazquez b,1, James Borneman c, Jonathan Braun a,b,*

a Molecular Biology Institute, David Geffen School of Medicine at UCLA, University of California,

Los Angeles, 10833 Le Conte Avenue, CHS 13-222, Los Angeles, CA 90095, USAb Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, University of California,

Los Angeles, 10833 Le Conte Avenue, CHS 13-222, Los Angeles, CA 90095, USAc Department of Plant Pathology, University of California, Riverside, CA 92521, USA

Received 12 November 2004; accepted with revision 27 September 2005

Available online 14 November 2005

Abstract

The epidemiologic risk of certain systemic immunologic diseases is affected by commensal or environmental microbiota, but the cellular basis

of the ‘‘hygiene hypothesis’’ is poorly understood. In this study, we demonstrate that composition of the commensal microbiota affects the

functional state of the peripheral naıve (CD62LhiCD44lo) T lymphocyte populations. Restricted flora (RF) mice (stably colonized with excess

nonpathogenic Clostridium sp., and changes in other bacterial and fungal taxa) were distinguished after the neonatal period by a progressive

deficiency in absolute numbers of naıve CD4+ and CD8+ T lymphocytes. SPF and RF mice had comparable levels of memory CD4+ and CD8+ T

cells. This phenotype was attributable to the altered levels of certain commensals and their products, since germ-free mice had normal absolute

numbers of splenic CD4+ and CD8+ T cells and their respective naıve and memory subsets. The naıve CD4+ T cell subset was functionally

distinguished in RF mice versus SPF mice by TCR hyperresponsiveness, pro-inflammatory cytokine production, and increased activation-induced

cell death. Biochemically, these traits were associated with higher basal phosphorylation of the TCR signaling proteins ZAP-70, Lck, and LAT.

These findings indicate that enteric microbial products, through unknown cellular circuitry, influence steps in CD4 T cell differentiation

moderating basal TCR signaling and immune responsiveness.

D 2005 Elsevier Inc. All rights reserved.

Keywords: T cell receptors; Bacteria; Fungi; Commensals; Signaling; T lymphocytes; Mucosal immunology; Hygiene hypothesis

Introduction

Mucosal lymphocytes require the colonization of commensal

bacteria to develop and function appropriately [1]. The reduced

size of gut-associated mucosal tissues and Peyer’s patch, and

reduced cell numbers of lamina propria or intraepithelial T cells

are the characteristics of germ-free rodents which lack

commensal microflora [2–6]. Restoration of the normal

microflora reverses the germ-free mucosal phenotype, further

1521-6616/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.clim.2005.09.012

Abbreviations: AICD, activation-induced cell death; RF, restricted flora;

SPF, specific pathogen free.

* Corresponding author. Department of Pathology and Laboratory Medicine,

CHS 13-222, UCLA School of Medicine, Los Angeles, CA 90095-1732, USA.

Fax: +1 310 825 5674.

E-mail address: jbraun@mednet.ucla.edu (J. Braun).1 Present address: Skirball Institute of Biomolecular Medicine, New York

University School of Medicine, 540 First Ave., New York, NY 10016, USA.

suggesting the requirement of commensal bacteria for mucosal

lymphocytes development. Germ-free mice also have decreased

and delayed immune responses after mucosal antigen challenge,

indicating that the presence of commensal bacteria in the

gastrointestinal tract helps to prime mucosal lymphocytes for

quicker immune responses to antigen exposure [7–11]. The

induction and/or maintenance of mucosal tolerance requires the

presence of bacteria in the intestine [12–15]. The loss of

commensal bacteria augments disease in certain models of

autoimmunity, including diabetes onset in NOD mice, collagen-

induced arthritis, and myelin basic protein-induced experimen-

tal autoimmune encephalomyelitis [16–20].

Attenuation of TCR activation is an important homeostatic

feature of mucosal CD4+ Tcells. However, the process directing

this mode of Tcell development and the biochemical basis of the

modified signaling pathway itself are poorly understood. Most

mucosal T lymphocytes have an activated or memory phenotype

7 (2005) 221 – 230

www.e

T. Huang et al. / Clinical Immunology 117 (2005) 221–230222

with CD45RBlo, CD62Llow, CD44hi, and CD69hi surface

markers. This state in part may reflect ongoing innate or

adaptive stimulation of the mucosal immune system by enteric

bacterial products. However, immune ignorance or tolerance

autologous enteric flora by these mucosal lymphocytes has also

been reported [21,22]. Mucosal T cells produce both pro- and

anti-inflammatory cytokines such as IFN-g and IL-10, indicat-

ing that homeostasis is maintained through an active immuno-

regulatory process.

In this study, we tested whether there is an effect of enteric

microbial colonization on the composition of the systemic T

cell population. Germ-free mice were comparable to SPF mice

in their abundance of the major splenic T cell subsets. In

contrast, RF mice (bearing an altered enteric microbiota) had a

selective numerical deficit in naıve CD4+ and CD8+ T cells,

accompanied by functional hyperresponsiveness and Th1

polarization. Collectively, these observations demonstrate a

role for commensal enteric bacteria in shaping the systemic T

cell population.

Materials and methods

Mice

Specific pathogen free (SPF) C57BL/6 mice were obtained

from Jackson Laboratories (Bar Harbor, ME) and monitored

for a panel pathogenic microorganisms based on serologic

and microbiologic screening by the UCLA Division of

Laboratory Animal Medicine. A restricted flora (RF) colony

was established 12 years ago in a separate facility maintained

by the UCLA Department of Radiation Oncology, by

Fig. 1. Normal abundance of splenic T cells in germ-free mice. Splenocytes fro

(lymphocyte size gate) for CD4+ and CD8+ T cells and tabulated for the absolute n

were calculated using the percentage of ‘‘naıve’’ (CD62Lhi CD44lo) and ‘‘memory’’

(CD44hi) in CD8+ T subsets (b). Each symbol represents data from an individual m

extensive antibiotic treatment and recolonization with 6

species of nonpathogenic Clostridium [23]. RF C57BL/6

mice were established 6 years ago by cesarean section

delivery of SPF fetal C57BL/6 mice and adoptive transfer

to RF foster mothers. RF mice were housed in enclosed racks

with filtered air, autoclaved bedding, food, and water. The

mice examined in this study were 8- to 10-week-old male in

both SPF and RF groups. All animal procedures were

performed in accordance with current UCLA institutional

review board-approved protocols.

C57BL/6 mice (Taconic Laboratories, Germantown, NY)

were maintained under SPF conditions free of Helicobacter

species. Germ-free C57BL/6 mice were derived by hysterecto-

my and maintained in the Gnotobiotic Animal Core at the

College of Veterinary Medicine, North Carolina State Univer-

sity, Raleigh. Absence of contamination by other bacterial

species was confirmed by periodic aerobic culture of stool

samples. Eight- to ten-week-old males were used from both

groups of mice. Animal use protocols were approved by the

Institutional Animal Care and Use Committee (IACUC), North

Carolina State University and the University of North Carolina

at Chapel Hill.

Antibodies and reagents

Except where indicated, monoclonal antibodies were

purchased as purified proteins from BD Bioscience (San

Diego, CA). These included unlabeled or fluorochrome-

conjugated antibodies for CD3( (145–2C11), CD28 (37.51),

PE- or CD4 (RM4–5), CD8 (53–6.7), CD62L (MEL-14),

CD44 (IM7), PCD178 (MFL3), and CD95 (Jo2). Anti-

m germ-free and age-matched SPF mice were analyzed by flow cytometry

umbers per mouse (‘‘total’’). The absolute numbers of naıve and memory cells

(CD62Llo CD44hi) in CD4+ T cells (a), and ‘‘naıve’’ (CD44lo) and ‘‘memory’’

ouse. P values are student’s t test comparing germ-free and SPF groups.

Fig. 3. Reduced splenic CD4+ T cell population in RF mice. (a) Splenocytes

from SPF and RF mice were analyzed by flow cytometry (lymphocyte size

gate) for CD4+ and CD8+ cells. (b) Tabulation of CD4+ T cells (%total

lymphocytes) in SPF and RF mice (each symbol represents one experiment

data). (c) Tabulation of the absolute number of CD4+ T cells. (d) Tabulation of

percentage of CD8+ T cells. (e) Tabulation of the absolute number of CD8+ T

cells. P values are student’s t test comparing SPF and RF groups.

Fig. 2. Comparison of enteric microbial taxa in RF and SPF mice. Molecular

clones isolated from small and large intestines of RF and SPF mice were

identified by OFRG analysis (24). The numbers of bacterial and fungal taxa in

each intestinal segment were tabulated for RF and SPF mice and expressed as

the RF:SPF ratio. The four major fungal phyla are indicated in bold.

T. Huang et al. / Clinical Immunology 117 (2005) 221–230 223

phospho-ZAP-70, anti-phospho-Lck, and anti-phospho-LAT

were purchased from Cell Signaling (Beverly, MA).

T cell stimulation and cytokine assays

Splenic CD4+ cells were purified using anti-CD4

microbeads (L3T4) according to the manufacturer’s instruc-

tions (Miltenyi Biotech, Auburn, CA). Purity was determined

by FACS analysis and ranged between 90 and 95% for

CD4+ expression. Cells were stimulated for 72 h in complete

medium (RPMI 1640 medium supplemented with 5% heat

inactivated FCS, 50 AM 2-ME, 1.0 mM sodium pyruvate,

100 U/ml penicillin, 0.1 mg/ml streptomycin) with plate-

bound anti-CD3 (1 Ag/ml) in the presence or absence of

soluble anti-CD28 (1 Ag/ml) at 37-C in a 5% CO2-

humidified atmosphere. Supernatants for cytokine measure-

ments were harvested after 72 h of cell culture and assayed

using the OptEIA ELISA kits according to manufacturer’s

instruction (BD PharMingen, San Diego, CA). Plates were

read on a microplate reader (BioRad, Richmond, CA).

Cytokine concentrations were calculated by MPM software

program based on the standard curves.

Flow cytometry

Cell surface staining was performed with splenocytes

depleted of erythrocytes. Data were collected on a LSR

flow cytometer (BD Biosciences) and analyzed using

CellQuest software. All staining profiles were based on

live gates, as determined by forward and side-scatter. Naıve

and memory subsets were distinguished by anti-CD44 and

anti-CD62L expression; in some cases, CD8+ T cell subsets

were also delineated with anti-CD122 and Ly6C. Intracel-

lular staining was performed with Cytofix/Cytoperm kit

(BD PharMingen, San Diego, CA) according to manufac-

ture’s instructions.

T cell proliferation with CFSE

For measurement of CFSE-incorporated proliferation, puri-

fied CD4+ T cells were incubated with CFSE (5 AM) in PBS for

10 min at 37-C, washed with cold media three times, and then

plated in anti-CD3 coated 96-well plates in the presence or

absence of anti-CD28 (1 Ag/ml) for 60 h. Cultures were

harvested and stained with PE-labeled anti-CD62L to distin-

guish naıve and memory subsets. The cells were collected on a

LSR flow cytometer (BD Biosciences) and analyzed using

CellQuest software.

T. Huang et al. / Clinical Immunology 117 (2005) 221–230224

Apoptosis assay

Purified CD4+ or CD8+ T cells (1 � 106 cells) were cultured

in 24-well plates coated with anti-CD3 and anti-CD28 as

described above, but also with anti-CD95. The cells were

cultured for 18 h, harvested, and stained with annexin-V and

propidium iodide (BD PharMingen). Cell death was measured

using a LSR flow cytometer as the %cells positive for annexin-

V or propidium iodide.

Results

Normal formation of systemic T cell populations in germ-free

mice

In germ-free mice lacking detectable enteric bacteria, B and T

cell populations associated with the intestinal mucosa are

dramatically altered in abundance and function. To evaluate

whether the changes in the mucosal compartment affected the

overall state of T cells in the periphery, we evaluated the

abundance of splenic CD4+ and CD8+ T cells in germ-free and

age-matched SPF mice. A minor but significant reduction was

observed in the absolute numbers of CD4+ T cells (Fig. 1a).

However, phenotypically identified naıve and memory subsets

of CD4+ T cells were within normal range (Fig. 1a), and no

differences from SPF mice were observed for CD8+ T cells and

their subsets (Fig. 1b). Analysis of these CD4+ and CD8+ T cell

subpopulations on a percent basis resulted in concordant

findings (data not shown). These results indicate that enteric

Fig. 4. Reduced naıve CD4+ and CD8+ T cell populations in RF mice. Splenocytes w

The percentage of‘ naıve and memory subsets was determined as a percentage of ga

SPF and RF mice (b). Tabulation of the absolute number of naıve and memory CD

subsets in SPF and RF mice (d). Tabulation of the absolute number of naıve and mem

RF and SPF groups.

bacteria are not required to drive the normal numerical

abundance of the peripheral T cell compartment.

RF mice are reduced in absolute numbers of CD4+ T cells

We next asked whether divergent enteric bacteria positively

or negatively influence the peripheral T cell compartment. For

this purpose, we evaluated RF mice, a long-term colony of

C57BL/6 mice originally produced by intestinal recolonization

with six species of nonpathogenic Clostridium (see Materials

and methods section). A recent study using oligonucleotide

fingerprinting of ribosomal RNA genes (OFRG) analyzed the

composition of bacterial and fungal entericmicrobiotamore than

50,000 molecular clones isolated from large and small intestines

of SPF and RF mice [24]. A summary of these findings is

presented in Fig. 2. As expected, Clostridium species were

overrepresented in both the large and small intestine. However,

substantial differences also were observed in the apparent

abundance of a variety of bacterial and fungal taxa.

Splenic T lymphocyte subpopulations from age-matched RF

and SPF mice were then analyzed. Compared to SPF mice, RF

mice had about half the number of total splenic lymphocytes,

reflecting a two-fold decrease in both T and B cell subpopula-

tions (Fig. 3 and data not shown). As a percent of total

lymphocytes, CD4+ T cells were similar in both SPF and RF

mice (Figs. 3a, b), whereas the CD8+ T cells were increased

about two-fold in RFmice (Figs. 3a, d). To understand the reason

for this relative change in T cell subsets, we determined their

absolute numbers (Figs. 3c, e). This analysis revealed that

ere analyzed by flow cytometry using naıve and memory markers (see Fig. 1).

ted CD4+ or CD8+ T cells. (a) Percentage of naıve and memory CD4+ T cells in

4+ subsets in SPF and RF mice (c). Percentage of naıve and memory CD8+ T

ory CD8+ T subsets in SPF and RF mice. P values are student’s t test comparing

Fig. 5. Ontogeny of naıve CD4+ and CD8+ T cell populations in RF and SPF

mice. Splenocytes from 4-, 6-, and 8-week-old SPF and RF were analyzed by

flow cytometry for total, naıve, and memory CD4+ and CD8+ T cells. Absolute

cell numbers (mean T SEM) were tabulated from 5 or more mice. (a) Total

CD4+ and CD8+ T cells; (b) naıve and memory CD4+ T cells. (c) Naıve and

memory CD8+ T cells. Asterisks denote P < 0.01 for student’s t tests comparing

RF and SPF groups.

T. Huang et al. / Clinical Immunology 117 (2005) 221–230 225

absolute numbers of CD4+ T cells were reduced in RF mice,

while CD8+ Tcells were comparable in SPF and RF mice. Thus,

altered commensal enteric microflora influenced the size of the

CD4+ T cell compartment, while the overall size of the CD8+ T

cell compartment was unaffected.

RF mice are reduced in the CD4+ and CD8+ naıve Tcell subsets

The immunological balance of naıve and memory subsets in

the periphery is actively maintained [25]. We therefore

wondered whether altered commensal colonization might

selective affect formation of naıve and memory T cell subsets.

In both CD4+ and CD8+ T cell compartments, the percent of

memory cells was increased about two-fold in RFmice (Figs. 4a,

c). The change in these compartments was further defined by

determining absolute numbers of these T cell subsets. Since RF

mice had fewer splenocytes, the absolute numbers of memory

CD4+ Tcell numbers were comparable in SPF and RFmice (Fig.

4b). Moreover, the absolute numbers of memory CD8+ T cells

were actually increased in RF compared to SPF mice (Fig. 4d).

These findings were confirmed using CD122 and Ly6C as

alternate markers to distinguish memory CD8+ T cells (data not

shown). These data indicated that RF mice were selectively

impaired in the formation or maintenance of naıve populations in

the CD4+ and CD8+ compartments. Moreover, despite the

altered antigen exposure in RF mice, their memory CD4+ and

CD8+ T cell compartments were preserved or even augmented.

Reduction of naive CD4+ and CD8+ T cells in RF mice occur

early in ontogeny

The size of the peripheral lymphocyte population expands

during the first weeks of life and stabilizes during the first month

after weaning [3,10]. To determine when the relative reduction in

naıve CD4+ and CD8+ T cells occurred, SPF and RF mice at 4

and 6 weeks old were examined for their Tcell profile. As shown

in Fig. 5a, the CD4+ Tcell numbers in RF mice lagged SPF mice

between the ages of 4 and 8 weeks. CD8+ T cell numbers lagged

at 4 weeks, but dramatically increased by 6 weeks to levels

comparable to age-matched SPF mice. Examination of the naıve

and memory subsets revealed that naıve CD4+ and CD8+ T cells

in RF mice lagged SPF mice at 4 and 8 weeks (Figs. 5b, c), and

the memory CD4+ Tcells were comparable between the SPF and

RF mice regardless of age (Fig. 5b).

The number of memory CD8+ T cells was reduced in RF

mice at 4 weeks old, but dramatically increased to exceed

levels in SPF mice at and beyond 6 weeks of age (Fig. 5c).

Although the mechanism of this episode of expansion is

uncertain, it is notable that this correlates with post-weaning

environmental antigenic stimulation of CD8+ T cells, or to

other elements of the mucosal environment involved in

memory CD8+ T cell formation.

RF CD4+ T cells are hyperresponsive to TCR stimulation

Since naıve CD4+ and CD8+ T cells were reduced in RF

mice but not germ-free mice, it appeared that certain

commensal microbiota impaired the formation or maintenance

of the naıve T cell population. We next assessed the

functional state of this T cell population. Purified CD4+ T

cells from SPF and RF mice were isolated and activated in

vitro with anti-CD3 in the absence or presence of anti-CD28

and evaluated after 72 h for cytokine secretion and

proliferative responses. Both SPF and RF CD4+ T cells

produced IL-2 and IFN-g in a dose-dependent manner with

TCR stimulation. However, RF CD4+ T cells displayed

elevated levels of both IL-2 and IFN-g production (Figs.

6a, b). IL-10 production by SPF and RF cells was minimally

detectable (data not shown). The higher levels of IL-2 and

IFN-g productions suggested that RF CD4+ T cells were

hyperresponsive to TCR stimulation.

Proliferation was assessed in these stimulated CD4+ T cells

by CFSE (Fig. 6c). At 60 h, CD4+ T cells from both RF and

SPF mice achieved four cell divisions with anti-CD3 alone or

with anti-CD28. However, a larger fraction of RF CD4+ T cells

achieved multiple divisions in RF CD4+ T cells (left shift in

CFSE intensity), again reflecting TCR hyperresponsiveness in

the RF-derived population. To compare the response in naıve

and memory T cells, we assessed the CFSE profile of

Fig. 6. TCR hyperresponsiveness of RF CD4+ T cells. CD4+ T cells were stimulated with anti-CD3 and anti-CD8, and analyzed after 72 h for cytokine production by

ELISA, and after 60 h for proliferation by flow cytometric CFSE dilution. (a) IL-2 secretion; (b) IFN-g secretion; (c) proliferation; (d) flow analysis of CFSE dilution

counterstained with anti-CD62L to identify naıve (CD62Lhi) and memory (CD62Llo) CD4+ T cells.

T. Huang et al. / Clinical Immunology 117 (2005) 221–230226

CD3+CD4+ gated cells that were additionally stained with anti-

CD62L (Fig. 6d). As expected, multiple cell divisions were

mainly observed in naıve (CD62L+) cells from SPF mice, since

memory cells are know to poorly respond to in vitro

stimulation. Proliferation also predominated in naıve cells

from RF mice, but these were again distinguished with a larger

fraction of cells with multiple divisions, and a greater

stimulation-induced decline in CD62L expression.

RF CD4+ but not CD8+ T cells are more sensitive to

activation-induced cell death (AICD)

In addition to proliferation, a notable consequence of

intense TCR activation is AICD. To evaluate the AICD

response, CD4+ T cells from RF and SPF mice were

stimulated with anti-CD3, anti-CD28, and anti-CD95. The

cells were harvested after 16 h of culture and stained with

annexin-V and propidium iodide to identify cells engaged in

the process of apoptosis and cell death. As predicted, greater

AICD was observed in RF versus SPF CD4+ T cells (Fig. 7a).

In contrast, AICD was comparable in CD8+ T cells from RF

and SPF mice (Fig. 7b). However, the sensitivity for

distinguishing RF versus SPF AICD rates in the CD8+

compartment may have been impaired by the high level of

AICD in all CD8+ T cells. These data provided independent

validation for the selective TCR hyperresponsiveness of RF

naıve CD4+ T cells.

Increased basal activation of RF naıve CD4+ T cells

To further characterize the RF naıve population with this

qualitative hyperresponsiveness, we analyzed the present cells

Fig. 7. Increasedactivation-inducedcelldeathinRFCD4+Tcells.CD4+andCD8+

T cells were cultured with anti-CD3, -CD28, and -CD95. After 18 h, cells were

stainedwith annexin-Vand propidium iodide and analyzed for%positive cells for

annexin-Vor propidium iodide staining. (a) %Positive CD4+ T cells; (b) %posi-

tive CD8+ Tcells. P values are student’s t tests comparing SPF and RF groups.

T. Huang et al. / Clinical Immunology 117 (2005) 221–230 227

expressing CD69, an early Tcell activation marker. As expected,

CD69 was low in naıve CD4+ Tcells from SPFmice. In contrast,

CD69 was elevated in cells from RF mice (Fig. 8a, left panel).

Fig. 8. Elevated basal protein phosphorylation of TCR signaling components in R

positive selection and stained on ice for naıve and memory markers (CD62L and CD

for phosphorylated forms of LAT, lck, and ZAP-70. (a) %CD69+ cells in naıve and m

(c) %P-LAT, P-lck, or P-ZAP-70 in naıve CD69+ CD4+ T cells. Symbols are data fro

CD69was high inmemory CD4+ Tcells, at comparable levels in

both SPF and RF mice (Fig. 8a, right panel).

To further evaluate this basally activated phenotype, RF and

SPF naıve CD4+ T cells were compared for basal levels of

activated (phosphorylated) forms of proximal TCR signaling

proteins. CD69+ cells displayed elevated phosphoprotein levels

in both SPF and RF mice, reflecting their in vivo-activated

state (Fig. 6b). As expected, phosphoprotein levels were low in

CD69� (quiescent) cells from SPF mice (Fig. 6c). However,

phosphoprotein levels were significantly elevated in CD69�

cells from RF mice. Thus, the hyperresponsiveness of RF naıve

CD4+ T cells involves not only a higher frequency of in vivo-

activated cells but also an elevated state of basal biochemical

signaling activity even in phenotypically quiescent cells.

Discussion

Studies with germ-free mice have established that normal

enteric microbiota are necessary for the abundance and

activation state of T lymphocytes resident in the intestinal

mucosa. However, the role of commensal microbiota in the

state of T cells in the periphery is less understood. In this study,

we find a surprising role for altered enteric microbiota on naıve

CD4+ and CD8+ T cells. RF mice, stably colonized with an

altered intestinal microbiota, have a selective numerical deficit

in naıve T cells. The residual naıve CD4+ T cell subset in these

F naıve CD4+ T cells. (a) Splenic CD4+ T cells were isolated and purified by

44) and the activation marker CD69. Cells were then permeabilized and stained

emory subsets. (b) %P-LAT, P-lck, or P-ZAP-70 in naıve CD69- CD4+ T cells;

m individual mice. P values are student’s t tests comparing SPF and RF groups.

T. Huang et al. / Clinical Immunology 117 (2005) 221–230228

mice are hyperresponsive, AICD-prone, and basally activated

in vivo. In this discussion, we consider the possible cellular

mechanisms for this aberrant T cell phenotype.

What properties of the commensal microbiota might

contribute to the abnormal peripheral T cell phenotype of RF

mice? RF mice differ from SPF mice in their composition of

commensal enteric bacterial and fungal taxa [24]. Microbial

composition accounts for the RF phenotype, since RF mice

conventionalized to SPF housing conditions at the time of birth

developed a normal, SPF-like T cell compartment (T. Huang

and B. Wei, unpublished data). It should be noted that

differences in microbial composition probably extend to other

commensal compartments (such as the skin or respiratory

system), so the biologically pertinent microorganisms may

reside in any of these anatomic sites.

Analysis of germ-free mice displayed a normal abundance

of the major splenic T cell subsets. This confirms that the

biologic niche required by the peripheral T cell compartment

forms independently of molecules derived from commensal

bacteria. Accordingly, the RF phenotype probably is not due to

the absence of certain microorganisms. Instead, it implies that

the increased action of unconventional commensals in RF mice

may impair formation or maintenance of the naıve T cell

compartment. While the microorganisms contributing to this

role are uncertain, candidates include microorganisms over-

represented in RF mice, including Clostridium and certain

fungi taxa.

What are the possible mechanisms that caused immunolog-

ically developmental defects in the RF mice? Various microbial

sensing systems, notably toll-like receptors (TLRs) in hemo-

poietic and epithelial cell types [26–28], play a critical role in

differentiating antigen presentation function by these cell types

[29–31]. Compared to memory T cells, naıve T cells are

particularly dependent on optimal functional state of antigen-

presenting cells [32–34]. Impairment of this differentiation

process is thus a plausible target for the RF phenotype,

exemplified by the microbial inactivation of NFkB signaling in

antigen-presenting cells [35–37]. As a second precedent, T cell

superantigens promote AICD in a process that is selective for

naıve T cells, and such molecules are produced by uncommon

enteric commensals [38,39].

Another possible cellular mechanism is the alterations of

cytokine/chemokine profiles in the RF mice, resulting in the

impaired recruitment and survival of naıve T cell subsets.

Innate microbial sensing through several receptor systems

elicits TNF-a and IL-12, which selectively differentiate naıve T

cells [29,40]. Dendritic cells produce a variety of chemokines

to recruit T cells [41–45], and molecules such as CXCL12 and

CCL21 increase the adhesion of naıve CD8 cells to the antigen

presenting cells in an antigen-independent manner [46].

Cytokines are also selective trophic for naıve versus memory

cell expansion, such as IL-2 versus IL-15 in CD8+ T cells

[33,34,47–55]. Mucosal homing and clonal expansion of

intraepithelial and mesenteric T cells are impaired in RF mice

[23,56]. This phenotype in part may be due to a deficiency in

the commensals producing innate and adaptive antigenic

stimuli required for normal mucosal immune system formation,

as observed in germ-free mice. However, the present study

suggests that the mucosal T cell deficiency also may be

compounded by the reduced abundance and aberrant functional

state of the systemic naıve T cell compartment.

A surprising feature of the naıve T cell population in RF

mice was their TCR hyperresponsiveness and elevated basal

state of activation in vivo. CD4+ T cells from mice loss of

function mutations in positive regulators such as ZAP-70 and

LAT exhibited hyporesponsiveness to TCR-mediated stimula-

tion, whereas overexpression of these molecules reduces in

hyperresponsiveness [57–59]. Conversely, deficiency of neg-

ative regulators such as c-cbl and CTLA-4 has a lymphopro-

liferative phenotype [60–63]. In RF T cells, an elevated basal

state of activation involved the entire proximal segment of

TCR signaling. This finding suggests that the RF T cell

disorder might involve abortive TCR signaling, such as

observed with partial peptide agonists, or due to aberrant T

cell expression of regulatory molecules affecting the phos-

phorylation state of the CD3 complex itself [64,65]. It is also

possible that this phenotype is augmented by some degree of

homeostatic expansion in these partially lymphopenic mice

[66,67].

In contrast to the phenotype of RF mice, increased AICD

might be expected to mainly impair formation of memory cells

and preserve naıve T cells. However, the ultimate size of the

memory pool is determined by many additional factors,

including the degree of recurrent antigenic challenge and

immunoregulatory processes; moreover, the maintenance of the

memory pool is relatively independent of TCR stimulation

[68,69]. With respect to naıve cells, premature activation and

AICD would be expected to deplete the naıve T cell pool. Thus,

a reduced antigenic threshold for activation and AICD in naıve

T cells is a plausible factor in the selective depletion of these

cells in RF mice.

The hyperresponsiveness of RF T cells reflects a discrete,

systemic T cell abnormality resulting from altered commensal

microbiota. The epidemiology of allergy, atopy, and certain

autoimmune diseases relates this immunologic susceptibility to

certain profiles of microbial colonization or antigenic exposure,

including products of helminths, bacteria, and fungi [70–75].

The cellular mechanisms underlying this ‘‘hygiene hypothesis’’

are relatively unaddressed, although particularly in the case of

helminths, model systems suggest that reduced TCR respon-

siveness is one important factor. The present study indicates that

the RF phenotype may provide an animal model for this

epidemiologic relationship and could be used to delineate the

microbial products and host traits resulting in the hyperrespon-

sive T cell trait. It also suggests a set of T cell biomarkers that

might be used to determine whether this phenotype is observed

in a subset of individuals at risk for atopy in the human

population.

Acknowledgments

Supported by NIH DK46753 (J.B.), DK69434 (J.B.),

DK43026 (CURE), AI07126 (T.H.), the Eli and Edythe L.

Broad Medical Research Foundation (J.B., J.B.), the Crohn’s

T. Huang et al. / Clinical Immunology 117 (2005) 221–230 229

and Colitis Foundation of America (B.W.), and the Jonsson

Comprehensive Cancer Center. Flow cytometry was performed

in the UCLA Jonsson Comprehensive Cancer Center for AIDS

Research Flow Cytometry Core Facility, supported by NIH

CA-16042 and AI-28697. Germ-free mice were produced

by and analyzed at the Center for Gastrointestinal Biology

and Disease, UNC-Chapel Hill School of Medicine (NIH

DK34987, R. Balfour Sartor, P.I.).

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