Differentiation of human single-positive fetal thymocytes in vitro into IL-4- and/or IFN-{gamma}-producing CD4+ and CD8+ T cells

Etsuro Yamaguchi2, Jan de Vries1,3 and Hans Yssel1,4

1 Department of Human Immunology, DNAX Research Institute, CA 94304, USA
2 Present address: First Department of Medicine, Hokkaido University, Sapporo 060, Japan
3 Present address: Novartis Research Institute, Vienna 1235, Austria
4 Present address: INSERM U454, 34259 Montpellier, France

Correspondence to: H. Yssel, INSERM U454, CHU Arnaud de Villeneuve, 371, Avenue Doyen Gaston Giraud, 34295 Montpellier Cedex 5, France


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we have investigated the capacity of human fetal thymocytes to differentiate in vitro into subsets of T cells with polarized Th1 or Th2 cytokine profiles. Stimulation of freshly isolated human fetal thymocytes with anti-CD3 mAb, cross-linked onto CD32,CD58,CD80-expressing mouse fibroblasts and subsequent culture in the presence of exogenous rIL-2 for 6 days, induced the production of both IL-4 and IFN-{gamma}, which was mainly produced by CD4+ single-positive (SP) and CD8+ SP cells respectively. Addition of rIL-4 during priming augmented IL-4 production in cultures of human fetal thymocytes, which was mainly due to an increased production of IL-4 by CD8SP cells. In contrast, addition of IL-4 to the cultures only slightly enhanced IL-4 production and had little effect on frequencies of IL-4-producing CD4SP cells. Both CD4SP and CD8SP cells produced IL-5, IL-10 and IL-13 at comparable levels, following priming in the presence of rIL-4. Priming in the presence of rIL-12 strongly enhanced the production of IFN-{gamma} in both CD4SP and CD8SP cells. No correlation between expression of CD27, CD30 and CD60, and a particular cytokine profile of differentiated thymocytes could be demonstrated. Together, these results demonstrate the full capacity of fetal human thymocytes to differentiate into cytokine-producing T cells in a priming milieu with appropriate stimulatory molecules and exogenous cytokines. In addition, CD4SP thymocytes rapidly differentiate into polarized Th2 cells following stimulation in vitro in the absence of exogenous rIL-4.

Keywords: cellular differentiation, cytokines, FACS, human, Th1, Th2, thymocytes


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Thymus-derived (T) lymphocytes play a central role in the regulation of the immune response, in part by their capacity to produce a wide array of cytokines. Differentiated human CD4+ T lymphocytes have been divided into functionally distinct subsets, based on their cytokine production profile. Activated Th1 cells produce mainly IL-2, IFN-{gamma}, but no IL-4 or IL-5, and induce cell-mediated responses, essential for the elimination of intracellular pathogens, whereas Th2 cells which produce IL-4 and IL-5, and no or low levels of IFN-{gamma} upon stimulation, give optimal help to B cells in antibody-mediated immune responses, and are involved in anti-inflammatory and allergic responses (reviewed in 1–3).

Many in vivo and in vitro studies have demonstrated that cytokines, present during early T cell activation by antigen, can profoundly influence the direction of Th phenotype differentiation. IL-4 is required for naive T cells to differentiate into Th2 cells (47), whereas IL-12, which acts in most systems as an inducer of IFN-{gamma}, favors the development of Th1 cells (810). In human in vitro models, umbilical cord blood T cells have been used as a source of immunological naive cells to study the role of cytokines in Th differentiation (11,12); however, even those cells may already contain Th cells committed to a particular phenotype. For example, the development of atopic eczema of new-borns strongly correlates with increased proliferative responses and defective IFN-{gamma} production of cord blood mononuclear cells to stimulation with ß-lactoglobulin (13). In addition, cord blood leukocytes contain small and variable, but significant, percentages of activated CD25+ T cells and therefore the status of these populations with respect to their immunological naiveté is questionable.

Differentiation of human T cells from immature CD3CD4CD8 triple-negative (TN) hematopoietic precursor cells takes place predominantly in the thymus, where they expand and differentiate into CD3+CD4CD8 double-negative (DN) and CD4+CD8+ double-positive (DP) T cells. DP thymocytes constitute ~90% of all thymocytes, and can be subdivided on basis of low, intermediate and high expression levels of the CD3–TCR complex. Most of the DP thymocytes die by apoptosis, whereas a small percentage of these cells undergo intrathymic positive and negative selection, eventually giving rise to phenotypically and functionally mature CD3highCD4+CD8 (CD4SP) and CD3highCD4CD8+ single-positive (CD8SP) T cells (reviewed in 14). Freshly isolated, non-stimulated, CD3low DP human fetal thymocytes express transcripts for IL-4, unlike CD3high and SP fetal thymocytes in which only IL-2 mRNA could be detected. Furthermore, stimulation of human CD3high DP and SP thymocytes resulted in the production of high levels of IL-2, but no or barely detectable levels of IL-4 and IFN-{gamma} (15). In addition, using a RT-PCR assay, transcripts for IL-4 and IFN-{gamma} were detected in CD3high CD1 CD4SP and CD8SP thymocytes respectively, albeit at very low levels and comparable to those detected in peripheral naive cells. The latter results indicate that human post-natal thymocytes have a cytokine production profile reminiscent of that of naive T cells (16), in contrast to mouse thymocytes which have been reported to produce IL-2, IL-4, IL-5 and IL-10 upon stimulation in vitro (17). Whereas much is known about the presence of various cytokines in the thymic microenvironment and the role of some cytokines, notably IL-7, in thymocyte development, no information is as yet available about the potential of the precursor cells of the T cell lineage to differentiate into mature effector populations with different cytokine production profiles.

In this study, we have examined the differentiation-inducing effects in vitro of IL-4 and IL-12 on naive human fetal thymocytes, as representative for the most immunologically naive T cells, using flow cytometry to simultaneously analyze production of intracellular cytokines and expression of cell surface antigens.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Fetal thymocytes
Human fetal thymuses were obtained between 16 and 24 weeks of gestation (Advanced Bioscience Resources, Alameda, CA) with informed consent, according to state and federal regulations. Fetal thymus tissue was homogenized by gently mincing with a glass homogenizer, and the resulting thymocyte population was washed twice with PBS, supplemented with 2% FCS and resuspended in culture medium (see below), supplemented with 5 µg/ml DNase (Sigma, St Louis MO), prior to culture. If possible, maternal serum IgE levels were determined by isotype-specific ELISA.

Mouse L cell transfectants
Mouse L cells, transfected with CD32 (Fc{gamma}RII), human CD58 (LFA-3) and human CD80 (B7.1), were described previously (12), and were kind gifts from Drs R. de Waal Malefyt and L. Lanier (DNAX Research Institute) respectively. The expression of CD58 and CD80 on the L cell transfectants was monitored periodically by flow cytometry to ensure identical expression levels during the experiments.

Culture conditions
All cultures were carried out in DMEM (JRH Biosciences, Lenexa, KS), supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µg/ml streptomycin and 100 U/ml penicillin (Life Technologies, Grand Island, NY), and 10% FCS (Sigma), referred to as culture medium. Five hundred thousand, freshly isolated, human fetal thymocytes were stimulated with anti-CD3 mAb (100 ng/ml), cross-linked onto 2x105 irradiated (70 Gy) L cell transfectants and rIL-2 (100 U/ml; a kind gift of Dr Satish Menon, DNAX Research Institute), in the presence or absence of rIL-4 (10 ng/ml; Schering-Plough Research Institute, Kenilworth, NJ) and/or rIL-12 (0.5 ng/ml; R & D Systems, Minneapolis, MS), in 24-well tissue culture plates (Linbro, ICN Biomedicals, Aurora, OH), in a final volume of 1 ml. After 4 days of culture, fresh culture medium was added, containing similar combinations of rIL-2, rIL-4 and/or IL-12, as used during the primary stimulation, and the cells were cultured for an additional 2 days after which their cell surface antigen expression and cytokine production profiles were analyzed.

Re-stimulation of thymocytes for cytokine production by cytokine-specific immunoassay
Fetal thymocytes were harvested, washed 3 times with PBS, supplemented with 2% FCS, and 2x105 fetal thymocytes were re-stimulated in wells of a flat-bottom 96-well culture plate (Falcon, Becton Dickinson, Lincoln Park, NJ), coated with anti-CD3 antibody (plates were coated overnight with 10 µg/ml anti-CD3 mAb diluted in PBS, and washed twice with PBS and once with culture medium prior to the addition of cells) and 1 µg/ml of anti-CD28 mAb, in the presence of 100 U/ml of rIL-2, in a final volume of 200 µl. After 48 h of activation, supernatants were collected and stored at –80°C until assay. Quantification of cytokines was performed by cytokine-specific immunoassay, as described previously (18). The sensitivity of the assay for production of IL-13 was ~100 pg/ml and the sensitivity of all other cytokine assays was 50 pg/ml.

Re-stimulation of thymocytes for analysis of intracellular cytokine production by flow cytometry
Fetal thymocytes (106/ml) in 24 well tissue culture plates were stimulated with plate-bound anti-CD3 mAb and anti-CD28 mAb or with 1 ng/ml of TPA and 500 ng/ml of A23187 (both purchased from Calbiochem, La Jolla, CA), as described above. After 3 h of activation, 10 µg/ml of Brefeldin A (Epicentre Technologies, Madison, WI) was added and the cells were cultured further. After a total of 5 h of activation, cells were harvested. Intracellular cytokines were detected by flow cytometry, using the method of Andersson et al. (19), with some modifications, as described previously (12). For simultaneous analysis of the expression of cell surface antigens and intracellular cytokine production, cells were stained with either FITC-conjugated anti-CD4 and TriColor [Cy5–phycoerythrin (PE)-tandem]-conjugated anti-CD8 mAb, prior to fixation with formaldehyde, after which the procedure for intracellular cytokine production analysis was continued, using PE-conjugated anti-IL-4 and FITC- or PE-conjugated anti-IFN-{gamma} mAb. Cells were analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA).

Flow cytometry and cell sorting
Methods of flow cytometry and data analysis were carried out as described by Lanier and Recktenwald (20). Sorting by positive selection of freshly isolated fetal thymocytes into CD4SP and CD8SP subpopulations was carried out using FITC-conjugated anti-CD4 (Leu-3a) and PE-conjugated anti-CD8 (Leu-2a) mAb, on a FACS Vantage flow cytometer (Becton Dickinson, San Jose, CA). Both sub-populations were >99% pure upon reanalysis, using the same gates as determined for sorting.

mAb
mAb used for activation of fetal thymocytes were the anti-CD3 mAb SPV-T3b (21) and the anti-CD28 mAb L293.1 (a generous gift of Dr Lewis Lanier, DNAX Research Institute). The following mAb were used for cell surface staining: anti-CD1a, anti-CD31 (Biosource International, Camarillo, CA), anti-CD2, anti-CD3, anti-CD4, anti-CD45RA, anti-CD45RO, anti-CD56, anti-CD69 (Becton Dickinson), anti-CD8ß, anti-CD30 and anti-CD60 mAb (generous gifts of Drs Ellis Reinherz, Dana-Farber Cancer Institute, Boston, MA, T. Ellis, Loyola University, Maywood, IL and C. Morimoto, IMSUT, Tokyo, Japan, respectively). For indirect cell surface staining, a FITC-conjugated goat anti-mouse Ig (Tago, Burlingame, CA) was used. Analysis of intracellular cytokine production was performed with the PE-conjugated anti-IL-4 mAb 25D2 (22) and the FITC- or PE-conjugated anti-IFN-{gamma} mAb B27 (23) (generously provided by Dr K. Davis, Becton Dickinson), in combination with FITC- or TriColor-conjugated anti-CD4 and anti-CD8 mAb respectively (Caltag, South San Francisco, CA).

Statistical analysis
Statistical analysis was carried out using the Wilcoxon signed rank test. P < 0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell growth
Freshly isolated human fetal thymocytes were stimulated with anti-CD3 mAb, cross-linked onto L cell transfectants, in the presence of combinations of rIL-2, rIL-4 and rIL-12, and their growth during in vitro differentiation was analyzed by measuring the recovery of viable cells after 6 days of culture. The number of viable fetal thymocytes cultured in the presence of rIL-2 alone was increased 3.4 ± 0.7-fold (SE), as compared to the number of cells at the onset of culture (n – 13). A maximum fold increase in the cell number was observed in the presence of rIL-2 and rIL-4 (7.2 ± 1.0), whereas cultures grown in the presence of rIL-2 and rIL-12 increased only 2.6 ± 0.7-fold in cell number. The recovery of viable cells was intermediate in cultures containing rIL-2, rIL-4 and rIL-12 (5.9 ± 1.1).

Phenotype of differentiated fetal thymocytes
Flow cytometric analysis of freshly isolated fetal thymocyte populations showed three populations of cells expressing low, intermediate and high levels of CD3, whereas the majority of the thymocytes (95%) were CD1a+, which is in agreement with reports in the literature (14). In addition, CD4SP (15%) and CD8SP (3%), DP (80%) and DN (1%) cells could be detected. All fetal thymocytes were CD30 and CD60, and ~20% of the cells expressed CD27 (results not shown). Upon stimulation of the cells with anti-CD3 mAb and L cell transfectants, all thymocytes expressed intermediate levels of CD3 after 6 days of culture, whereas CD1a could no longer be detected (data not shown). The absence or presence of expression of some cell surface molecules, such as CD27, CD30 and CD60, has been reported to be associated with a particular cytokine production profile (2426). However, as shown in Table 1Go, no changes in the expression of these molecules was observed on fetal thymocytes, stimulated and cultured in the various cytokine combinations. The CD69 antigen, which was expressed on a proportion of freshly isolated thymocytes, was induced on all cells after 24 h of activation (nor shown), but was down-regulated after 6 days of culture, irrespective of the presence of IL-4 or IL-12.


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Table 1. Expression of cell surface molecules on fresh and cultured human fetal thymocytes
 
Cytokine production profile of fresh and cultured fetal thymocytes
Freshly isolated thymocytes did not produce measurable levels of IL-4, IL-5 and IFN-{gamma}, whereas only very low levels of IL-10 and IL-13 were detected (data not shown). Fetal thymocytes, stimulated with anti-CD3 mAb cross-linked onto L cell transfectants and cultured for 6 days with rIL-2, produced low, but significant, levels of IL-4, IL-5, IL-13 and IFN-{gamma}, and high levels of IL-10 upon re-stimulation (Fig. 1Go). Differentiation of fetal thymocytes in the presence of both rIL-2 and rIL-4 resulted in a strong increase in the production of the Th2-associated cytokines IL-4, IL-5, IL-13 and IL-10, whereas these culture conditions suppressed the production of IFN-{gamma}. With the exception of IL-4 which was generally not detectable before day 6 of culture, other cytokines could be measured already at day 4, albeit at lower levels (data not shown). As expected, the production of IFN-{gamma} was strongly increased when fetal thymocytes were stimulated in the presence of rIL-2 and rIL-12. Total serum IgE levels detected in the mothers of the thymus donors were within the normal range in 12 cases and at an atopic level in four cases (results not shown). However, no correlation (P = 0.43) could be detected between maternal IgE levels and the levels of IL-4 production by the fetal thymocytes that had been stimulated and cultured in rIL-4.



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Fig. 1. Cytokine production by differentiated fetal thymocytes. Human fetal thymocytes were stimulated and cultured for 6 days with anti-CD3 mAb, L cell transfectants and 100 U/ml rIL-2 (–) in the presence or absence of combinations of 100 U/ml rIL-4 and 0.5 ng/ml rIL-12 respectively. Cytokine levels in culture supernatants, derived from thymocytes that had been stimulated for 48 h with plate-bound anti-CD3 and soluble anti-CD28 mAb, were determined by ELISA, as described in Methods. Results are expressed as the mean + SE of 13 independent experiments. rIL-2 was present in all culture conditions.

 
Fetal thymocytes that had been stimulated in the presence of rIL-2, rIL-4 and rIL-12 produced significantly higher levels of IL-4, IL-5 and IL-13 than those cultured in the presence of rIL-2 and rIL-4, representing an additive, but not synergistic effect of IL-12 on the IL-4-induced production of these cytokines. In contrast, the addition of rIL-12 in thymocyte cultures, together with rIL-2 and rIL-4, significantly inhibited IL-4-induced production of IL-10.

Cytokine production profile of differentiated fetal thymocytes analyzed at the single cell level
Thymocyte populations that had been cultured under different stimulation conditions were analyzed for their cytokine production profile, using an intracellular staining technique. As is shown in Fig. 2, Goa substantial proportion of thymocytes that had been stimulated and cultured for 6 days in the presence of rIL-2 alone were Th1-type cells, whereas fewer Th2 and Th0 cells could be detected. Differentiation of fetal thymocytes in the presence of rIL-2 and rIL-4 resulted in an increase in the frequency of IL-4-producing Th2 cells and a concomitant reduction of IFN-{gamma}-producing Th1 cells, whereas the frequency of Th0-type cells in these cultures was unchanged (Fig. 2 and 3GoGo). As expected, the presence of both rIL-2 and rIL-12 markedly increased the proportion of Th1 cells, as compared to that of rIL-2-grown cells. The enhanced production of IL-4, as observed in the culture supernatants of thymocytes that had been differentiated in the presence of rIL-2 and rIL-12, was found to be due to an increase in the frequency of Th0 cells, since rIL-12 did not induce the generation of IL-4-producing Th2 cells. Thymocyte populations differentiated in the presence of all three cytokines contained Th0, Th1 and Th2 cells. The proportion of IFN-{gamma}-producing Th1 cells under these culture conditions was significantly reduced, underscoring the dominant inhibitory effects of IL-4 on the generation of these cells, which is also consistent with reduced IL-4 levels detected in their culture supernatants.



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Fig. 2. Frequencies of cytokine-producing cells in differentiated human fetal thymocyte populations. Human fetal thymocytes, cultured as indicated under the legend to Fig. 1Go, were stimulated after 6 days of culture with anti-CD3 and anti-CD28 mAb (A) or with TPA and A23187 (B) for 6 h, and the production of IL-4 and IFN-{gamma} was analyzed by flow cytometry, using FITC-conjugated B27 (anti-IFN-{gamma}) and PE-conjugated 25D2 (anti-IL-4) mAb, as described in Methods. Data are displayed as dot blots of correlated FITC and PE fluorescence (four-decade log scales). Quadrant markers are positioned to include >98% of control Ig stained cells in the lower left quadrant (not shown).

 


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Fig. 3. Frequencies of IL-4- and IFN-{gamma}-producing cells in differentiated human fetal thymocytes. Human fetal thymocytes were stimulated with anti-CD3 and anti-CD28 mAb as described in the legend to Fig. 2Go. Results are expressed as mean + SD of 13 independent experiments. rIL-2 was present in all culture conditions.

 
In differentiated thymocytes, higher frequencies of cytokine-producing cells were detected, following stimulation with TPA and A23187, as compared to stimulation with cross-linked anti-CD3 and CD28 mAb (Fig. 2BGo). However, the overall cytokine production pattern was similar under both stimulation conditions, indicating that the profile of IL-4 and IFN-{gamma} production by differentiated thymocytes was not significantly affected by the mode of activation used in this in vitro differentiation system.

Highly purified SP fetal thymocytes can differentiate into IL-4- or IFN-{gamma}-producing T cells
To examine the capacity of individual subsets of thymocytes, as determined by the expression of surface CD4 and CD8 molecules, to differentiate in vitro into effector T cells with different cytokine production profiles, highly purified CD4SP, CD8SP and DP thymocyte populations were stimulated with anti-CD3 mAb and L cell transfectants and cultured in the presence of exogenous cytokines. DN thymocytes were not purified because of their low frequencies in the fetal thymocyte preparations. Purified DP cells did not proliferate in any of the stimulation conditions. In contrast, CD4SP and CD8SP cells expanded ~20- to 30-fold when stimulated and cultured in the presence of rIL-2, which was not further enhanced when rIL-4 or rIL-12 was added. Phenotypic analysis of purified CD4SP and CD8SP thymocytes, following a 6 day culture, showed heterogeneous populations, as defined by the expression of CD4 and CD8 (Fig. 4Go). Populations derived from CD4SP fetal thymocytes, following differentiation in the presence of rIL-2 alone, expressed substantial levels of CD8, which were further enhanced when rIL-4 was present in the cultures. Most of the CD8+ cells derived from CD4SP cells expressed CD8 {alpha}, as well as CD8 ß chains (data not shown). In contrast, the presence of rIL-12, but also of rIL-4, resulted in a strong increase in CD4 expression on CD8SP-derived thymocytes.



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Fig. 4. Phenotypic changes of purified human fetal thymocyte subsets following differentiation. Human fetal thymocytes were purified by negative selection, based on expression of CD4 and CD8, and cultured as indicated in the legend to Fig. 1Go. After 6 days of culture, expression of CD4 and CD8 was determined by flow cytometry.

 
Purified, rIL-2-cultured CD4SP fetal thymocytes produced higher levels of IL-4 and IL-13 than CD8SP cells (Fig. 5Go). In particular, CD4SP cells cultured in the presence of rIL-2 were able to produce high levels of IL-4 upon activation, which were not markedly enhanced when rIL-4 had been added during the culture period. CD8SP cells also produced low levels of IL-4, IL-5, IL-10 and IL-13, following differentiation in the presence of exogenous rIL-2 and rIL-4. The addition of rIL-12 strongly induced the production of IFN-{gamma} by both CD4SP and CD8SP cells (Fig. 5Go). Analysis of intracellular staining of these subsets by flow cytometry showed that IL-4-producing cells were more enriched in CD4SP-derived cells than in CD8SP-derived fetal thymocytes (Fig. 6Go). The highest proportion of IL-4-producing cells was detected in CD4SP thymocytes in which the expression of CD8 could not be induced upon culture. Notably, the frequency of IL-4-producing cells in the CD4SP population cultured in rIL-2 alone reached maximal levels following stimulation that were only slightly increased when IL-4 was added to the cultures (Fig. 6Go), which was in keeping with the results obtained by ELISA. In contrast, IFN-{gamma}-producing cells were more abundant in CD8SP than in CD4SP fetal thymocytes. The expression of CD4 and CD8, induced during culture of purified CD8SP and CD4SP thymocytes, respectively, had no effect on the frequencies of IL-4- and IFN-{gamma}-producing cells.



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Fig. 5. Cytokine production by purified, differentiated, CD4SP and CD8SP human fetal thymocytes. Purified CD4+ and CD8+ human fetal thymocytes were stimulated and cultured with cytokines for 6 days and cytokine concentrations in the culture supernatants were measured by ELISA after 6 days of culture, as indicated in the legend to Fig. 1Go. Open bars, cross-hatched bars and hatched bars indicate cultures with rIL-2 alone, rIL-2 and rIL-4, and rIL-2 and rIL-12, respectively. Representative results obtained in one of two experiments are shown.

 


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Fig. 6. Frequencies of cytokine-producing cells in purified human fetal thymocyte subsets following differentiation. Purified and cultured CD4+ and CD8+ human fetal thymocytes were analyzed for intracellular production of IFN-{gamma} and IL-4. Culture conditions as described in Fig. 1Go.

 
To rule out possible interactions between CD4SP and CD8SP populations which may affect their cytokine production profile, intracellular IL-4 and IFN-{gamma} production by these cells was analyzed in differentiated unseparated fetal thymocytes. The respective differences in the capacity to produce IL-4 (Fig. 7AGo) and/or IFN-{gamma} (Fig. 7BGo) between CD4SP and CD8SP cells, as determined in unseparated fetal thymocyte populations, were similar to those observed in purified cells. Together, these results indicate that a proportion of naive CD4SP fetal thymocytes seems to be committed to produce IL-4, irrespective of the presence of exogenous rIL-4, whereas CD8SP cells predominantly produce IFN-{gamma}.



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Fig. 7. Frequencies of differentiated cytokine-producing human fetal thymocyte populations. Unseparated human fetal thymocytes, cultured as indicated in legends of Fig. 1Go, were simultaneously stained for CD4 and CD8, respectively, as well as for intracellular IL-4 (A) or IFN-{gamma} (B) by flow cytometry. Only SP cells were gated and examined for cytokine staining. Data from six tissues.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The thymic microenvironment supports the development and commitment of bone marrow and fetal liver-derived pluripotent progenitors to the cells of the T cell lineage. In human, full differentiation of the thymus occurs around week 7 and 8 of gestation after which development of progenitor cells into CD4SP and CD8SP effector T cells occurs (reviewed in 14). Since antigenic exposure in utero immediately after the commitment to the T cell lineage is unlikely, thymocytes, especially in the embryonic stage, represent a population comprised primarily of naive T cells. Here, we show that a single stimulation of this unique population of truly naive cells with anti-CD3 mAb, cross-linked onto CD32+ murine fibroblasts, expressing CD80 and CD58, and subsequent culture in the presence of rIL-2, resulted in the generation of a population of cells which produces low, but significant, levels of IL-4, IL-5 and IL-13. In addition, fetal thymocytes differentiated under these conditions produce IFN-{gamma} and IL-10. CD4SP, but not CD8SP, thymocytes were the main producers of IL-4, as shown by intracellular staining and flow cytometry. Interestingly, whereas the addition of rIL-4 during priming and subsequent culture induced a strong increase in the production of IL-4, detected in the culture supernatants, the frequency of IL-4-producing cells within the subset of CD4SP thymocytes was only slightly enhanced by the presence of exogenous rIL-4. In addition, the production of IL-4 was restricted mostly to cells that were not susceptible to in vitro induction of CD8 by IL-4 and therefore remained true CD4+ cells. It has been reported previously that repetitive stimulation of CD4+ cord blood and peripheral blood T cells via CD3 and CD28 molecules, in the absence of exogenous rIL-4, resulted in the generation of IL-4- and IL-5-producing cells (27), suggesting that these cells are capable of releasing sufficient autocrine IL-4 to support their differentiation along the Th2 pathway. It is, however, unlikely that the CD3+ CD4SP and CD8SP thymocytes which differentiated into IL-4-producing cells in our culture system are themselves a source of endogenous IL-4, in view of the observation that freshly isolated, non-stimulated, CD3high DP, CD4SP and CD8SP human fetal thymocytes do not express transcripts for IL-4 (28). Moreover, postnatal CD3high CD4SP and CD8SP human thymocyte populations produce high levels of IL-2, following stimulation, but no detectable levels of IL-4 and IFN-{gamma} (15). It is, however, important to note that, in contrast to CD3high DP and SP cells, freshly isolated human fetal thymocytes at an earlier stage of differentiation, such as CD3low DP cells, have been reported to express transcripts for IL-4 (28). Since the latter population of pre-T cells is thought to be involved in the process of positive selection in the thymus, their constitutive expression of IL-4 mRNA might be the result of TCR–CD3 complex triggering and endogenous levels of IL-4 produced by this subpopulation upon stimulation may therefore be sufficient to drive Th2 differentiation of CD4SP cells. In this respect, as has been described for in vitro differentiated naive mouse CD4+ cells, a small, but distinct, population of IL-4-producing T cells may be present which is able to induce the differentiation of naive T cells into IL-4-producing cells (29). Another potential source for endogenous IL-4 in the human fetal thymus might be a population of cells described for murine thymocytes, characterized by the expression of the NK1.1 molecule, which are able to rapidly produce IL-4 upon stimulation (3033). This hypothesis could however not be tested, because of the lack of mAb that delineate the human equivalent of mouse NK1.1 cells.

The presence of exogenous rIL-4 also induced production of IL-4 by CD8SP cells. Both in experimental mouse and human models, CD8 cells with different cytokine production profiles, reminiscent of CD4+ Th1 and Th2 cells, have been described (3436). For example, CD8+ type 1 cytotoxic (Tc1) and Tc2 clones could be generated from peripheral blood mononuclear cells of cultures of healthy, as well as HIV-infected, donors. Moreover, allo-specific human CD8+ Tc1 and Tc2 cells which could lyse tumor target cells and bone marrow-derived target cells, and which may play a role in the regulation of allogeneic transplantation responses, could be generated in vitro (37). Our data indicate that CD8+ T cells with the potential to rapidly differentiate in subpopulations with different cytokine production profiles are present in the human fetal thymus.

Similar to what has been observed in differentiated CD4+ cord blood T cells (12), fetal thymocyte populations, stimulated and cultured in the presence of rIL-4, produced very high levels of IL-10, with both CD4SP and CD8SP thymocytes producing this cytokine. The high IL-10 production seems to be specific for in vitro differentiated (fetal) thymocytes and cord blood lymphocytes, since naive, CD4+CD45RA+, peripheral blood T cells, stimulated and cultured under identical experimental conditions, produced only low levels of IL-10 (data not shown). In contrast to the failure of rIL-12 to induce the IL-10 production by fetal thymocytes, IL-12 reportedly is able to induce the production of IL-10 by naive, peripheral blood T cells (38) and it has been argued that this reflects a negative feedback loop, since IL-10 is know to down-regulate the production of IL-12 by monocytes (39). The reason for the discrepancy between the latter results and those described in this study with respect to the failure of IL-12 to induce the production of IL-10 in fetal thymocytes and cord blood T cells is not clear, but the strong enhancement of IL-10 production by IL-4, may reflect its property as a Th2-like cytokine. Although no information about the usefulness of fetal thymocytes for allogeneic transplantation purposes has been reported yet, our observation may of interest, in view of the reported property of cord blood T cells which induce little graft-versus-host disease following allogenic transplantation which has been contributed, at least in part, to their high production levels of IL-10 (40).

The expression of a number of cell surface molecules has been associated with a particular cytokine production profile. In particular, the Reed–Sternberg-specific surface antigen CD30 has been reported to be expressed exclusively on Th0 and Th2 T cell clones, but not on Th1 clones (25). This observation however has been disputed by others (41,42), and also in the present study, using in vitro differentiated fetal thymocytes, no positive correlation between CD30 expression and a Th2 type cytokine production could be demonstrated. In addition, the expression of two other cell surface molecules on fetal thymocytes, CD27 and CD60 described to be expressed on distinct subsets of T lymphocytes (24,26), did not change following in vitro differentiation of fetal thymocytes.

The activation marker CD69, a C-type lectin with unknown function, was found to be expressed on ~30% of total thymocytes, but present on only 10% at the end of the 6 day culture period. This seemingly paradoxical situation can be explained by the observation that CD69 is up-regulated on thymocytes following positive selection and expressed at the cell surface of DP, as well as CD45RA+CD3high CD4SP or CD8SP thymocytes (15), but is down-regulated on these cells, when they leave the thymus. Therefore, in our culture system, the expression of CD69 is strongly up-regulated on all thymocytes, including those already expressing CD69, following CD3-mediated activation, but its expression is lost upon subsequent culture. It is of note that the kinetics of induction and down-regulation of CD69 expression on stimulated fetal thymocytes followed similar kinetics, as those described for mature T cells (unpublished data).

In vitro culture of CD4SP fetal thymocytes in the presence of rIL-4 resulted in the induction of CD8{alpha} as well as CD8ß. This results confirm previous reports demonstrating that IL-4 is capable of inducing CD8{alpha}, but not CD8ß, on the surface of peripheral blood T cells upon in vitro culture (42,43). Since IL-4-cultured CD4+ T cells express only CD8{alpha}, but not CD8ß, in contrast to CD8+ T cells which express both molecules (43), it has been speculated that the CD8 ß chain may be involved in polymerization and stabilization of CD8 which enhances the efficiency of HLA class I-restricted antigen recognition. Interestingly, however, as we show in the present study, rIL-4-induced CD8 on thymocytes consists of CD8{alpha} and CD8ß and therefore could be functionally active.

We have no explanation for the observation that ~25% of purified CD8SP cells, cultured for 6 days in the presence of exogenous rIL-2, expressed CD4 and the percentage of CD4-expressing CD8 cells augmented to ~50% following culture of purified thymocytes in rIL-4, as well as rIL-12. An inducing effect of both cytokines on CD4 expression has not been reported yet, although it seems to be specific for fetal thymocytes and no induction of CD4 on CD8+ peripheral blood T cells was observed under identical experimental conditions (data not shown). As purified DP fetal thymocytes did not proliferate in our in vitro culture system, it is likely that the generation of CD4+CD8+ T cells is the result of de novo induction of CD4 or CD8 molecules on the cell surface of SP cells rather than an outgrowth of pre-existing DP cells. However, although CD4SP, as well as CD8SP-derived, populations of CD4+CD8+ T cells showed some differences in their capacity to produce cytokines, as compared to the parental CD4SP or CD8SP fetal thymocytes, the significance of induction of these molecules on the cell surface following in vitro culture remains to be determined. Interestingly, in line with our results, it was reported recently that stimulation of CD8+ T cells through the TCR complex leads to de novo expression of the CD4 antigen on the cell surface, rendering these cells susceptible to infection with HIV-1 (45). Therefore, if an activation- or cytokine-mediated induction of CD4 on CD8 cells occurs in vivo as well, it can be speculated that CD8+ thymocytes might be a target for infection with HIV.

In conclusion, our results show that a short-term primary stimulation with anti-CD3 mAb, in the presence of relevant co-stimulatory signals and exogenous cytokines, is sufficient to induce the differentiation of SP fetal thymocytes into cytokine-producing CD4+ or CD8+ effector cells, indicating that following encounter with antigen functional differentiation of SP thymocytes with respect to their capacity to produce cytokines may occur in the thymus. Although the overall profile of cytokines produced by either CD4SP or CD8SP thymocytes showed similarities to that their peripheral counterparts, a comparatively high production of IL-4 in the absence of exogenous IL-4 in the cultures was observed for CD4SP cells. It remains, however, to be demonstrated whether this phenomenon could be responsible for the development of atopic diseases shortly after birth in some infants.


    Acknowledgments
 
We would like to thank Jim Cupp, Eleni Callas and Josephine `Dixie' Polakoff for their expert assistance with FACS, Patricia Larenas for technical assistance, and JoAnn Katheiser for secretarial help. DNAX Research Institute Inc. is supported by the Schering-Plough Corp.


    Abbreviations
 
DPdouble positive
PEphycoerythrin
SPsingle positive
TNtriple negative

    Notes
 
Transmitting editor: K.-i. Arai

Received 16 June 1998, accepted 8 January 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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