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Peripheral Immature CD2^-/low T Cell Development from Type 2 to Type 1 Cytokine Production¹

Kimmel Cancer Center, Department of Microbiology and Immunology, Jefferson Medical College, Philadelphia, PA 19107

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immature myeloid and NK cells exist, and undergo cytokine-induced differentiation, in the periphery. In this study, we show that also immature CD2^-/low T cells exist in peripheral blood. These cells produce the type 2 cytokines IL-13, IL-4, and IL-5, but not IFN- or IL-10, and, upon culture with IL-12- and TCR-mediated stimuli, differentiate to IL-13⁺IFN-⁺ cells producing high IL-2 levels, and finally IL-13^-IFN-⁺ cells. The monokine combination IL-12, IL-18, and IFN- substitutes for TCR-mediated stimulation to induce the same differentiation process in both immature CD2^-/low and primary mature CD2⁺ IL-13⁺ T cells. IFN- is needed to maintain high level IL-2 production, which is confined to type 2 cytokine-producing cells and lost in the IFN-⁺ ones. Upon TCR-mediated stimulation, IFN-⁺ cells are then induced to produce IL-10 as they undergo apoptosis. These data indicate that peripheral type 2 cytokine⁺ T cells are immature cells that can differentiate to effector IFN-⁺ cells following a linear monokine-regulated pathway identical with that previously described for NK cells. They define the cellular bases to support that cell-mediated immune responses are regulated not only via Ag-induced activation of mature effector cells, but also via bystander monokine-induced maturation of immature T cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In addition to IFN-⁺ and/or cytotoxic T cells, efficient inflammatory immune responses require the participation of cells of innate immunity, namely myeloid and NK cells. A common mechanism ensuring increased numbers of these effector cells involves differentiation of immature myeloid cells, their progenitors, and CD34⁺ hemopoietic stem cells present in minor proportions in the periphery (1), and mobilization of their bone marrow-resident counterparts (2). The demonstration that peripheral immature CD56^-CD2^-CD161⁺ NK cells (3, 4) producing type 2 cytokines only (IL-13 and IL-5) undergo terminal differentiation to type 2 cytokine^- IFN-⁺ cytotoxic effector cells upon IL-12 induced differentiation (5) supports the contention that a cytokine-regulated differentiation of these cells may also contribute to increase immediate and adaptive inflammatory responses to pathogens. Whether a similar mechanism operates to increase the numbers of IFN-⁺ and/or cytotoxic effector T cells in cell-mediated immunity is unknown.

T cells develop from their bone marrow-derived T lineage-committed (CD2^-CD3^-) progenitors in the thymus. CD2 expression is acquired early during development, before or at the time of CD3 (reviewed in Ref. 6). Only mature single-positive (SP)³ helper (CD4⁺) or cytotoxic (mostly CD8⁺) T cells expressing productively rearranged Ag-specific TCR are believed to be exported to the periphery after thymic selection. However, extrathymic maturation has been proposed for minor T cell subsets (e.g., TCR /⁺ cells (7)). Our finding of CD3⁺ peripheral blood T cells with the same high proliferative potential and cytokine production capabilities of immature NK cells (5) supports the possibility that this may occur, also for T cells of the major TCR /⁺ subset, depending on differentiation of peripheral hemopoietic progenitor cells or of few T lineage-committed cells that either escaped the thymus or exited it at a preterminal differentiation stage.

In this study, we report that immature CD2^-/low T cells producing only type 2 cytokines are indeed present in neonatal and adult peripheral blood, and are inducible to mature to terminally differentiated IFN-⁺ cells under the sole influence of monokines, following the same maturation pathway defined for NK cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lymphocyte populations and T cell clones

Peripheral lymphocytes, CD2^-/low, and CD2⁺ T cells were separated from neonatal (umbilical cord) or adult PBLs (5) and cultured in RPMI 1640 medium with 5% autologous plasma and 50 U/ml rIL-2 (Biological Response Modifiers Program, National Cancer Institute) (control conditions: IL-2 only, to support T cell survival and/or proliferation). Other factors were added as specified.

CD2⁺ and CD2^-/low T cells

Sheep E treated with 2-aminoethylisothiouronium bromide (AET; Sigma-Aldrich, St. Louis, MO) were used to deplete most T and NK cells (8). CD3⁺ lymphocytes from the E_AET⁺ cell pellet (99.9% CD2⁺ on reanalysis) are referred to as CD2⁺ T cells. Mature and CD2⁺ cells remaining in the E_AET^- cell fraction after one round of rosetting were depleted by indirect anti-Ig rosetting (9) with mAb to CD2, CD56, and HLA-DR. This technique cannot ensure complete depletion of cells expressing low CD2 levels, and the lymphocytes obtained are referred to as CD2^-/low T cells.

To obtain sufficient numbers of cells for analysis, CD2^-/low lymphocytes (10⁵ cells/ml) were cultured in control medium with added rIL-4 (10 ng/ml; R&D Systems, Minneapolis, MN) (unless otherwise noted) and anti-IL-12 mAb C8.6 (5 µg/ml; cells provided by G. Trinchieri, Schering-Plough, Dardilly, France), plate-bound CD3 mAb (OKT3, 5 µg/ml), and soluble CD28 mAb (9.3, 2 µg/ml), or the Staphylococcus aureus enterotoxins SEC3 + SEE (0.5 µg/ml each; Toxin Technology, Sarasota, FL). The cells were transferred to a new plate after 3 days, and subcultured as needed with IL-2 only without CD3-mediated stimuli. When indicated, CD2⁺ T cells were cultured under the same conditions.

IL-13⁺ T cell clones

CD2^-/low lymphocytes (one to five cells/well microtiter plates) were cultured in control medium with added IL-4, anti-IL-12 mAb, SEC3 + SEE, or PHA-P (1 µg/ml; Sigma-Aldrich), and 50 Gy-irradiated autologous mononuclear cells (10⁴:1). After 1 wk, the cells were subcultured with IL-2 only. Cloning efficiency was 5%.

Secondary culture conditions

Further cultures to induce differentiation of the polyclonal and monoclonal T cell populations (1–2 x 10⁶ cells/ml) were in control conditions and with added other cytokines, individually or combined, with or without CD3/TCR ligands. Cytokines used were: rIL-12 (2 ng/ml; provided by S. Wolf, Genetics Institute, Andover, MA); purified human rIFN-, 500 U/ml (IFN-A/D (BglII), sp. act. 1.17 x 10⁸ antiviral U/mg; PBL Biomedical Laboratories, New Brunswick, NJ); rIFN- from Escherichia coli, 500 U/ml (sp. act. 7 x 10⁷ antiviral U/mg; provided by H. M. Shepard, Genentech, South San Francisco, CA); or rIL-18, 50 ng/ml (R&D Systems). CD3-mediated stimuli were those listed above. When indicated, anti-TNF- mAb (10) were added. The cell cycle inhibitor mimosine was added (300 µM; Sigma-Aldrich) at and for the indicated times.

Induction of cytokine production and intracellular cytokine detection

Cells (5 x 10⁶/ml) were stimulated (5 h, 37°C, brefeldin A added, 10 µg/ml, during the last 3 h) with Ca²⁺ ionophore A23187 (0.1 µg/ml), PMA (10^-9 M) (all reagents from Sigma-Aldrich), and rIL-2 (100 U/ml). Surface phenotype and intracellular cytokine accumulation were detected in three- and four-color immunofluorescence, as described (11); specificity and sensitivity were discussed previously (12). Viable lymphocytes were gated based on forward and side angle light scatter, and analysis was performed on gated CD3⁺ (biotin OKT3 or PE-Texas Red (PE-TR) S4.1; Caltag Laboratories, Burlingame, CA), CD2⁺ (biotin B67.1 + B67.6 detected with CyChrome streptavidin (BD PharMingen, San Diego, CA)), CD4⁺ or CD8⁺ T cells (FITC B66.6 and B116.1) (13), or PE-TR CD4 clone S3.5 and PE-TR CD8 clone 3B5 (Caltag), as indicated. GATA-3 was detected with the anti-GATA-3 mAb HG3-31 (Santa Cruz Biotechnologies, Santa Cruz, CA) and FITC goat anti-mouse IgG + IgM (intracellular immunofluorescence as for cytokine detection). Samples were analyzed on an XL-MCL automated analytical cytometer (Beckman Coulter, Hialeah, FL). Listmode data were analyzed with the WinMDI Flow Cytometry Application (J. Trotter, Scripps Research Institute, La Jolla, CA; http://facs.scripps.edu/). Percentages of positive cells are reported after background subtraction.

Cell-mediated cytotoxicity

The CD32⁺ THP-1 cells were used as targets in 4-h ⁵¹Cr release assays. CD3-induced redirected cytotoxicity was measured, as described (14), in the presence of the CD3 mAb OKT3, or CD56 mAb B159.5 as control.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phenotype and functions of peripheral blood CD2^-/low T cells

T and NK cells share expression of CD2, an early T cell differentiation marker. The presence of CD2^- T cells, unlike that of the equivalent NK cells (4, 5), could not be resolved (<0.1\% by surface immunofluorescence) within PBLs (Fig. 1>A). However, 1% CD3⁺ CD2^-/low cells were detected in the freshly separated E_AET^- fraction after partial depletion (one round rosetting) of CD2⁺ cells from freshly separated PBLs (Fig. 1B, right panels). They contained 20% CD4^-CD8^- double-negative (DN), high proportions of CD4⁺ SP and IL-13⁺ T cells, and almost no CD8⁺ SP cells. Instead, the CD3⁺ T cells in the freshly separated E_AET⁺ and E-_AET^- CD2⁺ fractions (left panels) contained T cell subsets with the same phenotype and cytokine production ability of freshly separated T cells (see Fig. 1A) (4), except for small proportions of double-positive (DP) CD4⁺CD8⁺ and DN CD4^-CD8^- cells in the E_AET^- CD2⁺ cells.

View larger version (39K): [in this window] [in a new window]   FIGURE 1. Phenotypic and functional characterization of immature peripheral T cells. Multiple color immunofluorescence analysis was performed in freshly separated neonatal (cord blood) lymphocytes (A, total), and in their EAET+ (left) and EAET- subsets obtained after one round of EAET rosetting (B). CD2+ and CD2- cells were detected within the different populations using biotin-labeled B67.1 and B67.6 mAb, detected with CyChrome streptavidin. CD3 and CD2 (A, top), and CD4 and CD8 surface expression (B, top), and IFN- and IL-13 production upon stimulation of the freshly separated cells (A and B, bottom panels) were analyzed (four-color immunofluorescence) in the total lymphocytes (A) and the gated CD3+ T cells (CD3-PE, B). Isotype-matched Ig were used as negative controls (not shown). Cytokine production was induced as described in Materials and Methods. Data are displayed as density plots; the percentages of cells in each quadrant are indicated (background subtracted). Experiment representative of two with similar results. C—E, Neonatal CD2-/low (C and D) and CD2+ lymphocytes (E) obtained after the two-step procedure described in Materials and Methods were cultured for 10 days, as described in Materials and Methods. Surface phenotype and cytokine production, as indicated, were analyzed in gated CD3+ lymphocytes as in A and B. Similar results were obtained in three experiments with neonatal and two with adult peripheral lymphocytes. F, Cytotoxicity of CD2-/low T cells obtained as in C, but from adult blood, was analyzed against THP-1 target cells. Effector cells were lymphocytes from 5-day cultures of identical aliquots of the cells with IL-2 without (left) or with PHA added (right). Redirected cytotoxicity assays were performed in the presence of CD3 (OKT3) () or control CD56 mAb B159.5 (). Experiment representative of three performed with similar results (two neonatal and one adult T cells).

Most expanded CD2^-/low T cells were IL-13⁺ (72 ± 13%, n = 5) (Fig. 1D), and none produced detectable IFN-. About half of the IL-13⁺ cells were IL-4⁺, and these included IL-5⁺ cells. About 50% of the cells produced IL-2. Most IL-2⁺ cells were IL-13⁺ >70% of them were IL-4⁺, and <10% IL-5⁺. Parallel cultures of nonseparated or CD2⁺ T cells contained, instead, a major IFN-⁺ population, lower proportions of IL-13⁺ T cells (15 ± 14, n = 6), and some IL-13⁺IFN-⁺ cells (Fig. 1E). TNF- and GM-CSF were produced at similar levels by most CD2⁺ and CD2^-/low T cells (not shown). After priming with PHA for 5 days, the CD2^-/low lymphocytes mediated significant levels of CD3-induced redirected cytotoxicity against nonsensitive target cells (Fig. 1F), indicating functional expression of CD3.

Generation of IFN-⁺ cells from CD2^-/low T cells

Cells from the polyclonal CD2^-/low T cell populations expanded with IL-4 were further cultured with IL-12- and TCR-mediated stimuli in the presence of mimosine to inhibit cell cycle progression (Fig. 2A, top). This resulted in the appearance of IFN-⁺ cells (right panels), significantly decreased proportions of IL-13⁺ (and IL-4⁺) IFN-^- cells, and proportionally increased percentages of IL-13⁺ (and IL-4⁺) IFN-⁺ cells. About two-thirds of the IFN-⁺ cells produced IL-13, less than one-quarter of which were IL-4⁺. The same results were obtained after a 5-day culture under the same conditions without mimosine (bottom panels). No significant changes occurred in cultures with IL-2 for 15 h with mimosine added (Fig. 2A, top left); the proportions of IL-13⁺ and IL-4⁺ cells were only slightly decreased after culture with PHA (middle); those of TNF-⁺ and GM-CSF⁺ cells were unchanged (not shown). Like in immature hemopoietic cells (16) and thymocytes (17, 18, 19), most CD2^-/low T cells were GATA-3⁺. Consistent with the role of GATA-3 to control transcription of most type 2 cytokine genes (20) and proliferation of immature hemopoietic progenitors (21) and T cells (18), the percentages of GATA-3⁺ cells in the above experiments remained 99% after culture with IL-2, and decreased to that of the IL-13⁺ cells after culture with IL-12 (Fig. 2E). Only nonlabeled mAb are available for human GATA-3 detection. Although direct analysis of GATA-3 and IL-13 coexpression was, therefore, not possible, the overall data, together with the fact that GATA-3 is not a transcription factor for IFN-, strongly support the conclusion that GATA-3 is lost in cells that produce IFN- only. The proportions of TNF-⁺ and GM-CSF⁺ cells remained similar to the original ones under any culture condition (not shown). Analysis performed on the CD2⁺ T cells cultured under the same conditions gave similar results (Fig. 2B).

View larger version (50K): [in this window] [in a new window]   FIGURE 2. Cytokine production by immature T cells after culture with IL-12. Neonatal CD2-/low (A) and CD2+ T cells (B) were purified and cultured for 10 days as in Fig. 1C. Identical cell aliquots were then cultured with IL-2 alone (left) or with added PHA-P without (middle) or with IL-12 (right). After 15-h culture with mimosine (A, top; B) or 5-day culture without it (A, bottom), the cells were stimulated as in Fig. 1, and intracellular accumulation of IFN- (FITC, y-axis), IL-13, and IL-4 (PE, x-axis) was analyzed on gated CD3+ cells. On day 0, IFN-+, IFN-+IL-13+, and IL-13+ T cells were 0.1, 0.4, and 84.1%, respectively. The T cell numbers after culture with mimosine were 10% (IL-2 only) and 20% lower (both conditions with PHA) than those on day 0. Experiment representative of six performed with similar results. C, Identical aliquots of a CD2-/low T cell population obtained as in A were further cultured in the presence of IL-2 alone (left) or with added IL-12 and irradiated autologous monocytes without (middle) or with SEE + SEC3 as superantigens (right), as described in Materials and Methods. Induction of cytokine production, detection, and analysis of intracellular IFN- and IL-13 were as in A. Experiment representative of two performed with similar results. D, Identical aliquots of an IL-13+ T cell clone (representative of 10 derived from CD2-/low T cells in the presence of IL-4) were cultured, stimulated, and analyzed as in A. E, GATA-3 expression (indirect immunofluorescence) in the experiment in A.

Monokine-induced maturation to IFN- production

TCR engagement may contribute to the differentiation process by inducing functional IL-12R expression. We tested the possibility that IFN- and IL-18, which affect type 1 polarization of T cell responses (22) and enhance IL-12R₂ expression (23), promote maturation of the immature T cells in the absence of TCR-mediated stimulation. Cultures for up to 7 days with IL-2 (or IL-15, not shown) alone or with added IFN-, IL-18, or IFN- contained proportions of IL-13⁺ cells similar to those in the original cell populations (Fig. 3) and undetectable IFN-⁺ cells. Only addition of IL-18 to the cultures with IL-12 induced appearance of IL-13⁺IFN-⁺ T cells, increased proportions of IL-13^-IFN-⁺ cells, and decreased proportions of IL-13⁺IFN-^- cells. The combination of IL-18 and IFN- with IL-12 resulted in the highest proportion of IFN-⁺ T cells. Slightly increased, unchanged, and significantly decreased numbers of cells were recovered, respectively, from the cultures with IL-2, IL-12, and monokines combined (Table I), indicating differentiation and lack of proliferation only in the cultures with all relevant monokines.

View larger version (61K): [in this window] [in a new window]   FIGURE 3. Monokine-induced generation of IFN-+ cells from immature T cells. Identical cell aliquots from neonatal CD2-/low T cells obtained as in Fig. 1 were cultured for 18 h (A, mimosine added) or 7 days (B and C, without mimosine) with IL-2 alone (none) or with the added indicated cytokines without (top) or with IL-12 (bottom). Intracellular accumulation of IFN- and IL-13 (A and B), and of IL-2 (C) was analyzed in stimulated cells within gated CD3+ cells. Experiment representative of five performed with similar results.

IL-2 production during maturation of type 2 cytokine⁺ T cells

The above data established a correlation between IL-2 and type 2 cytokine production. To determine whether IFN- and IL-2 can be produced concomitantly, we attempted to generate greater proportions of IL-13⁺IFN-⁺ cells from the CD2^-/low T cells upon primary culture without IL-4. Under these conditions (Fig. 4A), most T cells were still IL-13⁺ (30% IL-4⁺), but both IL-13⁺IFN-⁺ and IL-13^-IFN-⁺ cells were present. Only 15% of the IL-2⁺ cells were IL-5⁺, and 20% of them overlapped with the cells producing low IFN- levels; most IFN-^+/high T cells did not produce IL-2. This phenotype was unchanged after secondary cultures with IL-2, with or without added PHA or IL-12 (Fig. 4B, top). In cultures with IL-12 and PHA, instead, the proportions of the IL-2⁺IL-13⁺ T cells decreased by 20%, and 60% of the IL-2⁺ cells, all IL-13⁺, were IFN-⁺ (Fig. 4B, bottom). All IL-4⁺ cells, like in the cultures of the CD2^-/low T cells with IL-4, were IL-13⁺ (not shown). Thus, during differentiation, both the immature CD2^-/low and the more mature CD2⁺ IL-13⁺ T cells acquire the ability to produce IL-2 when IL-4, and more so IL-5, production is almost completely lost, IL-13 production is partially reduced, and ability to produce low levels of IFN- starts being acquired. IL-2 and type 2 cytokines are no longer produced as the cells become capable of producing high IFN- levels. The phenotypic and functional characteristics of the IFN-⁺ cells derived from the immature T cells are identical with those of the mature primary CD4⁺ or CD8⁺ peripheral T cells, the majority of which produce low IFN- levels with no detectable type 2 cytokines, and only a minority of which produces high IFN- levels (4).

View larger version (71K): [in this window] [in a new window]   FIGURE 4. IL-2 production in differentiating immature T cells. A, IFN-, IL-13, IL-4, and IL-5 production was analyzed (see Fig. 1) within gated CD3+ cells from cultures of neonatal CD2-/low T cells as in Fig. 1, but without IL-4. B and C, Identical aliquots of neonatal CD2-/low lymphocytes obtained as above (B) or of two (CD3+CD2+) clones generated from the CD2-/low T cells and containing IL-13+, IFN-+, and IL-13+IFN-+ cells (C) were cultured for 5 days with IL-2 alone (top) or with added PHA + CD28 mAb and IL-12 (bottom). Accumulation of the indicated cytokines in stimulated cells was analyzed within gated CD3+ cells. The experiments in A and B are representative of three of each type with similar results. Top panels in C are isotype-matched negative controls.

Final step of T cell maturation

The observations that apoptotic bodies from T cells contain TGF- (24), our previous data in NK cells (5), and reports using IFN-⁺ Th1 cells (25) or superantigen-activated Th cells (26) position inhibitory cytokine production (including IL-10) at the very terminal stages of T cell maturation. The T cells derived from both the immature CD2^-/low (Fig. 5A) and the mature CD2⁺ T cells after culture with IL-2 (Fig. 5B) did not produce IL-10. In the cultures with IL-12, a minor proportion of IL-10⁺ cells, mostly IL-4^-, was detected. A greater percentage of viable IL-10⁺ T cells, all IFN-^high and distinct from the IL-13⁺ cells, was detected after culture of the CD2⁺ T cells with IL-12 (Fig. 5B). IFN- and IL-18, alone or in combination, neither induced increased proportions nor enhanced the IL-12-mediated induction of IL-10⁺ cells in CD2⁺ T cells (not shown). Most IL-10⁺ cells had light scatter characteristics of dying cells, supporting association between its production and programmed cell death. Thus IL-10, which is neither induced as a result of type 2 responses (reviewed in Ref. 27) nor is expressed in NK (5), and T cells producing only type 2 cytokines, cannot be considered a type 2 cytokine.

View larger version (64K): [in this window] [in a new window]   FIGURE 5. IL-10 production by immature and mature T cells. CD2-/low (A) and CD2+ (B) neonatal lymphocytes were cultured with IL-2 and the combination of CD3 and CD28 mAb for 8 days, and for 5 additional days with IL-2 and anti-TNF- mAb, without or with PHA-P + CD28 mAb and/or IL-12, as indicated. After stimulation, IL-10 and IL-4 (A), IFN- (top three columns), and IL-13 accumulation (bottom three columns) (B) was analyzed within gated CD3+ cells. In B, IL-10 (y-axis) and side angle light scatter (x-axis) were analyzed in cells, including nonviable ones, from cultures with IL-12 (right end plots). Quadrant markers were set to include viable lymphocytes (low side angle light scatter) in the left quadrants. Experiments representative of three with similar results.

IL-13 and IFN- production were analyzed in T cells (99.9% CD2⁺) in freshly purified lymphocytes after a 5-day culture with IL-2 without or with monokines alone (Fig. 6) in the presence of mimosine to prevent cell cycle progression. In the cultures with IL-12- and TCR-mediated stimulation used as control, and more so in those with IL-12 and monokines, the numbers of IL-13⁺ T cells decreased, IL-13⁺IFN-⁺ cells were generated, and both proportions and numbers of the IFN-⁺ cells increased. These were greatest in the cultures with TCR-mediated stimulation without mimosine. These results are identical with those obtained using CD2^-/low T cells as the starting population, indicating that the monokine combination suffices to induce most, if not all, peripheral IL-13⁺ (type 2) T cells to differentiate to IFN-⁺ (type 1) cells.

View larger version (32K): [in this window] [in a new window]   FIGURE 6. Monokine-induced differentiation of peripheral blood IL-13+ T cells. Cytokine production was induced in neonatal lymphocytes freshly purified (day 0), or after a 5-day culture with IL-2 and anti-TNF- mAb, with or without IFN-, IL-12, and IL-18 with mimosine added (second and third panel) or with added PHA and CD28 mAb without mimosine (right panel), as indicated. Intracellular accumulation of IFN- and IL-13 was analyzed within gated CD3+ cells (99.9% CD2+) as in Fig. 1. Numbers in each quadrant are percentages and, in parentheses, absolute number (x105) of positive cells. Experiment representative of three with similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Neonatal and adult blood CD2^-/low T cells are immature T cells. Like CD2^- thymocytes, they expressed CD2 in short-term culture under conditions (IL-4) supporting proliferation of immature lymphocytes and contained lymphocyte populations identical with those in the thymus. A high percentage of the CD4⁺ cells, all CD2^-, was CD4^+/dim, like developing cells; the same population was not generated in similar cultures of CD2⁺ T cells from the same individual (not shown); and the proportions of CD8⁺ T cells were much lower than those in peripheral blood. Preliminary evidence also indicates that the DN CD2^-/low T cells are CD45RA⁺CD45RO⁺, like cortical thymocytes (29), and contain a minor CD5^-CD3⁺ T cell population, consistent with pre-TCR⁺ DN T cells (30, 31). Furthermore, CD4⁺CD8⁺ and CD4^+/lowCD8^- cells are generated from these cells in cultures with CD3-mediated stimuli (our unpublished data). Also, consistent with the notion that CD8⁺ cells derive from CD4^+/dim cells in the thymus (31, 32), SP CD8⁺ T cells can be generated in the same cultures only when a relevant proportion of CD4^+/dim cells is present.

CD3 was functional on the immature cells to mediate signal transduction events for cytokine production (not shown) and redirected cytotoxicity. Only cells cultured with PHA, however, mediated significant cytotoxicity levels, indicating that cytokine production and cytotoxicity are developmentally uncoupled and that TCR-mediated priming in the immature cells is most likely needed to increase the levels of cytotoxic mediators.

Consistent with the expression of GATA-3 in most cells, nearly all immature peripheral T cells produced IL-13. GATA-3 is expressed in CD34⁺ hemopoietic progenitor cells (16, 33) and triple-negative thymocytes (17, 34), plays a role in the proliferation of immature lymphocytes (18), and is required for T cell development (18, 19). This suffices to exclude that its expression was induced de novo in previously negative cells under the culture conditions used. The observation that this transcription factor is expressed in all immature IL-13⁺ CD161⁺ human NK cells (5) provides a precedent for and an analogy with our findings in the immature T cells, and refutes the common belief that GATA-3 serves as master switch gene for differentiation of type 0 to type 2 cells upon expression in previously negative cells.

Primary culture of the immature CD2^-/low lymphocytes resulted in acquisition of CD2 expression. In the absence of the proliferative stimulus provided by IL-4, the cultures contained lower numbers of IL-13⁺ T cells and increased numbers of IL-13⁺IFN-⁺ and mature IFN-⁺ cells. In this case, like in the cultures of the CD2^-/low cells with IL-12, low levels of IL-2 were produced, concomitant with loss of IL-4, by the IL-13⁺IL-4⁺ T cells, and only these cells transiently expressed the highest IL-2 levels upon acquisition of the intermediate phenotype reminiscent of type 0 cells (35). The loss of IL-4⁺ and IL-5⁺ T cells from the CD2^-/low T cells upon culture with IL-12, with or without TCR-mediated stimulation, was more rapid than that of the IL-13⁺ cells. IL-4, but not IL-13, production is regulated by c-Maf (36, 37). Interestingly, c-Maf has been implicated in the differentiation of erythroid and myeloid cells (38, 39) via a mechanism related to the formation of c-Maf/c-myb complexes (39) and to other yet to be identified effects. The possibility that similar mechanisms are involved in regulating T cell differentiation is not excluded. Loss of ability to produce IL-4 may correspond more closely to the transition of T cells to terminal differentiation, whereby c-Maf expression is lost before that of GATA-3, which regulates primarily proliferation of immature hemopoietic cells (18). This possibility is supported by the observation that IFN- is detected in the IL-13⁺ cells only when IL-4 production is almost completely waned, but both GATA-3 and IL-13 are still expressed. Analysis of c-Maf expression in the cells defined in this study will allow testing this hypothesis. Consistent with the role of GATA-3 in proliferation is also the fact that the proliferative potential of both IL-13⁺ NK and T cells is greater than that of the IFN-⁺ and overall NK and T cell populations and that their accumulation depends exclusively on it (5). The loss of IL-4⁺ and IL-5⁺ T cells from the CD2^-/low T cells upon culture with IL-12 with or without TCR-mediated stimulation was more rapid than that of the IL-13⁺ cells, indicating a progressive loss of ability to produce type 2 cytokines during maturation.

IFN-, although inhibiting proliferation of most T cells (unpublished data), allowed generation of a greater proportion of IL-2⁺IL-13⁺IFN-⁺ cells by preventing the IL-12-induced loss of IL-2 production as IFN- expression is induced. Thus, if produced early during an immune response before Ag recognition takes place, IFN- may prime immature T cells for IL-2 production, allowing them to progress along their maturation pathway. The withdrawal of the inhibitory effect of IFN- on lymphocyte proliferation as its levels wane in the responding T cells upon Ag recognition would then allow them to produce IL-2 to support their own proliferation and that of other bystander type 2 and type 0 cells. This response is transient because, upon IL-12-induced progression to the mature type 1 phenotype, the cells lose the ability to both produce IL-2 and to proliferate. The kinetics of monokine production by dendritic cells (DC), the control of their production by IFN-, and the fact that distinct stimuli may regulate production of different cytokines/chemokines by DC and monocytes/macrophages (33, 40, 41, 42) make the above scenario likely and important to be carefully dissected. IFN- simultaneously induces IL-15 production by monocytes (43) and primes T cells for IL-2 production; the timing of IFN- and IL-15 production could likewise modulate T cell proliferation. The same mechanism may operate to sustain, in a bystander fashion, the increased lymphocyte numbers in viral infections (44), in which plasmacytoid DC are expected to produce high IFN- levels (45), and may explain the beneficial role of IFN- in therapeutic attempts to promote T cell activation and expansion (46).

The fact that the monokine combination IL-15 + IL-18 + IFN- alone can substitute TCR-mediated stimulation in the presence of IL-12 to induce terminal differentiation of both immature IL-13⁺ T cells and primary IL-13⁺ PBLs (containing CD2⁺ T cells and proportions of CD2^-/low cells below the detection limit of the commonly used techniques) to effector IFN-⁺ T cells establishes the relevance of this maturation process to all peripheral T cells and needs to be considered in vaccine formulations. Potential adjuvants inducing IL-12 production by accessory and APC may be efficient because, inducing also IL-18 production, they affect maturation of both Ag-specific and bystander T cells. This is a likely scenario because: accessory cells, including monocytes/macrophages and DC, produce IL-12, IL-15, and IFN-, and the latter, produced with faster kinetics than IL-12, can induce IL-15 and IL-18 production (43).

Mature IFN-⁺ NK (5) and T cells (this study) are induced by IL-12 to produce IL-10 as they die. Release of TGF- has also been reported by T cells at this terminal, apoptotic stage (24), which is induced rapidly following activation upon TCR/CD3 engagement. Thus, the positive feedback between IL-12 and IFN- (47) eventually leads to activation- and/or death receptor-induced apoptosis of the mature Ag-specific T cells and to the release of anti-inflammatory cytokines as they die, possibly reflecting an active attempt to down-regulate a predominantly type 1 immune response. Based on their known major functions, it is likely that IL-10 acts primarily to suppress production of inflammatory (TNF-, IL-1) and type 1 cytokines (IL-12 and IL-18) by myeloid cells (48), while TGF- functions to suppress proliferation of more immature lymphocytes (49), preventing an increase in the pool of the cells that could terminally differentiate to type 1 responders.

Compared with the total and the CD2⁺ cells, greater proportions of the CD2^-/low T cells were type 2 cytokine⁺, suggesting that ability to produce these cytokines is acquired relatively early during T cell development. Like the cells derived from the immature T cells early after IL-12 stimulation, most naive peripheral T cells produce low to high IFN- levels, but not type 2 cytokines. This is consistent with the hypothesis that at least part of the type 2 cytokine⁺ T cells in peripheral blood are immature T cells that exited the thymus at the beginning of their terminal stage of differentiation. IL-4 and IL-12 have effects on early thymocytes similar to those discussed in this study on immature peripheral T cells (15), and the same differentiation sequence can be induced in thymic lymphocytes (31, 50). The hypothesis that intrathymic maturation, possibly promoted by, but not necessarily associated with the process of positive and negative selection, may obey the same rules discussed above for the differentiation of immature peripheral T cells, and the role played in the process by TCR engagement by self Ag deserve analysis. The possibility should also be considered that thymus-independent development, or dysregulated differentiation of these immature T cells may participate in autoimmune situations.

The similarity between human immature NK and T cells at their latest developmental stages is striking. Also, accumulation of both CD4⁺ and CD8⁺ type 2 cytokine⁺ T cells upon culture of lymphocytes in vitro under type 2-polarizing conditions depends exclusively, and irrespective of TCR-mediated stimulation, on proliferation of pre-existing peripheral type 2 cytokine⁺ cells (5).⁴ This, and the results reported in this study with T cell clones with intermediate phenotype and the primary T cells strongly support the conclusion that most T cells undergo the pathway of differentiation described in this work for the immature peripheral T cells.

The cumulative data stand against an interpretation of the type 1-type 2 paradigm based on TCR-induced de novo differentiation of type 0 T cells to either type 2 or type 1 cells, proposed for both helper and cytotoxic T cells. Rather, they support the alternative interpretation that, analogous to NK cells (5), type 2 T cells are precursors to type 1 cells. Proliferation-dependent accumulation of type 2 T cells occurs under type 2-polarizing conditions, whereas their terminal differentiation to mature IFN-⁺ T cells is induced by monokines, including IL-12, that allow/induce progression through an intermediate type 2 cytokine^+/low/IFN-^+/low (type 0) differentiation stage (Fig. 7). This process is independent from, although it can be modulated by, TCR-mediated stimulation. In this scenario, efficient cell-mediated immune responses involving IFN- depend on the monokines produced by the pathogen-stimulated accessory cells and on the possibility of both relatively mature (IFN-^low) and immature (type 2 cytokine⁺, CD2^-/low and CD2⁺) Ag-specific T cells to undergo differentiation in response to them. IL-12 would induce terminal maturation of the relatively mature T cells that become responsive to it upon Ag-mediated TCR-induced expression of functional IL-12R, activation of their effector functions, and eventually TCR-mediated activation-induced death of the effector cells. Concomitantly, increased numbers of effector cells can be recruited upon differentiation of the immature Ag-specific T cells induced by IL-12 and the other monokines consequently produced by the APC. Timing of stimulation and Ag concentration may not allow complete maturation of all Ag-specific IL-2⁺ type 0 cells, part of which would remain as memory cells after Ag elimination. The type 1 response would terminate as soon as sufficient levels of IL-10 and TGF- are produced by the effector cells undergoing apoptosis. Differentiation of bystander immature T cell would lead also, temporarily, to accumulation of the type 0 (IL-2⁺) and eventually IFN-⁺ Ag-nonspecific T cells that may contribute inflammatory effector functions. Accumulation of type 2 cells, responsible for allergic and autoimmune pathologies, would instead occur only under conditions in which their differentiation is dysregulated.

View larger version (32K): [in this window] [in a new window]   FIGURE 7. Developmental stages in peripheral immature CD2- T cells. The developmental sequence of T cells during the IL-12-dependent maturation process is summarized. Immature CD2-/low IL-13+IL-5+IL-4+ (type 2) T cells undergo terminal differentiation to IFN-+ (type 1) cells through the intermediate IL-13+IL-2+IFN-+ (type 0) stages. The relative proportions of IL-2+, IL-4+, IL-5+, IL-13+, IFN-+, and IL-10+ cells at each stage are indicated by the height of the bars. Indicated are also: the factors (IL-2, others) that maintain T cell survival/proliferation; those (monokines, e.g., IL-12) allowing terminal differentiation (curved arrow); those (e.g., TCR:Ag interaction) that, although not required, may participate (– – –) in it, and the stages at which CD2, functional IL-12R, and death receptors are expressed. ?, Lack of direct information on the exact timing of expression. The contribution of additional factors is not excluded.

	Acknowledgments

 
We thank Dr. M. Colombo and T. Manser for critically reading the manuscript; C. Croce for continuous support; Dr. V. Berghella and the staff of the Obstetrics and Gynecology Division, Thomas Jefferson Hospital, for providing the umbilical cord blood samples; and P. Hallberg and J. Faust for assistance with flow cytometry.

	Footnotes

 
¹ Supported, in part, by U.S. Public Health Service Grants CA45284, CA77401 (to B.P.), and T32-CA09683 (to M.J.L.).

² Address correspondence and reprint requests to Dr. Bice Perussia, Thomas Jefferson University, KCC, BLSB 750, 233 South 10th Street, Philadelphia, PA 19107. E-mail address: Bice.Perussia{at}mail.tju.edu

³ Abbreviations used in this paper: SP, single positive; AET, 2-aminoethylisothiouronium bromide; DC, dendritic cell; DN, double negative; DP, double positive; PE-TR, PE-Texas Red; SEC, Staphylococcus aureus enterotoxin C; SEE, Staphylococcus aureus enterotoxin E.

⁴ M. J. Loza and B. Perussia. Accumulation of type 2 cytokine⁺ T cells depends on differentiation-independnet proliferation of pre-existing type 2 T cells. Submitted for publication.

Received for publication May 9, 2002.

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