© Rockefeller University Press,
0022-1007/2003/11/1403 $5.00
The Journal of Experimental Medicine, Volume 198, Number 9, 1403-1414
Age-dependent Requirement for 
T Cells in the Primary but Not Secondary Protective Immune Response against an Intestinal Parasite
Elizabeth Ramsburg1,
Robert Tigelaar1,2,
Joe Craft1,3 and
Adrian Hayday4
1 Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06511
2 Department of Dermatology, Yale University School of Medicine, New Haven, CT 06511
3 Section of Rheumatology, Yale University School of Medicine, New Haven, CT 06511
4 Peter Gorer Department of Immunobiology, Guy's King's St. Thomas' Medical School, University of London, London, SE1 9RT UK
Address correspondence to Adrian Hayday, Peter Gorer Dept. of Immunobiology, Guy's King's St. Thomas' Medical School, University of London, London, SE1 9RT UK. Phone: 44-20-7955-4355; Fax: 44-20-7955-8894; email: adrian.hayday{at}kcl.ac.uk
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Abstract
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Between weaning (3 wk of age) and adulthood (7 wk of age), mice develop increased resistance to infection with Eimeria vermiformis, an abundant intestinal parasite that causes coccidiosis. This development of resistance was perturbed in T cell receptor (TCR)
-/- mice, which at 4 wk of age remained largely susceptible to infection and prone to infection-associated dehydration. These phenotypes were rescued by the repopulation of 
cells after adoptive transfer of lymphoid progenitors into newborn recipients. Because
ß T cells are necessary and sufficient for the protection of adult mice against E. vermiformis, the requirement for 
cells in young mice shows a qualitative difference between the cellular immune responses operating at different ages. An important contribution toward primary immune protection in young hosts may have provided a strong selective pressure for the evolutionary conservation of 
cells. This notwithstanding, the development of effective, pathogen-specific immunity in young mice requires
ß T cells, just as it does in adult mice.
Key Words: neonatal immunology mucosal immunology infection adoptive transfer knockout mice
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Introduction
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The neonatal period in humans and mice is characterized by heightened susceptibility to numerous microbial infections. In 2001, >5 million children worldwide died of infectious diseases to which more robust immune responses might have provided protection. Hence, attempts to improve vaccination and antimicrobial therapies for children and neonates are critically important. Ideally, such efforts can be set against an improved understanding of the basic cellular immune mechanisms that operate in young children and animals (1, 2).
Although T and B cell development begins in the fetus (3, 4), most mammals initially rely on passively acquired immunoglobulin transmitted transplacentally and/or via breastmilk. By adulthood, diverse repertoires of
ß T and B cells establish efficient primary clearance of pathogens, and a reservoir of immunological memory toward rechallenge. By contrast, the transition from dependence on maternal factors to the immune autonomy of adults is relatively poorly understood.
For many years, immunodeficiency in young animals was associated with the
neonatal tolerance
model based on observations by Billingham et al. that neonatal T lymphocytes react to antigen encounter by becoming tolerized, rather than activated (5). Subsequently, three separate groups demonstrated that neonatal T cells can make adult-type responses under particular conditions of antigen presentation and cytokine milieu (68). Despite this, the physiologic situation in vivo remains quite unclear. Compelling data demonstrate that naive T cells enter and are tolerized by the tissues of neonatal mice (9), and young mice and humans rarely mount antipathogen responses, particularly Th1 responses, with adult efficacy (10, 11). Thus, conditions provoking conventional immune responsiveness seem de facto to be suboptimal in the young animal. Consistent with this, weaknesses in the
ß T cell response have been attributed to intrinsic defects in the T cells themselves (e.g., poor cytokine responsiveness and
helper
or cytolytic activity; references 1216) and to defects in the antigen processing and presentation systems on which
ß T cells depend (6, 1719). Therefore, the question arises as to which immune response mechanisms are functionally available to young animals.
One candidate is the 
cell compartment. In all species in which their development has been extensively studied, 
cells are highly represented in young animals (2025). Many of the early-emerging 
cells localize to epithelial surfaces, such as that of the gut (26), where they may be well placed to encounter the first waves of pathogens entering the body. Moreover, although 
cells are clearly pleiotropic (27), they are commonly Th1 biased (28, 29), and their unconventional antigen specificities do not rely on MHC-restricted APCs that may function sub-optimally in young animals (3032).

cells can contribute to the primary clearance of pulmonary infections (33, 34). Furthermore, even in the many cases where the development of immunity seems largely intact in 
T celldeficient mice (25), an immunoprotective potential of 
cells is evident in the observations that mice lacking 
cells and
ß T cells are often more susceptible to infection than mice lacking
ß T cells alone (35, 36). For example, although the combined actions of CD4+
ß T cells and IFN
largely account for the primary response of adult mice to Eimeria vermiformis, a natural protozoan pathogen of intestinal epithelial cells (3739), mice lacking 
T cells as well as
ß T cells are markedly more susceptible to primary infection than those lacking only
ß T cells (40). This partial protection provided by 
cells can be neutralized by anti-IFN
antibodies (40).
The uncovering of a protective role for 
cells in the experimentally orchestrated absence of
ß T cells suggested that there may be physiologic instances in which 
T cells in part compensate for deficiencies in the
ß T cell compartment. One such case may be the young animal. An indication of this was provided by Waters and Harp's studies of Cryptosporidium parvum, a human pathogen related to E. vermiformis (41). In brief, most T cellintact mice challenged orally at 7 d of age cleared infection by 3 wk. By contrast, TCR
-/- mice shed detectable oocysts until 7 wk of age, whereas TCR
-/- mice developed chronic infection (41). Strikingly, it appeared that
ß celldeficient mice (that contain 
cells) initially controlled infection better than 
celldeficient mice. Although these findings suggested a nonredundant involvement of 
cells in responses to infection in early life, the experiments were limited in scope, and the authors could not rule out that there were other differences (e.g., IFN
levels, between the strains used). In particular, they did not assess whether the reconstitution of genetically identical TCR
-/- mice with 
cells would revert the transient immunodeficiency. Added to this, the use of TCR
-/- mice was a confounding variable, as these animals contain T cells bearing a
ß-only
TCR (42, 43).
Therefore, to test directly the hypothesis that 
cells make a critical contribution to immunological protection in young mice, a set of infections with E. vermiformis was undertaken. In the wild, eimerian coccidiosis of the gut is commonly contracted during early life, primarily through exposure to low but chronic inocula of parasites in contaminated feeding areas. In the laboratory, gavage administration of different stocks of parasites, passaged in vivo, can be used to mimic natural infection. We show here, across a broad spectrum of infectivity, that 
cells are essential for the effective primary protection of young mice shortly after weaning, and that, as a result, the age-related development of increasing immunocompetence toward E. vermiformis is temporally perturbed in the TCR
-/- strain. This exposes a clear difference between the cell-mediated immune responses of young mice as opposed to adults, and argues for a strong selective pressure for the evolutionary conservation of 
cells. At the same time, the development of pathogen-specific immunological memory in young mice was fully dependent on
ß T cells, as it is in adults.
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Materials and Methods
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Mice
Mice, bred locally or obtained from The Jackson Laboratories, were maintained under specific pathogen-free conditions. For comparisons of the susceptibility of different strains to infections, breedings of the different genotypes of mice were set up synchronously, so that precisely age-matched animals could be infected at the same time.
Parasite and Oocyst Enumeration
E. vermiformis (maintained in our laboratory) was passaged in mice, and oocysts were purified and sporulated. After microscopically scoring stocks for sporulation, mice were infected with either 102 or 12 x 103 E. vermiformis oocysts in sterile water via oral gavage. For the duration of infection, mice were caged singly on wire racks above sand from which feces were collected daily. Oocysts were enumerated in McMaster counting chambers after salt flotation. To assess fluid loss during infection, mice were weighed daily.
Preparation of Cells
Splenocytes.
Mice were killed and spleens were placed in sterile media (Click's medium with 5% fetal calf serum [CMF]) or PBS on ice. Tissues were mashed between two frosted slides to obtain a single cell suspension. Red blood cells were removed by incubation in lysis buffer (Sigma-Aldrich). Residual cells were washed and counted before use.
Intraepithelial Lymphocytes (IELs).
The method used was based on that published by Goodman and Lefrancois (44). In brief, the small intestine was removed and flushed with ice cold CMF to remove intestinal contents. Peyer's patches were cut out and removed to a separate container. The intestine was cut longitudinally, scraped lightly to remove mucus, and cut into 1-cm pieces that were rinsed several times in cold CMF to remove debris and placed into a clean 50-ml conical tube with 20 ml CMF-1 mM DTE. The tube was shaken at 37° for 20 min to disrupt the epithelial layer. The gut pieces were vortexed briefly and allowed to settle. The cell-rich supernatant was collected to a clean tube and the shaking process was repeated. After collection of the second supernatant (containing both IELs and enterocytes), the cells were pelleted (600 g, 10 min) and resuspended in ice-cold RPMI 1640 media (5 ml per gut). This solution was passed over a nylon wool column to remove contaminating enterocytes. The resulting effluent was collected and spun down (600 g, 10 min) to pellet the cells. Finally, the pellet was resuspended in 24 ml of 44% Percoll and layered on top of 5 ml of 67% Percoll. The resulting gradients were spun at 600 g for 20 min, and the intraepithelial lymophocytes were harvested from the interface. Cells were washed extensively in a large volume of RPMI 1640 medium and counted before use.
Reconstitution of Neonatal Mice with Fetal Liver Cells
Pregnant mice were killed at gestational day 14 by anesthetic overdose (HalothaneTM; Roche Labs) and fetuses were removed. Fetal livers were dissected and pooled in sterile Click's medium on ice. Liver tissue was disrupted by mashing between sterile frosted slides and passed through a 20-gauge needle to obtain a single cell suspension. The whole suspension was spun (600 g, 5 min) to pellet the cells. Red blood cells were lysed, and the residual cells were washed, counted, and resuspended at a concentration of 107 cells/30 µl. Recipient mice were 13 d old at the time of transfer, and each recipient was given 107 cells by intraperitoneal injection. Total volume of the injection was 30 µl. Mock-reconstituted control mice received a 30-µl injection of either buffer alone or an irrelevant cell type.
Flow Cytometry Analysis
Between 5 x 104 and 106 freshly isolated cells were preincubated with an antibody to block nonspecific labeling of FcRII/III (Fc BlockTM; BD Biosciences). Cells were washed and reacted with fluorescently tagged monoclonal antibodies for 15 min in 100 µl of staining buffer (1x PBS with 2% FCS) followed by three washes in 1x PBS. Cells were analyzed immediately (FACScanTM flow cytometer) or fixed in 2% formaldehyde (pH 7.0 in PBS). Data were analyzed using CELLQuestTM software. Antibodies used in these studies were obtained from BD Biosciences.
Molecular Analysis
Cells were harvested from chosen tissues into cell lysis buffer plus ß-mercaptoethanol (RNAeasy mini-kit; QIAGEN), from which RNA was ultimately eluted in 3060 µl of sterile water and stored at -80°C until use. Reverse transcriptase coupled polymerase chain reaction (RT-PCR) was undertaken exactly as described previously (45), using the primers as follows: IFN
, 5'-CATTGAAAGCCTAGAAAGTCTG-3', reverse, 5'-CTCATGAATGCATCCTTTTTCG-3'; and HPRT, 5'-GTTGGATACAGGCCAGACTTTGTTG-3', reverse, 5'-GAGGGTAGGC-TGGCCTATAGGCT-3'.
Statistics
Statistical analysis was performed using the Wilcoxon-Rank Sum test for unpaired data, from which a two-tailed p-value was obtained. Differences between tested groups were considered significant when P < 0.05. Statistical tests were made using InStatTM software for statistical analysis. Except where otherwise noted, error bars on graphs reflect ±SEM. Graphs with error bars were generated using PrismTM graphing software.
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Results
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The Capacity of T Cellintact Mice to Resolve Primary E. vermiformis Infection Increases with Age.
Young T cellintact C57.BL/6 mice infected between 21 d of age (1 d after weaning) and 40 d of age (after the onset of sexual maturity) gained immunological resistance to E. vermiformis as measured by a declining yield of infectious oocysts after infection with a uniform dose (1,000 oocysts) of a single stock (Fig. 1
A and Table I, Experiment 1). This development of immunocompetence did not proceed normally in young TCR
-/- mice, which remained significantly immunocompromised when infected at
2830 d of age (Fig. 1 A and Table I, Experiment 1). Although 40-d-old TCR
-/- mice also showed increased susceptibility, the difference was no longer statistically significant (Fig. 1 A and Table I). Moreover, consistent with previous experiments conducted in our laboratory (46, 47), 10-wk-old adult TCR
-/- mice displayed no enhanced susceptibility to primary infection with E. vermiformis (Fig. 1 B and Table I). These experiments point to a
window of susceptibility
between 28 and 40 d of age in which 
cells provide the young mouse with an essential, nonredundant contribution to primary protection. After this,
ß T cells are necessary and sufficient to provide protection (37, 46, 47). Because the difference in susceptibility between wild-type and TCR
-/- mice was greatest at 2830 d of age, additional experiments focused on 30-d-old mice.

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Figure 1. Age-dependent gain of immunocompetence is delayed in mice lacking  T cells. (A) Infection of T cellintact (shaded bars, wild type) and  T celldeficient (white bars, TCR 2/2) mice at 21 d old (wild type, n = 7; TCR 2/2, n = 10); 28 d old (wild type, n = 4; TCR 2/2, n = 3); and 40 d old (wild type, n = 4; TCR 2/2, n = 3). (B) Infection of adult T cellintact (shaded bar; wild type) and  T celldeficient mice (white bar, TCR 2/2). Mice were gavage infected with 1,000 (A) or 2,000 (B) sporulated E. vermiformis oocysts. Bars reflect average total oocysts shed over the patent period ±SEM. (C and D) 30-d-old T cellintact (wild type, open squares) and  T celldeficient (TCR -/-, open triangles) mice were gavage infected with 100 (C, wild type, n = 8 and TCR -/-, n = 11) or 1,000 (D, wild type, n = 7 and TCR -/-, n = 3) sporulated E. vermiformis oocysts. Plots depict average daily oocyst yields. (E) Weight change during days 710 after infection (1,000 oocysts) of infected T cellintact (wild type, shaded bar, n = 7),  T celldeficient (TCR -/-, white bar, n = 3), and mock-infected TCR -/- mice (diagonally striped bars).
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Dependence on 
Cells for Primary Protection Is Independent of Pathogen Dose or Virulence of Inoculum.
The infectivity and dose of pathogen encountered in the wild varies considerably. To mimic natural infection as closely as possible, sets of mice were infected with different doses of several different stocks of E. vermiformis derived by passage through different live hosts. The range of infectivity of these stocks is reflected in the broad spectrum of oocyst yields from infected C57.BL/6 mice (Table I). Nonetheless, the oocyst yields within individual experiments showed low standard error, permitting a clear comparison with the yields from 
celldeficient C57.BL/6 mice. The results show that young TCR
-/- mice infected at around 4 wk of age were invariably compromised relative to control mice, regardless of virulence or dose (Table I). Although this susceptibility was first evident in mice infected at 24 d of age (Table I, Experiment 3), the differences at that time point, like those at 40 d, were not statistically significant.
Analysis of daily oocyst yield across the patent period is presented in Fig. 1 (C and D) for representative low and high dose infections, respectively. Among young TCR
-/- mice, the margin of susceptibility was consistently greatest between days 9 and 13 after infection (913 days after infection [DAI]), with full resolution of the infection occurring at about the same time as control mice (usually 1517 dpi). In other systems, the role of 
cells has likewise been reported to be most crucial in the early phase of the T cell response (48, 49). By contrast, the effects of
ß T cell deficiency reported in adult mice extend beyond 13 DAI (47).
Susceptibility of Young TCR
-/- Mice Is Confirmed by Fluid Loss during Infection.
An established clinical manifestation of coccidiosis is a reproducible fluid loss after the start of oocyst shedding (710 DAI). Hence, daily weight measurements provide a robust and independent assessment of susceptibility among infected animals. C57.BL/6 mice infected at 30 d of age rarely lost >35% of total body mass. By contrast, TCR
-/- mice lost up to 12% of total body mass in a 2448-h period, a clinically significant dehydration (Fig. 1 E). Reflecting this, infected TCR
-/- mice appeared sicker than infected C57.BL/6 mice, commonly displaying ruffled fur, hunched posture, and listlessness. Despite severe dehydration, most TCR
-/- mice returned to normal body weight by 14 DAI.
Reconstitution of 
Cells Restores Resistance to Young 
Celldeficient Mice.
As an independent means to determine whether 
cells provide immunoprotection in the period between weaning and sexual maturity, the potential of 
cell reconstitution to restore immunocompetence in young TCR
-/- mice was assessed. Because E. vermiformis is a gut-tropic pathogen, it was important to reconstitute gut TCR
+ IELs as well as systemic 
cells. Both compartments can be reconstituted by adoptive transfer of embryonic day 14 (E14) fetal liver cells to newborn recipients, up until 4 d of age (50). The use of neonatal TCR
-/- recipients accommodates the early development of many 
cell compartments (21) and obviates the need for radiation to ensure graft survival. Although all three lymphocyte cell types,
ß T, 
, and B cells will develop from inocula of unfractionated C57.BL/6 fetal liver, the lymphocyte progenitor potential of fetal liver from TCRß-/- mice is limited to 
and B cells.
18 neonatal recipients of E14 liver cells were assessed for their resistance to E. vermiformis infection after reaching 30 d of age, and, after recovery, were killed and assessed for cellular reconstitution. Fig. 2
depicts the analysis of splenocytes isolated from wild-type unmanipulated mice (Fig. 2 A), mock-reconstituted TCR
-/- mice (Fig. 2 B), or TCR
-/- mice reconstituted with E14 liver from C57.BL/6 (Fig. 2, C and D) or TCRß-/- donor mice (Fig. 2, E and F). In unmanipulated wild-type mice, 
cells commonly comprise 0.52.0% of the total lymphocytes in the spleen; in mice reconstituted with E14 liver from either C57.BL/6 or TCRß-/- donor mice, splenic 
cells accounted for 0.20.8% of total lymphocytes. Intestinal TCR
+ IELs were similarly reconstituted to varying degrees, composing 445% of CD3+ IELs, compared with 2556% in unmanipulated wild-type mice (Fig. 2, GJ).
The heightened levels of oocyst shedding shown by young TCR
-/- mice across the patent period reverted to wild-type levels in mice receiving E14 liver cells from either C57.BL/6 or TCRß-/- donors (Fig. 3
A). The rescue of the TCR
-/- susceptibility phenotype was also evident in measures of body weight from 7 to 10 DAI (Fig. 3 B). Because functional reconstitution from TCRß-/- donors was as effective as that from C57.BL/6 mice (Fig. 3), the result cannot be attributed to a bolstered
ß T cell compartment. Likewise, it is unlikely that functional reconstitution can be attributed to an altered B cell compartment, because although E14 liver can give rise to B cells, recipients of neither type of inoculum showed an altered representation of B cells relative to TCR
-/- mice (Fig. 2, K and L).

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Figure 3. Replacement of  cells restores resistance to infection among 30-d-old TCR -/- mice. Reconstituted and control mice were gavage infected with 1,000 E. vermiformis oocysts. (A) Oocysts shed by TCR -/- mice (open triangles, n = 3; same group as in Fig. 1 D), wild-type mice (open squares, n = 7; same group as in Fig. 1 D), TCR -/- mice reconstituted with fetal liver from wild-type donors (closed squares, n = 11), and TCR -/- mice reconstituted with fetal liver from ß T celldeficient donors (closed circles, n = 7). (B) 30-d-old wild-type mice (shaded bars, n = 7), TCR -/- mice mock reconstituted (white bar, n = 3), and TCR -/- mice reconstituted with E14 fetal liver cells from either wild-type mice (horizontally striped bar, n = 11) or ß celldeficient mice (vertically striped bar, n = 7) were weighed daily at the peak of infection. Bars represent average weight loss in grams ± SD among members of a group during days 710 after infection. Age-matched control mice were mock infected with sterile water (diagonally striped bar, n = 7).
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Variable Susceptibilities to Infection of Young Cytokine-deficient Mice.
IFN
inhibits the growth of E. vermiformis within infected cells (38), and IFN
-deficient mice infected as adults are severely susceptible to primary E. vermiformis infection, showing higher parasite yields and longer patent period, and generally becoming more ill than wild-type control mice (38, 39). Furthermore,
ß T cells but particularly 
cells harvested from the splenic and intestinal IEL compartments of infected 30-d-old mice show increased levels of RNA for a spectrum of immunological mediators, including IFN
and perforin (Fig. 4
E and not depicted). To test whether the immunoprotective effects of 
cells in young mice are attributable to IFN
, the susceptibilities of wild-type and IFN
-deficient mice infected at 30 d of age were compared. Although the IFN
-/- mice shed more oocysts, their increased susceptibility was largely confined to 910 DAI (Fig. 4 A), whereas that shown by young TCR
-/- mice was more extensive (Fig. 1, C and D; and Fig. 3 A). To investigate this further, infections of age-matched 30-d-old wild-type, IFN
-/-, and TCR
-/- mice were directly compared (Table I, Experiment 5). Although IFN
-/- mice again shed many more oocysts on 9 DAI (7,800 compared with 2,300 by wild-type mice), the aggregate yield over the full course of infection was not significantly greater than that of wild-type mice, unlike the TCR
-/- mice (Table I). Therefore, although IFN
production is a parameter of host resistance, it alone cannot account for the protection provided by 
cells in the young mouse.

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Figure 4. IFN -/- mice partially phenocopy susceptibility of TCR -/-mice. The susceptibility of 30-d-old wild-type and various cytokine deficient mice was compared as follows: (A) 30-d-old IFN -/- mice (closed triangles, n = 4) and wild type (closed squares, n = 4); (B) 30-d-old IL-4-/- mice (open circles, n = 4) and wild type (closed squares, n = 3); (C) perforin-/- mice (inverted open triangles, n = 4) and wild type (closed squares, n = 3); and (D) IL-10-/- mice (open squares, n = 4) and wild type (closed squares, n = 6). (E) RT-PCR analysis of IFN (top) and HPRT (bottom) RNA expression in splenic and intestinal ß T (lane A) and  cells (lane B) harvested from mice that 6 d previously, at the age of 30 d, were either uninfected (left) or E. vermiformis infected (right). S, 100 bp size marker. IFN , bottom marker = 100 bp, product = 267 bp. HPRT, bottom marker = 200 bp, product = 351 bp. C, positive control (activated adult mouse spleen cells). The bona fide band is in each case the lower of a doublet; the fast running band in the top panel for the uninfected samples corresponds to primer dimers. The  cell sample from infected mice shows an IFN signal comparable to the HPRT signal, whereas the signal in uninfected mice was much weaker than the HPRT signal.
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Consistent with this, recent gene profiling analyses showed that gut-associated 
cells express a wide variety of conventional and unconventional effector and regulatory genes (45), (51), any or each of which may contribute to 
cell function in young mice. To begin to assess the potential contributions of some of these molecules, separate infections were undertaken in 30-d-old mice lacking IL-4, perforin, and IL-10, respectively. Neither 30-d-old IL-4-/- mice nor 30-d-old perforin-/- mice showed enhanced susceptibility to E. vermiformis infection (Fig. 4, B and C). Conversely, 30-d-old IL-10-/- mice showed enhanced resistance to primary infection with E. vermiformis (Fig. 4 D). This implies that under normal circumstances, infection of young mice is followed by secretion of IL-10 by cells that effectively dampen the primary immune response. Were these cells to be in the
ß T cell compartment (e.g., T-reg cells), young mice lacking
ß cells might actually be more resistant to infection (see Fig. 6).
ß T Cells Are Not Critical to the Primary Response of Young Mice to E. vermiformis.
ß T cells are critical to the efficacious protection of adult mice against E. vermiformis infection. To assess their role in young mice, infections were undertaken in TCRß-/- mice. First, as with TCR
-/- mice (Fig. 1 A), freshly weaned, 2124-d-old TCRß-/- mice showed no increased susceptibility to infection (Fig. 5
A). Together, these data demonstrate that neither T cell subset contributes significantly to primary protection in very young mice. However, in contrast to the susceptibility of 2830-d-old mice lacking 
cells (Fig. 1 A), TCRß-/- mice were commonly no more susceptible to infection than were wild-type mice (Fig. 5 B), and in some cases, they actually showed enhanced resistance to the early stages of infection (see Fig. 6). In contrast, adult TCRß-/- mice were highly susceptible to infection (Fig. 5 C), consistent with all previous analyses (37, 46, 47). These works provide further evidence for the qualitative differences between the cellular immune responses of young and adult mice.

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Figure 5. Young ß T celldeficient mice display variable susceptibility to infection. (A) Wild-type (closed squares, n = 7) and TCRß-/- mice (open circles, n = 7) were infected (1,000 oocytes) at 2124 d of age. (B) Wild-type mice (closed squares, n = 6), TCRß-/- mice (open circles, n = 7), and TCRßx -/- mice (inverted triangles, n = 6) were infected (1,000 oocysts) at 30 d of age. (C) Wild-type (shaded bar) and TCRß-/- mice (gray bar, n = 7) were infected (2,000 oocysts) as adults. Bars reflect total oocyst production ±SEM over the patent period.
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ß T Cells Are Critical to the Development of Immunological Memory in Young Mice.
Some of the T cells that effect a primary immune response acquire immunological memory and protect the host against subsequent infection with the same organism. Because 
cells and not
ß cells are the primary immunoprotective T cells in 30-d-old mice, it was important to determine whether 
cells could also protect against rechallenge. Fig. 6
A shows the result of an experiment in which wild-type, TCR
-/-, or TCRß-/- mice were initially infected at 30 d of age (1° infection) and rechallenged at 8 wk of age (2° infection). Consistent with previous results, TCR
-/- mice but not TCRß-/- mice showed enhanced susceptibility to primary infection. However, upon rechallenge, all
ß cellsufficient mice (wild-type and TCR
-/- mice) were completely immune. In contrast, TCRß-/- mice, which were comparatively resistant to primary challenge, developed no protective immunity and were highly susceptible to rechallenge. Thus, although 
cells are integral to the primary antiE. vermiformis response in 30-d-old mice, they do not provide pathogen-specific protective immunity.

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Figure 6. ß cells are required for the acquisition of memory. (A) 30-d-old wild-type mice (shaded bars, n = 5), TCR -/- mice (white bars, n = 3), and TCRß-/- mice (striped bars, n = 2) mice were infected with 1,000 E. vermiformis oocysts (1° infection), and rechallenged at 8 wk of age with 10,000 oocysts (2° infection). (B) 11-d-old wild-type (white bars, n = 6) and TCRß-/- mice (shaded bars, n = 4) were infected with 100 oocysts and rechallenged at 28 d of age with 1,000 oocysts. In both cases, only ß T cellintact mice were immune to reinfection.
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One concern regarding this result was that
ß T cells might not be acquiring memory in very young mice, but rather through exposure to antigen retained in adult mice in which
ß T cells have gained full immunocompetence. To test this, both primary and secondary infections were performed before 30 d of age (Fig. 6 B). C57.BL/6 mice and TCRß-/- mice were initially infected with 100 oocysts at 11 d of age. As in some infections of 30-d-old mice, the TCRß-/- mice were more resistant to infection than their T cellintact counterparts. However, upon reinfection at 30 d of age, only the C57.BL/6 mice and not the TCRß-/- mice were immune to reinfection (Fig. 6 B). This result confirms that even when
ß T cells are not critical for the resolution of primary infection, they are essential to the development of pathogen-specific immunity.
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Discussion
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The vertebrate immune system is a dynamic entity, changing to respond to the unique environment and sets of challenges that confront animals at each stage of life. For mammals, the period between weaning and the onset of sexual maturity is characterized by the need to discriminate between newly encountered pathogens, ongoing colonization by nonpathogenic, symbiotic organisms, and the expression de novo of self antigens specific to particular developmental stages. Some of these challenges will be particularly relevant at specific anatomical sites (e.g., the gut).
The results presented here demonstrate that when young mice were infected with a natural gut pathogen, their capacity to resolve infection increased with age, ranging from relative immunodeficiency at weaning (21 d) to full immune competence at adulthood (10 wk). This schedule of developing immunologic autonomy was perturbed in mice lacking 
cells, which remained highly susceptible to infection at around 1 mo of age. In most experiments, mice lacking
ß T cells were not similarly compromised. Instead, it was only after
6 wk that
ß T celldeficient mice showed an invariable and severe immunodeficiency, whereas mice lacking 
cells were less affected by then. Collectively, these results permit us to propose a revised
time line
for the development of primary responses to E. vermiformis infection (Fig. 7)
.
The time line depicted raises the question of whether, as mice reach adulthood, there is a developmental loss of function of 
cells or a gain of function of
ß cells. Several results are germane to this issue. First, in E. vermiformisinfected adult mice,
ß and 
cell double-deficient mice are more susceptible than TCRß-/- mice alone (39, 40). This suggests that 
cells retain function in the adult, but that their immunoprotective role is largely redundant in the presence of a fully mature and expanded
ß T cell compartment. This conclusion is consistent with several other papers that confirm a continued role for 
cells in adult animals, including humans. Thus, 
cells protect adult mice against epithelial tumors (52, 53), appear effective against mycobacteria in adult humans (54), and may protect against various viruses in mice and humans alike (55, 56).
Second, many works have demonstrated the immunodeficiency de facto of neonatal
ß T cell responses in both humans and mice (1012, 57). Our own studies have confirmed reports that cord blood
ß T cells are distinct from their adult counterparts being highly compromised in IFN
production, even after activation (unpublished data). The results presented here would indicate that vis-a-vis a natural gut infection of the mouse, this immunodeficency persists into the postweaning period, rendering the animal more dependent on 
cells for immune protection than is the case in the adult.
Third, the level of protection provided by 
celldependent mechanisms in 1-mo-old mice is substantially lower than that provided by
ß T celldependent mechanisms in adults. In sum, it is as if 
cells provide a contingency protection until
ß T celldependent mechanisms have fully developed. That the protection mediated by 
cells differs qualitatively from that mediated by
ß T cells is supported by the observation that it fails to confer long-term, pathogen-specific immunity toward E. vermiformis.
The different schedules by which 
cells and
ß T cells may reach immunocompetence in the gut may be affected by several factors, including microbial colonization. Thus, colonization with segmented filamentous bacteria (SFB) is rapidly followed by a marked expansion of intraepithelial T cells (58), production of IgA by lamina propria B cells (59), and formation of Peyer's patch germinal centers (60). In mice, SFB are first detectable in the small intestine at 16 d of age, peaking at 32 d of age, and rapidly declining thereafter (61). This schedule clearly overlaps the development of functional 
cell immunocompetence as measured by the increasing resistance of normal mice to primary infection between 21 and 30 d of age (Fig. 1). Interestingly, withdrawal of maternal IgA is associated with increased colonization by SFB, and likewise, SFB colonization and expansion occurs earlier (13 d of age), peaks sooner (28 d of age), and persists longer in animals in which either the host or the mother is immunodeficient (e.g. scid/scid; references 61, 62). These observations raise the possibility that some cellular components of the gut immune system, including 
cells, may develop precociously in TCRß-/- colonies, in which there would be less maternal Ig, and thereby confer enhanced resistance to primary infections experienced by young
ß T celldeficient mice (e.g., Fig. 6 and not depicted).
The relative resistance of young TCRß-/- mice to infection demonstrates that the actions of 
cells are independent of
ß T cells. Second, the susceptibility of IFN
-/- mice during the early stages of infection is consistent with the general Th1 bias of 
cells, and with the vulnerability of E. vermiformisinfected cells to IFN
. Nonetheless, the failure of IFN
-/- mice to fully phenocopy TCR
-/- mice suggests that 
cells are pleiotropic, making multiple contributions to immunoprotection, as do B and T cells in adult mice. For example, TCR
+ IELs express high levels of several other immunological effectors, including granzymes (45, 51). Although we show that perforin-deficient mice are no more susceptible to infection than are wild-type mice, perforin-independent effects of granzymes have been described previously (63). TCR
+ IELs also highly express RNA for chemokines that may have a strong influence over innate immunoprotective mechanisms. Clearly, further experiments will determine the full mechanistic basis for the immunoprotective role of 
cells.
It will be important to determine the generality of the results presented here, and the time line that has been developed (Fig. 7). To this end, rather similar data were obtained in a single experiment by Waters and Harp for young mice infected by C. parvum (41), although because no adoptive transfers were used, an immunoprotective function could not be assigned unequivocally to 
cells. It might be argued that the generality of the results will be limited to specific tissues in animals that show a reasonably high representation of 
cells. Thus, beyond mice, the data may be relevant to intestinal infections of chickens, where coccidiosis is a major economic problem.
Alternatively, 
cells have been proposed to be prototypes of a broader range of cells that include 
cells and nonconventional
ß T cells (64). Were this coclassification to prove appropriate, the generality of the results presented here may extend to species in which 
cells are less abundant, but that harbor sets of unconventional TCR
ß+ T cells (e.g., CD8
TCR
ß+ IELs). In that case, the proposal would be that young animals progress through a developmental window during which primary responses to gut infections are dependent largely on unconventional T cells, whereas memory to rechallenge would remain dependent on conventional T cells.
The reasons why 
cells fail to develop pathogen-specific immunity toward E. vermiformis remain to be elucidated. Possibly, the nature of the signaling events that follow TCR
engagement do not provoke memory cell differentiation. This cannot be resolved until the general nature of the ligands directly recognized by TCR
is elucidated. Currently, the murine MHC class IB molecule, T10/T22, is the only ligand shown biochemically to bind TCR
(65), and analyses are underway to determine whether such stress-induced self antigens are induced on epithelial cells by E. vermiformis infection (66). Likewise, any of several mechanisms may explain why
ß T cells in young mice contribute strongly to memory, but not primary effector responses. Among those mechanisms is the possibility that
ß T cells can adopt memory status without prior transit through an effector stage (67). The improved understanding of these various aspects of cell signaling can hopefully enhance the design of prophylactic and therapeutic strategies for the protection of young animals and children. At the same time, the data presented in this paper may provide some clue as to the selective advantage that may have been conferred by the presence of 
cells over >400 million yr of vertebrate evolution.
 |
Acknowledgments
|
---|
We acknowledge important contributions from R. McCord and Drs. S. Roberts, J. Lewis, and J. Shires.
We acknowledge the support of a Wellcome Trust University grant to A. Hayday, and the National Institutes of Health predoctoral training grant support to E. Ramsburg.
Submitted: January 13, 2003
Revised: August 8, 2003
Accepted: September 26, 2003
 |
Footnotes
|
---|
E. Ramsburg's present address is Dept. of Pathology, Yale University School of Medicine, Cedar St., New Haven, CT 06511.
Abbreviations used in this paper: CMF, Click's medium with 5% fetal calf serum; DAI, days after infection; E14, embryonic day 14; IEL, intraepithelial lymphocyte; SFB, segmented filamentous bacteria.
 |
References
|
---|
- Adkins, B. 2000. Development of neonatal Th1/Th2 function. Int. Rev. Immunol. 19:157171.[Medline]
- Marshall-Clarke, S., D. Reen, L. Tasker, and J. Hassan. 2000. Neonatal immunity: how well has it grown up? Immunol. Today. 21:3541.[CrossRef][Medline]
- Haynes, B.F., and C.S. Heinly. 1995. Early human T cell development: analysis of the human thymus at the time of initial entry of hematopoietic stem cells into the fetal thymic microenvironment. J. Exp. Med. 181:14451458.[Abstract]
- Cuisinier, A.M., F. Fumoux, D. Moinier, L. Boubli, V. Guigou, M. Milili, C. Schiff, M. Fougereau, and C. Tonnelle. 1990. Rapid expansion of human immunoglobulin repertoire (VH, V kappa, V lambda) expressed in early fetal bone marrow. New Biol. 2:689699.[Medline]
- Billingham, R., L. Brent, and P.B. Medawar. 1953. Actively acquired tolerance of foreign cells. Nature. 172:603606.
- Ridge, J.P., E.J. Fuchs, and P. Matzinger. 1996. Neonatal tolerance revisited: turning on newborn T cells with dendritic cells. Science. 271:17231726.[Abstract]
- Sarzotti, M., D.S. Robbins, and P.M. Hoffman. 1996. Induction of protective CTL responses in newborn mice by a murine retrovirus. Science. 271:17261728.[Abstract]
- Forsthuber, T., H.C. Yip, and P.V. Lehmann. 1996. Induction of TH1 and TH2 immunity in neonatal mice. Science. 271:17281730.[Abstract]
- Alferink, J., S. Aigner, R. Reibke, G.J. Hammerling, and B. Arnold. 1999. Peripheral T-cell tolerance: the contribution of permissive T-cell migration into parenchymal tissues of the neonate. Immunol. Rev. 169:255261.[Medline]
- Adkins, B., Y. Bu, E. Cepero, and R. Perez. 2000. Exclusive Th2 primary effector function in spleens but mixed Th1/Th2 function in lymph nodes of murine neonates. J. Immunol. 164:23472353.[Abstract/Free Full Text]
- Barrios, C., P. Brawand, M. Berney, C. Brandt, P.H. Lambert, and C.A. Siegrist. 1996. Neonatal and early life immune responses to various forms of vaccine antigens qualitatively differ from adult responses: predominance of a Th2-biased pattern which persists after adult boosting. Eur. J. Immunol. 26:14891496.[Medline]
- Burchett, S.K., L. Corey, K.M. Mohan, J. Westall, R. Ashley, and C.B. Wilson. 1992. Diminished interferon-gamma and lymphocyte proliferation in neonatal and postpartum primary herpes simplex virus infection. J. Infect. Dis. 165:813818.[Medline]
- Seghaye, M.C., W. Heyl, R.G. Grabitz, K. Schumacher, G. von Bernuth, W. Rath, and J. Duchateau. 1998. The production of pro- and anti-inflammatory cytokines in neonates assessed by stimulated whole cord blood culture and by plasma levels at birth. Biol. Neonate. 73:220227.[CrossRef][Medline]
- Suarez, A., L. Mozo, A. Gayo, A. Simo, and C. Gutierrez. 2000. Induction of functional CD154 (CD40 ligand) in neonatal T cells by cAMP-elevating agents. Immunology. 100:432440.[CrossRef][Medline]
- Nonoyama, S., L.A. Penix, C.P. Edwards, D.B. Lewis, S. Ito, A. Aruffo, C.B. Wilson, and H.D. Ochs. 1995. Diminished expression of CD40 ligand by activated neonatal T cells. J. Clin. Invest. 95:6675.[Medline]
- Moser, J.M., J.D. Altman, and A.E. Lukacher. 2001. Antiviral CD8 T cell responses in neonatal mice: susceptibility to polyoma virus-induced tumors is associated with lack of cytotoxic function by viral antigen-specific T cells. J. Exp. Med. 193:595605.[Abstract/Free Full Text]
- Arulanandam, B.P., V.H. Van Cleave, and D.W. Metzger. 1999. IL-12 is a potent neonatal vaccine adjuvant. Eur. J. Immunol. 29:256264.[CrossRef][Medline]
- Lu, C.Y. 1984. The delayed ontogenesis of Ia-positive macrophages: implications for host defense and self-tolerance in the neonate. Clin. Invest. Med. 7:263267.[Medline]
- Tasker, L., and S. Marshall-Clarke. 1997. Immature B cells from neonatal mice show a selective inability to up-regulate MHC class II expression in response to antigen receptor ligation. Int. Immunol. 9:475484.[Abstract]
- Bucy, R.P., C.L. Chen, J. Cihak, U. Losch, and M.D. Cooper. 1988. Avian T cells expressing gamma delta receptors localize in the splenic sinusoids and the intestinal epithelium. J. Immunol. 141:22002205.[Abstract/Free Full Text]
- Havran, W.L., and J.P. Allison. 1988. Developmentally ordered appearance of thymocytes expressing different T-cell antigen receptors. Nature. 335:443445.[CrossRef][Medline]
- Parker, C.M., V. Groh, H. Band, S.A. Porcelli, C. Morita, M. Fabbi, D. Glass, J.L. Strominger, and M.B. Brenner. 1990. Evidence for extrathymic changes in the T cell receptor
/
repertoire. J. Exp. Med. 171:15971612.[Abstract]
- Wilson, R.A., A. Zolnai, P. Rudas, and L.V. Frenyo. 1996. T-cell subsets in blood and lymphoid tissues obtained from fetal calves, maturing calves, and adult bovine. Vet. Immunol. Immunopathol. 53:4960.[CrossRef][Medline]
- McVay, L.D., S.S. Jaswal, C. Kennedy, A. Hayday, and S. Carding. 1998. The generation of human gammadelta T cell repertoires during fetal development. J. Immunol. 160:58515860.[Abstract/Free Full Text]
- Hayday, A.C. 2000. [gamma][delta] cells: a right time and a right place for a conserved third way of protection. Annu. Rev. Immunol. 18:9751026.[CrossRef][Medline]
- Itohara, S., A.G. Farr, J.J. Lafaille, M. Bonneville, Y. Takagaki, W. Haas, and S. Tonegawa. 1990. Homing of a gamma delta thymocyte subset with homogeneous T-cell receptors to mucosal epithelia. Nature. 343:754757.[CrossRef][Medline]
- Wen, L., D.F. Barber, W. Pao, F.S. Wong, M.J. Owen, and A.C. Hayday. 1998. Primary

cell clones can be defined phenotypically and functionally as Th1/Th2 cells and illustrate the association of CD4 with Th2 differentiation. J. Immunol. 160:19651974.[Abstract/Free Full Text]
- Yin, Z., D.H. Zhang, T. Welte, G. Bahtiyar, S. Jung, L. Liu, X.Y. Fu, A. Ray, and J. Craft. 2000. Dominance of IL-12 over IL-4 in gamma delta T cell differentiation leads to default production of IFN-gamma: failure to down-regulate IL-12 receptor beta 2-chain expression. J. Immunol. 164:30563064.[Abstract/Free Full Text]
- Yin, Z., C. Chen, S.J. Szabo, L.H. Glimcher, A. Ray, and J. Craft. 2002. T-Bet expression and failure of GATA-3 cross-regulation lead to default production of IFN-gamma by gammadelta T cells. J. Immunol. 168:15661571.[Abstract/Free Full Text]
- Bigby, M., J.S. Markowitz, P.A. Bleicher, M.J. Grusby, S. Simha, M. Siebrecht, M. Wagner, C. Nagler-Anderson, and L.H. Glimcher. 1993. Most gamma delta T cells develop normally in the absence of MHC class II molecules. J. Immunol. 151:44654475.[Abstract/Free Full Text]
- Grusby, M.J., H. Auchincloss, Jr., R. Lee, R.S. Johnson, J.P. Spencer, M. Zijlstra, R. Jaenisch, V.E. Papaioannou, and L.H. Glimcher. 1993. Mice lacking major histocompatibility complex class I and class II molecules. Proc. Natl. Acad. Sci. USA. 90:39133917.[Abstract]
- Crowley, M.P., Z. Reich, N. Mavaddat, J.D. Altman, and Y. Chien. 1997. The recognition of the nonclassical major histocompatibility complex (MHC) class I molecule, T10, by the

T cell, G8. J. Exp. Med. 185:12231230.[Abstract/Free Full Text]
- King, D.P., D.M. Hyde, K.A. Jackson, D.M. Novosad, T.N. Ellis, L. Putney, M.Y. Stovall, L.S. Van Winkle, B.L. Beaman, and D.A. Ferrick. 1999. Cutting edge: protective response to pulmonary injury requires gamma delta T lymphocytes. J. Immunol. 162:50335036.[Abstract/Free Full Text]
- Moore, T.A., B.B. Moore, M.W. Newstead, and T.J. Standiford. 2000. Gamma delta-T cells are critical for survival and early proinflammatory cytokine gene expression during murine Klebsiella pneumonia. J. Immunol. 165:26432650.[Abstract/Free Full Text]
- Sciammas, R., P. Kodukula, Q. Tang, R.L. Hendricks, and J.A. Bluestone. 1997. T cell receptor
/
cells protect mice from herpes simplex virus type 1induced lethal encephalitis. J. Exp. Med. 185:19691975.[Abstract/Free Full Text]
- Mombaerts, P., J. Arnoldi, F. Russ, S. Tonegawa, and S.H. Kaufmann. 1993. Different roles of alpha beta and gamma delta T cells in immunity against an intracellular bacterial pathogen. Nature. 365:5356.[CrossRef][Medline]
- Rose, M.E., H.S. Joysey, P. Hesketh, R.K. Grencis, and D. Wakelin. 1988. Mediation of immunity to Eimeria vermiformis in mice by L3T4+ T cells. Infect. Immun. 56:17601765.[Medline]
- Rose, M.E., D. Wakelin, and P. Hesketh. 1989. Gamma interferon controls Eimeria vermiformis primary infection in BALB/c mice. Infect. Immun. 57:15991603.[Medline]
- Smith, A.L., and A.C. Hayday. 2000. Genetic dissection of primary and secondary responses to a widespread natural pathogen of the gut, Eimeria vermiformis. Infect. Immun. 68:62736280.[Abstract/Free Full Text]
- Smith, A.L., and A.C. Hayday. 2000. An alpha beta T-cell-independent immunoprotective response towards gut coccidia is supported by gamma delta cells. Immunology. 101:325332.[CrossRef][Medline]
- Waters, W.R., and J.A. Harp. 1996. Cryptosporidium parvum infection in T-cell receptor (TCR)-alpha- and TCR-delta-deficient mice. Infect. Immun. 64:18541857.[Abstract]
- Mombaerts, P., A.R. Clarke, M.A. Rudnicki, J. Iacomini, S. Itohara, J.J. Lafaille, L. Wang, Y. Ichikawa, R. Jaenisch, M.L. Hooper, et al. 1992. Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages. Nature. 360:225231.[CrossRef][Medline]
- Viney, J.L., L. Dianda, S.J. Roberts, L. Wen, C.A. Mallick, A.C. Hayday, and M.J. Owen. 1994. Lymphocyte proliferation in mice congenitally deficient in T-cell receptor alpha beta + cells. Proc. Natl. Acad. Sci. USA. 91:1194811952.[Abstract/Free Full Text]
- Goodman, T., and L. Lefrancois. 1988. Expression of the gamma-delta T-cell receptor on intestinal CD8+ intraepithelial lymphocytes. Nature. 333:855858.[CrossRef][Medline]
- Shires, J., E. Theodoridis, and A.C. Hayday. 2001. Biological insights into TCRgamma delta+ and TCRalpha beta+ intraepithelial lymphocytes provided by serial analysis of gene expression (SAGE). Immunity. 15:419434.[Medline]
- Smith, A.L., and A.C. Hayday. 1998. Genetic analysis of the essential components of the immunoprotective response to infection with Eimeria vermiformis. Int. J. Parasitol. 28:10611069.[CrossRef][Medline]
- Roberts, S.J., A.L. Smith, A.B. West, L. Wen, R.C. Findly, M.J. Owen, and A.C. Hayday. 1996. T-cell alpha beta + and gamma delta + deficient mice display abnormal but distinct phenotypes toward a natural, widespread infection of the intestinal epithelium. Proc. Natl. Acad. Sci. USA. 93:1177411779.[Abstract/Free Full Text]
- Selin, L.K., P.A. Santolucito, A.K. Pinto, E. Szomolanyi-Tsuda, and R.M. Welsh. 2001. Innate immunity to viruses: control of vaccinia virus infection by gamma delta T cells. J. Immunol. 166:67846794.[Abstract/Free Full Text]
- Maloy, K.J., B. Odermatt, H. Hengartner, and R.M. Zinkernagel. 1998. Interferon gamma-producing gamma delta T cell-dependent antibody isotype switching in the absence of germinal center formation during virus infection. Proc. Natl. Acad. Sci. USA. 95:11601165.[Abstract/Free Full Text]
- Girardi, M., J. Lewis, E. Glusac, R.B. Filler, L. Geng, A.C. Hayday, and R.E. Tigelaar. 2002. Resident skin-specific

T cells provide local, nonredundant regulation of cutaneous inflammation. J. Exp. Med. 195:855867.[Abstract/Free Full Text]
- Fahrer, A.M, Y. Konigshofer, E.M. Kerr, G. Ghandour, D.H. Mack, M.M. Davis, and Y.H. Chien. 2001. Attributes of gammadelta intraepithelial lymphocytes as suggested by their transcriptional profile.Proc Natl. Acad Sci USA. 98:1026110266.[Abstract/Free Full Text]
- Girardi, M., D.E. Oppenheim, C.R. Steele, J.M. Lewis, E. Glusac, R. Filler, P. Hobby, B. Sutton, R.E. Tigelaar, and A.C. Hayday. 2001. Regulation of cutaneous malignancy by gammadelta T cells. Science. 294:605609.[Abstract/Free Full Text]
- Groh, V., A. Steinle, S. Bauer, and T. Spies. 1998. Recognition of stress-induced MHC molecules by intestinal epithelial gammadelta T cells. Science. 279:17371740.[Abstract/Free Full Text]
- Dieli, F., M. Troye-Blomberg, S.E. Farouk, G. Sirecil, and A. Salerno. 2001. Biology of gammadelta T cells in tuberculosis and malaria. Curr. Mol. Med. 1:437446.[Medline]
- Dechanet, J., P. Merville, A. Lim, C. Retiere, V. Pitard, X. Lafarge, S. Michelson, C. Meric, M.M. Hallet, P. Kourilsky, et al. 1999. Implication of gammadelta T cells in the human immune response to cytomegalovirus. J. Clin. Invest. 103:14371449.[Abstract/Free Full Text]
- Sciammas, R., and J.A. Bluestone. 1999. TCRgammadelta cells and viruses. Microbes Infect. 1:203212.[CrossRef][Medline]
- Reen, D.J., and E. Early. 1998. Cord blood naive T cells demonstrate distinct immunological properties compared with their adult counterparts. Bone Marrow Transplant. 22:S35.[Medline]
- Umesaki, Y., Y. Okada, S. Matsumoto, A. Imaoka, and H. Setoyama. 1995. Segmented filamentous bacteria are indigenous intestinal bacteria that activate intraepithelial lymphocytes and induce MHC class II molecules and fucosyl asialo GM1 glycolipids on the small intestinal epithelial cells in the ex-germ-free mouse. Microbiol. Immunol. 39:555562.[Medline]
- Klaasen, H., P. Van der Heijden, W. Stok, F. Poelma, J. Koopman, M. Van den Brink, M. Bakker, W. Eling, and A. Beynen. 1993. Apathogenic, intestinal, segmented, filamentous bacteria stimulate the mucosal immune system of mice. Infect. Immun. 61:303306.[Abstract]
- Talham, G.L., H.Q. Jiang, N.A. Bos, and J.J. Cebra. 1999. Segmented filamentous bacteria are potent stimuli of a physiologically normal state of the murine gut mucosal immune system. Infect. Immun. 67:19922000.[Abstract/Free Full Text]
- Jiang, H.Q., N.A. Bos, and J.J. Cebra. 2001. Timing, localization, and persistence of colonization by segmented filamentous bacteria in the neonatal mouse gut depend on immune status of mothers and pups. Infect. Immun. 69:36113617.[Abstract/Free Full Text]
- Cebra, J.J., S.B. Periwal, G. Lee, F. Lee, and K.E. Shroff. 1998. Development and maintenance of the gut-associated lymphoid tissue (GALT): the roles of enteric bacteria and viruses. Dev. Immunol. 6:1318.[Medline]
- Pinkoski, M.J., M. Hobman, J.A. Heibein, K. Tomaselli, F. Li, P. Seth, C.J. Froelich, and R.C. Bleackley. 1998. Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis. Blood. 92:10441054.[Abstract/Free Full Text]
- Hayday, A., E. Theodoridis, E. Ramsburg, and J. Shires. 2001. Intraepithelial lymphocytes: exploring the Third Way in immunology. Nat. Immunol. 2:9971003.[CrossRef][Medline]
- Crowley, M.P., A.M. Fahrer, N. Baumgarth, J. Hampl, I. Gutgemann, L. Teyton, and Y. Chien. 2000. A population of murine gammadelta T cells that recognize an inducible MHC class Ib molecule. Science. 287:314316[Abstract/Free Full Text]
- Janeway, C.J., B. Jones, and A.C. Hayday. 1988. Specificity and function of cells bearing

T cell receptors. Immunol. Today. 9:7376.[CrossRef][Medline]
- Manjunath, N., P. Shankar, J. Wan, W. Weninger, M.A. Crowley, K. Hieshima, T.A. Springer, X. Fan, H. Shen, J. Lieberman, and U.H. von Andrian. 2001. Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes. J. Clin. Invest. 108:871878.[Abstract/Free Full Text]