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Peripheral CD4⁺ Lymphocytes Derived from Fetal versus Adult Thymic Precursors Differ Phenotypically and Functionally ¹

Department of Microbiology and Immunology, University of Miami Medical School, Miami, FL 33136

	Abstract

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There is growing evidence that the differentiation processes in the fetal and adult thymus are not identical. However, there is little information on whether these developmental differences influence the properties of mature cells that exit the thymus and seed peripheral lymphoid organs. We have addressed this issue by comparing the development of Ag-specific Th1/Th2 function by fetal vs adult thymic derived CD4⁺ cells in the same adoptive adult hosts. Host mice were irradiated and transplanted with 14- to 15-day fetal thymic lobes from Thy-1 congenic mice. Ag (keyhole limpet hemocyanin)-specific Th1/Th2 responses of fetal-derived (donor) or adult-derived (host) CD4⁺ cells were analyzed by ELISA following primary or secondary immunization. Fetal-derived cells produced up to 10-fold more of both Th1 (IFN-) and Th2 (IL-4) cytokines than did adult-derived cells. Comparisons of the IL-4:IFN- ratios showed that the responses of fetal-derived cells were Th2-skewed in an Ag dose-dependent manner. At low doses of Ag, the fetal-derived ratio was 5 times higher than the adult-derived ratio. As the Ag dose was increased, the differences between the ratios of the fetal- and adult-derived responses were minimized. These relative responses were established initially during the primary effector phase but were maintained for weeks, into the memory phase of the immune response. Importantly, fetal-derived CD4⁺ cells showed these properties whether the fetal thymic precursors matured within the fetal or adult thymic microenvironment. These results demonstrate that cells arising from fetal thymic precursors are functionally different both qualitatively and quantitatively from adult-derived cells.

	Introduction

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During gestation in the mouse, the thymic rudiment is first colonized by hematopoietic precursors around day 12–14 of fetal life (1, 2, 3). The colonizing precursors are thought to originate in the aorta-gonad-mesonephros and/or the fetal liver (4). Over the next 5–7 days of fetal life, these cells proliferate and differentiate, giving rise to the four major classes of thymocytes, CD4^-8^-, CD4⁺8⁺, CD4⁺8^-, and CD4^-8⁺ cells. These single positive CD4⁺8^- and CD4^-8⁺ thymocytes constitute the first populations of cells to exit the thymus and populate the peripheral lymphoid tissues. Near birth, 19–21 days of gestation in inbred mice, the thymus is again colonized by a wave of precursor cells (1, 5). These cells probably originate in the bone marrow and represent the first entry of postfetal or adult thymopoietic progenitor cells. The newly arrived precursors also begin to proliferate and differentiate, ultimately largely replacing the cells of fetal origin.

The major pathways of TCR cell differentiation appear to be similar in both the fetal and adult stages of life. However, there are also many differences indicating that there are properties or processes unique to each developmental age. First, fetal thymic precursors give rise to some TCR-bearing progeny that are not detectably produced in the adult thymus (6, 7, 8, 9). Second, a relatively large percentage of T-lineage cells undergo the D-J recombination step in Ig gene rearrangement in the adult, but not the fetal, thymus (10). Experiments with genetically manipulated mice have also revealed effects selective for either fetal or adult thymopoiesis. For example, a null mutation of the Ikaros gene results in defective fetal thymopoiesis while adult thymopoiesis is intact (11). In contrast, IL-7R^-/- or 4 integrin^-/- mice show severe defects in thymopoiesis in adult mice while fetal thymopoiesis is less affected (12, 13, 14).

It is clear that differences between fetal and adult thymopoiesis exist. Whether these differences influence the properties of mature cells once they have exited the thymus is less clear. Previously, we addressed this issue by analyzing the phenotypic properties of fetal- or adult-derived peripheral T cells. Those studies showed that the peripheral progeny of fetal thymic precursors demonstrated a depressed CD4⁺:CD8⁺ ratio, relative to the progeny of adult precursors (15, 16). More indirect evidence has come from studies comparing newborn peripheral T cells, which are derived from the fetal thymus, with adult T cells. A great body of work over the last decade has shown that, under standard conditions of immunization or activation, the responses of neonatal T cells are biased to Th2 function (17, 18, 19). However, in these experimental designs, the relative contribution of the peripheral neonatal environment to conditioning or regulating fetal-derived thymic emigrants is unknown. To determine definitely whether fetal- and adult-thymic derived cells differ functionally, it is critical to evaluate their relative activities within the same environmental context. Using adoptive transfer into adult host animals, we have now directly compared the development of Ag-specific Th1/Th2 function in vivo by cells which originated either in the adult or fetal thymus. These experiments have shown that fetal-derived CD4⁺ cells mount robust responses of both Th1- and Th2-type. However, relative to the responses of adult-derived cells, the fetal responses show a Th2-bias that is dependent on the Ag concentration. Thus, fetal thymocytesgenerate peripheral CD4⁺ cells with functional properties that are distinct from adult-thymic derived cells. The Th2-skewed nature of the responses indicates that the Th2-bias of T cells in neonates in situ is due, in part, to their derivation from fetal thymic precursors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

C57BL/6 (Thy-1.2) and B6.PL (Thy-1.1) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred and housed under barrier conditions in the Division of Veterinary Resources at the University of Miami Medical School (Miami, FL). Seven- to 10-wk-old adult mice were used for host animals. Fetuses were removed from pregnant mice 14–15 days after observation of the vaginal plug (day 0).

Preparation of donor fetal thymocyte and adult bone marrow cell suspensions

Cell preparation medium consisted of HBSS containing 1% calf serum and 10 mM HEPES buffer (pH 7.0). Fetal thymus cell suspensions were prepared by pressing the lobes through a fine wire mesh. For bone marrow cells, femurs and tibias from 7- to 10-wk-old mice were excised and the marrow was extruded with a flow of cell preparation medium through a 25-gauge needle. All cells were washed two to three times with HBSS before injection.

Preparation and immunization of chimeric animals

Host mice were sublethally (575 rad) or lethally (900 rad) irradiated, as indicated, with an AECL Cobalt 60 panoramic irradiator. Lethally irradiated animals were reconstituted i.v. with 5 x 10⁶ syngeneic bone marrow cells. For transplantation of fetal thymic lobes, the left kidney was exposed dorsally and a small incision was made in the capsule. Four to six whole thymic lobes were gently placed between the capsule and the body of the kidney, using fine, curved forceps. The skin wound was then closed with surgical staples. For intrathymic injection of fetal thymocyte suspensions, animals were anesthetized with a mixture of xylazine (200 µg/g body weight, Vetus Animal Health, Rockville Center, NY) and ketamine HCl (1 mg/g body weight, Fort Dodge Animal Health, Overland Park, KS). A mid-line incision was made in the upper thoracic region to expose the sternum. A small (approximately one-fourth-inch long) longitudinal incision of the sternum exposed the tops of both thymic lobes. Each lobe was injected with 1–5 x 10⁵ thymocytes in 10 µl of HBSS, using a gas-tight Hamilton syringe with a 30-gauge needle. The wound was then closed using surgical staples. Lethally irradiated mice were maintained on water containing neomycin sulfate (1.1 g/L; Sigma-Aldrich, St. Louis, MO) and polymyxin B sulfate (10⁶ U/L; Sigma-Aldrich) for the duration of the experiments.

Chimeric animals were rested for 1 mo and then immunized i.p. and s.c. with 100 µg of keyhole limpet hemocyanin (KLH)³ (Calbiochem, San Diego, CA) in PBS (20). Intact 1-day-old neonatal or adult mice were immunized in parallel. Neonatal mice were immunized with 10 µg KLH; adults with 100 µg. One month later, all animals were reimmunized with 100 µg KLH in PBS.

Cell staining

To assess chimerism, PBL were prepared at the indicated time points and stained as previously described (15, 16). Anti-Thy-1.1, anti-Thy-1.2, anti-CD4, and anti-CD8 mAb were purchased from BD PharMingen (San Diego, CA). For propidium iodide (PI) staining of cultured cells, the cells were first stained with fluorescein conjugated anti-Thy-1.1 or anti-Thy-1.2 mAb, then processed for PI staining as described previously (21). Briefly, cells were suspended in HBSS containing 50% FCS, fixed by the addition of drops of ice-cold 70% ethanol, to a final concentration of 50%, and held on ice for at least 1 h. After extensive washing, the cells were suspended in HBSS containing 50 µg/ml PI (Sigma-Aldrich) and 50 µg/ml RNase A (F. Hoffman-La Roche, Basel, Switzerland) and incubated for 1 h at room temperature. Debris and doublets were eliminated from the analyses using pulse width/area discrimination.

Preparation of fetal- and adult-derived CD4⁺ cells for culture

A pool of cells was prepared from lymph nodes and spleen and treated with anti-Thy-1.1 or anti-Thy-1.2 plus complement. Anti-Thy-1.2 was purchased from BD PharMingen and used at a dilution of 10 µg/ml. Anti-Thy-1.1 mAb, clone 19XE5 (22), ascites was used at a final concentration of 1/25. Cells (1 x 10⁸/ml in RPMI 1640 medium containing 5% FCS) were incubated with the appropriate anti-Thy-1 mAb for 30 min at 4°C. Rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada) in RPMI 1640 was added to a final concentration of 1/10; a sufficient volume of RPMI 1640/5% calf serum was added to achieve a cell concentration of 5 x 10⁷/ml. The cells were then incubated at 37°C for 1 h, washed, and subjected to positive selection for CD4⁺ cells using the Miltenyi Biotec (Auburn, CA) system, as previously described in detail (20).

Cell culture for analyses of cytokine production by ELISA

To prepare APC, total spleen cells from naive adult mice were treated with anti-Thy-1 (mAb 42-21) plus complement, followed by treatment with 50 µg/ml mitomycin C, as described previously (23).

Thy-1.1⁺ or Thy-1.2⁺ CD4⁺ cells (2 x 10⁵ in 200 µl) were cultured with 4 x 10⁵ APC in 96-well culture dishes in the presence or absence of the indicated concentrations of KLH or anti-CD3 ascites (145-2C11 mAb) (24). Culture medium consisted of RPMI 1640 (Life Technologies, Grand Island, NY) containing 1 mM sodium pyruvate (Life Technologies), 2 mM L-glutamine (Life Technologies), 5 x 10^-2 mM 2-ME (Life Technologies), 1% penicillin-streptomycin (Life Technologies), and 10% heat-inactivated (56°C, 30 min) FCS (HyClone Laboratories, Logan, UT). Culture supernatants were harvested at 48 h (anti-CD3) or 72 h (KLH) and IFN- and IL-4 contents were assessed using mouse-specific cytokine ELISA kits (Pierce, Rockford, IL).

Cell culture for PI staining

Thy-1.1⁺ or Thy-1.2⁺ CD4⁺ cells (2 x 10⁵ in 200 µl) were cultured in wells precoated (16 h, 4°C) with purified anti-CD3 mAb (10 µg/well). Where indicated, soluble anti-CD28 mAb (BD PharMingen) was added at a final concentration of 5 ng/ml.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Development of Ag-specific Th1/Th2 function by fetal- vs adult-derived peripheral CD4⁺ cells in sublethally irradiated adoptive adult hosts: comparison to the responses seen in intact neonatal and adult mice

The responses of CD4⁺ cells in neonatal animals are often biased to Th2 function (17, 18, 19). Recent evidence suggests that cell intrinsic properties may contribute substantially to this Th2 bias (20). The cells found within the first week of life in peripheral organs are largely derived from fetal thymic precursors. Together, these observations raised the possibility that the functional properties of neonatal T cells are due to their derivation from fetal thymic precursors. To test this idea, we took advantage of an adoptive transfer system previously used successfully in the phenotypic analyses of fetal- vs adult-thymic derived cells (15, 16). Adult Thy-1.2⁺ host mice were sublethally irradiated and transplanted under the kidney capsule with 14- to 15-day fetal thymic lobes from Thy-1.1⁺ embryos (Fig. 1A). The transplanted animals were rested for 1 mo to allow sufficient time for 1) proliferation, differentiation, and exit from the adoptive fetal thymus or from the endogenous host thymus, and 2) the normal processes of postthymic maturation to occur. One mo post transplant, the mice were bled to assess chimerism and immunized with 100 µg KLH in PBS. Following an additional 4 wk, the mice were again bled and reimmunized with KLH. Three days later, Thy-1.1⁺CD4⁺ (donor) and Thy-1.2⁺CD4⁺ (host) cells were prepared from pools of spleen and lymph node cells, stimulated with different concentrations of KLH, and supernatants were collected for cytokine ELISA. In parallel, intact 1 day neonatal and adult animals were similarly immunized and CD4⁺ cells were cultured and supernatants harvested.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. Experimental scheme for preparing chimeric animals and relative colonization and CD4:CD8 ratios of fetal vs adult thymic-derived peripheral T cells. A, Experimental setup: Thy-1.2+ adult mice were sublethally irradiated and transplanted (kidney capsule) with Thy-1.1+ fetal thymic lobes. Four weeks later, the mice were immunized i.p. and s.c. with 100 µg KLH in PBS (1°) and bled for assessment of chimerism. After an additional 4 wk, the mice were reimmunized (2°) and bled again. Three days later, Thy-1.1+ or Thy-1.2+ CD4+ cells were enriched, as described in Materials and Methods, and cultured with different concentrations of KLH for assessment of Th1/Th2 production by ELISA. B, Chimeric animals prepared as in A, above, were bled 4 and 8 wk after chimera formation. Peripheral blood cells were stained with anti-Thy-1.1 (anti-fetal derived cells) or anti-Thy-1.2 mAb (anti-adult derived cells), together with anti-CD4 and anti-CD8 mAb. The percent of fetal- or adult-derived CD4+ cells at the two time points is shown in the left panel. The ratios of CD4+:CD8+ cells among the fetal-derived or adult-derived populations are shown in the right panel. Each symbol represents the average ± SD of three to four individual mice in four separate experiments.

Culture supernatants were tested for the Th1 signature cytokine IFN- and the Th2 cytokine IL-4 (Fig. 2). As we have previously reported (25), the secondary responses of CD4⁺ cells from animals initially immunized as neonates were Th2-skewed (Fig. 2A). Neonatal cells made 3- to 4-fold less IFN- and 4- to 6-fold more IL-4 than did adult cells. This relative cytokine production resulted in a ratio of IL-4:IFN- (pg/ml:pg/ml x 10³) that was higher among neonatal than adult cells. However, the degree of skewing was dependent on Ag concentration. At low KLH concentrations, neonatal IL-4:IFN- ratios were up to 40-fold higher than those of adult cells. As the concentration of Ag was increased, this ratio declined among both neonatal and adult T cells. At the highest KLH concentration, the neonatal ratio was 20-fold greater than the adult ratio (IL-4:IFN- = 467, neonatal; 23, adult).

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Comparisons of the Ag-specific responses of fetal-derived vs adult-derived cells in sublethally irradiated chimeric animals with responses in intact neonatal and adult animals. Chimeric animals were prepared and treated as described in the legend for Fig. 1. At the time of primary immunization of the chimeras, normal 1-day-old neonatal and adult C57BL/6 mice were also given primary immunization. All mice received secondary immunizations at the same time and were analyzed for Th1/Th2 responses, as described in the legend for Fig. 1. Depicted are the IFN- and IL-4 levels in supernatants collected after 72 h, which was determined to be the optimal time for cytokine production (data not shown). One experiment typical of four individual setups is shown. A, Intact neonatal/adult mice. B, Fetal thymic chimeras.

Different functional responses by fetal- and adult-derived CD4⁺ cells also develop in lethally irradiated host animals

The experiments described in the previous section were designed to test whether there are functional differences in the progeny of fetal vs adult thymocytes. However, with sublethal irradiation, it remained possible that radio-resistant peripheral host T cells contributed to the responses of adult-derived cells. To minimize the potential impact of radio-resistant peripheral T cells, Thy-1.2⁺ adult animals were lethally irradiated (900 rad) and reconstituted with 5 x 10⁶ syngeneic bone marrow cells. Two days later, animals were transplanted with 14- to 15-day fetal thymic lobes. The animals were then treated experimentally in a manner similar to those for sublethal irradiation (Fig. 1A). Fig. 3A shows the colonization by fetal- and adult-derived cells at the time of primary and secondary immunization. Unlike in the case of sublethal irradiation, fetal- and adult-derived cells were similarly represented in the PBL of chimeric mice at the time of primary immunization. However, in both approaches, adult-derived cells became 5 times as abundant as fetal-derived cells by the time of secondary immunization.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. Fetal-derived and adult-derived cells also show different Ag-specific responses in lethally irradiated hosts. Thy-1.2+ adult mice were lethally irradiated (900 rad) and reconstituted with 5 x 106 Thy-1.2+ bone marrow cells. Two days later, animals were transplanted with Thy-1.1+ fetal thymic lobes. The animals were then treated as described in the legend for Fig. 1 with two immunizations and assays for Ag-specific Th1/Th2 function. A, Percentages of fetal- and adult-derived CD4+ cells in the PBL of chimeric mice at the times of immunization. Each symbol represents the average ± SD of three to four individual mice in four separate experiments. B, Depicted are the IFN- and IL-4 levels detected by ELISA in cultures stimulated for 72 h with the indicated concentrations of KLH. One representative experiment among four total experiments is depicted.

Different responses of fetal- and adult-derived cells arise during the development of primary effector cells

Functional responses in long-term chimeras are dependent on the proliferation and development of primary effectors, their transition to the memory state, and their survival. To understand better how functional differences between fetal- and adult-derived cells arise, we wished to eliminate many of these variables. For this purpose, animals were prepared as described in Fig. 1A, with the exception that Th1/Th2 primary responses were analyzed one wk following a single immunization with KLH. In these experiments, colonization by fetal- and adult-derived CD4⁺ cells differed less than twofold (20.2 ± 7.5% fetal-derived vs 15.6 ± 4.5% adult-derived cells in the PBL of 14 individual experimental animals at the time of immunization). The Ag specific responses of fetal- and adult-derived CD4⁺ cells showed patterns very similar to that previously observed for the long-term, secondary responses. In particular, fetal-derived cells produced more of both IFN- and IL-4 than did adult-derived cells and the IL-4:IFN- ratio of fetal-derived cells showed an Ag concentration-dependent Th2-skewing (Fig. 4A).

View larger version (24K): [in this window] [in a new window]   FIGURE 4. Different responses of fetal- and adult-derived cells develop during the generation of primary effectors. Chimeric animals were created as described in the legend for Fig. 1. In this case, however, the animals were immunized only once, 4 wk following chimera creation and fetal- and adult-derived CD4+ cells were prepared 1 wk later. Cells were stimulated with the indicated concentrations of either KLH or anti-CD3 ascites. Forty-eight hours (anti-CD3) or 72 h (KLH) culture supernatants were then tested by specific ELISA. One experiment typical of five individual setups is shown. A, Ag-specific responses. B, Anti-CD3 responses.

Peripheral CD4⁺ cells with fetal-like properties are produced in the absence of fetal thymic stroma and are stably maintained in the adult peripheral environment

In our earlier phenotypic studies, we found reduced CD4⁺:CD8⁺ ratios among the peripheral progeny of fetal thymocytes whether differentiation occurred within the fetal or adult thymic microenvironment (15, 16). However, in some cases, the combination of fetal precursors and fetal thymic stroma appear to be necessary to produce cells with fetal-like properties (26, 27). Thus, we next tested whether the fetal-like functional properties observed in our experiments required maturation to occur within the fetal thymus. For these experiments, a suspension of fetal thymocytes was injected directly into the adult host thymus. The animals were then treated as outlined in Fig. 1A, with two immunizations before analyses of functional responses. Ag-specific cytokine production appeared very similar to that observed following transplantation of whole fetal thymic lobes. In this case as well, fetal-derived CD4⁺ cells produced more of both IFN- and IL-4 and showed an Ag dose-dependent Th2-skewing (Fig. 5). Therefore, the generation of fetal-like peripheral CD4⁺ cells is due to intrinsic properties of fetal thymic precursors and does not require the influence of fetal thymic stroma.

View larger version (12K): [in this window] [in a new window]   FIGURE 5. The distinct properties of fetal-derived peripheral CD4+ cells also develop when fetal thymocytes mature within the adult thymus. Thy-1.2+ adult mice were sublethally irradiated and injected intrathymically with a cell suspension containing 5 x 105 Thy-1.1+ fetal thymocytes/lobe. The animals were then treated as described in the legend for Fig. 1 with two immunizations and assays for Ag-specific Th1/Th2 activity. One experiment representative of three individual experiments is presented.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our results support the idea that fetal and adult thymic precursors have distinct developmental potentials that are maintained when the cells differentiate to peripheral T cells. This proposal fits well with numerous observations that the hematopoietic system overall has different developmental potentials in fetal and adult life. Within the lymphoid compartments, there is striking evidence of these differences for both B and T cell lymphopoiesis. As mentioned earlier, there are T cell maturation events or processes, such as the generation of some T cells, which appear to be unique to either fetal or adult life. In B cell generation, fetal liver precursors give rise to B cells that differ phenotypically and functionally from adult bone marrow-derived B cells (28, 29). Thus, our experiments add to the growing body of evidence that point to fetal vs adult lineages of hematopoietic precursors.

These experiments were performed to address the specific question, "are the properties of peripheral neonatal CD4⁺ cells entirely due to their derivation from fetal thymic precursors?" In many settings, in vivo neonatal CD4⁺ responses are Th2-biased, relative to adult responses (17, 18, 19). In a typical experiment, neonatal cells make 3 to 4-fold less IFN- and 4 to 6-fold more IL-4 than do adult cells in response to secondary immunization (30). Fetal-derived cells resembled neonatal cells in that there was a greater production of the Th2 cytokine IL-4, compared with adult cells. However, fetal-derived cells differed from neonatal cells in that they also produced more IFN- than did adult-derived cells. We recently found (20) that adoptively transferred CD4⁺ cells from day 7 lymph nodes were highly deficient in the production of Ag-specific IFN-, compared with adult CD4⁺ cells. High level IL-4 production, in contrast, was retained in the adoptive hosts. Taking all of these results into account, we propose the following model of the developmental regulation of Th1/Th2 function: Fetal thymic precursors generate peripheral CD4⁺ cells with high potential for the development of Th2 function. This property appears to be retained out to 7 days post birth, both in intact neonates and in adoptively transferred adult hosts. Fetal thymic precursors similarly give rise to peripheral CD4⁺ cells with high potential for the development of Th1 function. This potential is realized within the adult environment in vivo. However, within the intact neonatal animal, there are peripheral influences that irreversibly downmodulate this Th1 potential, leading to reduced Ag-specific Th1 activity both in neonates and in adoptive adult hosts. The question that then arises is, "what occurs postthymically in vivo in neonates to down-regulate Th1 potential?" There are several recent reports (31, 32) that at least some CD4⁺ cells undergo spontaneous proliferation during the first week of life in the mouse. This process appears to be unique to the neonatal period and, in ways not well understood, may act to influence the Th potential of newly emigrated cells. We are beginning experiments to try to determine the role of this proliferation and of the neonatal environment, in general, in regulating Th potential.

These data show that fetal and adult thymocytes impart stable and distinct functional characteristics to their mature progeny. To try to understand how this occurs, it is important to consider regulation at both the cellular and molecular levels. Since fetal-derived cells made more of both Th1 and Th2 cytokines, it seemed possible that fetal, but not adult, precursors may be deficient in the development of regulatory T cell populations which act to downmodulate responses. However, the CD4⁺25⁺ population of regulatory T cells were equally represented among the progeny of fetal and adult precursors (data not shown). In addition, the expression of the CD44, CD69, CD122, and Ly-6C activation/memory markers was similar within the two populations and equivalent to the expression seen in unmanipulated mice (data not shown). Thus, the differential responses of fetal- and adult-derived cells may largely arise due to differences in intracellular molecular processes. Our laboratory is currently investigating three possible molecular mechanisms. First, in adult cells, the expression of cytokine genes is subject to epigenetic control. During Th1/Th2 differentiation, the cytokine loci undergo a remodeling process, leading to the efficient transcription of the cytokine genes and production of the effector cytokine proteins (reviewed in (33, 34). The epigenetic programs of fetal- and adult-derived cells may be different. For example, cytokine loci may be more accessible at the outset of activation in fetal- vs adult-derived cells. This could potentially lead to the development of higher frequencies of Th1/Th2 effectors within the fetal population. Second, the up-regulation of factors thought to be important in Th1/Th2 lineage development may be more rapid or, again, occur among greater proportions of fetal-compared with adult-derived cells. Lastly, at low, but not high, Ag concentrations, fetal-derived responses are Th2-skewed, relative to adult-derived responses. This indicates that the strength of the antigenic signal is "interpreted" differently by fetal- and adult-derived effector cells. Triggering of the TCR by Ag is followed by signaling cascades that ultimately lead to cytokine production (reviewed in (33, 35, 36, 37). Our current working hypothesis is that there may be differences in these signals (either quantitative or qualitative) between fetal- and adult-derived cells.

The observation that the progeny of fetal thymocytes make more of both Th1 and Th2 cytokines raises an interesting question: is this due to a change in the population of cells making either cytokine alone or a change in the ability of each cell to make both cytokines? A straightforward approach to this issue would be to perform intracellular staining for both cytokines simultaneously. However, with normal Ag-specific cells, this is difficult if not impossible because of the low frequencies of cytokine producing cells. In our system, ELISPOT analyses showed that the frequencies of Ag-specific cytokine secreting cells were less than 1% in all cases (IFN- = 0.02% adult-derived, 0.05% fetal-derived; IL-4 = 0.1% adult-derived, 0.3% fetal-derived). These levels are generally considered to be below the limit of detection for flow cytometry. It could be argued that the use of TCR transgenic animals for donors and hosts would increase the level of responding cells above the threshold of detection. However, reconstitution such that all cells (both donor and host) have a single specificity would likely lead to Ag-specific non-responsiveness. There are two technically challenging but potentially feasible approaches that could be taken: 1) two color ELISPOT analyses allowing the detection of frequencies of cells secreting a single type or both types of cytokine, and 2) enrichment of cytokine secreting cells followed by counterstaining with Abs for the other cytokine. We are currently conducting feasibility experiments to determine which of these methods can be most readily applied to answer this question.

The results presented here may have important implications for the treatment of allergy. Allergies often develop in early life. Indeed, Holt and colleagues (38, 39, 40) have shown that sensitization to common allergens can occur during fetal life; the responses of cord blood cells to allergens are universally Th2-skewed and prolonged Th2-biased responses are associated with the subsequent development of allergy in children. Currently, a common treatment regimen for IgE-mediated allergies is Ag-specific immunotherapy. The most effective and frequently used form of this therapy involves the repeated s.c. injections of increasing doses of adjuvant-bound allergen extracts (41). Although the suggested immunological mechanism(s) regulating the efficacy of this treatment are numerous, immunotherapy can decrease the production of IL-4 by CD4⁺ cells and shift cytokine responses from a Th2 to a Th1 pattern (41). Here, we have shown that the Ag-specific responses of murine fetal-derived cells are Th2-skewed at low concentrations of Ag but that this Th2-skewing is substantially downmodulated with increasing Ag dose. Thus, it is tempting to speculate that human allergen-specific CD4⁺ cells primed in utero are similar to fetal-derived murine CD4⁺ cells, thus providing one potential explanation for the action of Ag-specific immunotherapy. Nonetheless, our results suggest that the murine fetal-adoptive transfer system may be extremely useful for modeling the development and immunotherapy of human allergy.

	Acknowledgments

 
We are extremely grateful to Yurong Bu for outstanding technical assistance. We would also like to acknowledge Dr. Christa Muller-Sieburg and Shawn Rose for critical evaluation of the manuscript and stimulating discussions.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant RO1 AI44923-02.

² Address correspondence and reprint requests to Dr. Becky Adkins, Department of Microbiology and Immunology, R[hypehn]138, 1600 NW 10th Avenue, RMSB Room 3152A, University of Miami Medical School, Miami, FL 33136. E-mail address: radkins{at}med.miami.edu

³ Abbreviations used in this paper: KLH, keyhole limpet hemocyanin; PI, propidium iodide.

Received for publication May 5, 2003. Accepted for publication September 9, 2003.

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