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The Journal of Immunology, 2002, 168: 1113-1122.
Copyright © 2002 by The American Association of Immunologists

CD2 Facilitates Differentiation of CD4 Th Cells Without Affecting Th1/Th2 Polarization1

Tetsuro Sasada, Hailin Yang and Ellis L. Reinherz2

Laboratory of Immunobiology, Dana-Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of CD2 in murine CD4 helper T cell differentiation and polarization was examined using TCR-Cyt-5CC7-I transgenic recombination activating gene-2-/- H-2a mice on CD2+/+ or CD2-/- backgrounds. In the absence of CD2, thymic development was abnormal as judged by reduction in the steady state number of total, double-positive, and CD4 single-positive (SP) thymocytes, as well as a defect in their restorative dynamics after peptide-induced negative selection in vivo. In addition, in CD2-/- animals, lymph node CD4 SP T cells manifest a 10- to 100-fold attenuated activation response to cytochrome c (CytC) agonist peptides as judged by induction of CD25 and CD69 cell surface expression or [3H]TdR incorporation; differences in the magnitude of responsiveness and requisite molar peptide concentrations were even greater for altered peptide ligands. Although the presence or absence of CD2 did not impact the final Th1 or Th2 polarization outcome, CD2 expression reduced the CytC peptide concentration threshold necessary to facilitate both Th1 and Th2 differentiation. In vivo administration of CytC peptide to CD2-/- animals yielded an impaired CD4 SP T cell effector/memory phenotype compared with similarly treated CD2+/+ mice. Analysis of TCR-Cyt-5CC7-I human CD2 double-transgenic mice similarly failed to reveal a preferential Th1 vs Th2 polarization. Collectively, these results indicate that CD2 is important for the efficient development of CD4 SP thymocytes and TCR-dependent activation of mature CD4 lymph node T cells, but does not direct a particular helper T cell subset polarity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tcell differentiation and activation are highly orchestrated processes that begin with the specific recognition by TCRs (3) of peptides associated with major histocompatibility complex molecules (pMHC)3. Because the monomeric binding affinity between a TCR and a pMHC ligand is low with a relatively fast off-rate (1), the specificity and sensitivity of this recognition process is fine-tuned by a number of costimulatory and accessory T cell surface molecules including CD2, CD4, CD8, CD28, and LFA-1 (2, 3, 4, 5) among others.

Naive CD4+ T cells can differentiate into Th type 1 cells (Th1) which secrete IL-2, IFN-{gamma}, and lymphotoxin or Th type 2 cells (Th2), which produce IL-4, IL-5, and IL-10. Th1 cells play a role in proinflammatory cellular immunity to intracellular pathogens, whereas Th2 cells mediate humoral immunity against extracellular parasites (6, 7). The selective differentiation of either subset can be significantly influenced by a variety of factors, including cytokine environment, APCs, strength of TCR signaling, and costimulation (6, 8). IFN-{gamma} and IL-12 are thought to be the major cytokines promoting Th1 differentiation; IL-12 directly augments Th1 differentiation while IFN-{gamma} may inhibit naive T cell differentiation into Th2 cells. In contrast, IL-4 is the major cytokine contributing to Th2 differentiation (reviewed in Refs. 9 and 10). The in vivo sources of relevant cytokines are thought to be various APCs or alternatively, the activated T cells themselves. For example, different subsets of dendritic cells may regulate Th1 and Th2 differentiation (11, 12), and B cells can also be classified into Be1 and Be2 populations, which influence Th differentiation (13). In addition to these factors, TCR signals are also important for Th1 and Th2 polarization (reviewed in Refs. 6 and 8). In vitro differentiation experiments demonstrate that, in general, higher doses of specific Ag skew naive T cells to Th1 cells while lower doses of Ag produce Th2 cells.

The transmembrane T cell surface protein CD2 is an adhesion molecule with costimulatory/accessory activity expressed on virtually all T cells, thymocytes, and NK cells (5). CD2 promotes the physical interaction of T and NK lineage cells with APCs, stromal elements, and a variety of target cells bearing the ubiquitously expressed human CD2 ligand, CD58 (2, 5, 14, 15). CD2 initiates T cell/APC contact even before TCR recognition of pMHC complex (5, 16, 17). Moreover, the extremely fast on- and off-rates of the CD2-CD58 interaction (18) enable a TCR to scan APC surfaces searching for the specific agonistic pMHC complex. Although the monomeric CD2-CD58 Kd is weak (see references in Ref. 19), relatively strong binding of CD2-CD58 can be achieved by 2D clustering at cell-cell junctions (16, 20). CD2 functions to mediate T cell adhesion as well as to facilitate signal transduction (21). Human CD2 signaling serves to augment T cell activation (16), enhances the IL-12 responsiveness of activated T cells (22), and reverses T cell anergy (23). In the mouse, the CD2 ligand is CD48, a structurally related ancestor of CD58 with likewise rapid binding kinetic parameters (Ref. 19 and references therein).

Initially, CD2 was thought to be dispensable in the murine system because no specific immune phenotype was observed in CD2-deficient mice generated by targeted mutagenesis (24). However, double knockouts involving either LFA-1 (25) or CD28 (26) plus CD2 showed profound defects in mature peripheral T cell functions, including proliferation induced by either specific pMHC or anti-CD3 stimulation. These findings suggest that the adhesion/costimulatory receptors CD2, LFA-1, and CD28 collectively coordinate T cell activation, and the absence of a single molecule could be reasonably compensated for by the remaining pair of receptors in polyclonal T cell populations. Recently, we reported that when CD2-/- mice were bred with N15 TCR-transgenic (tg) recombination activating gene-2 (RAG-2-/-) H-2b mice, there were dramatic abnormalities in the resultant N15tg RAG-2-/-CD2-/- H-2b animals (27). Thymocyte development was blocked at the DN stage, especially in mice older than 6 wk of age, as a result of defective preTCR signaling. Responsiveness of the mature CD8 T cells bearing this vesicular stomatitis virus octapeptide/H-2Kb-restricted TCR was diminished. In particular, peripheral mature T cells showed reduced proliferation and IFN-{gamma}production upon Ag (vesicular stomatitis virus peptide) stimulation, consistent with the analyses of mature T cell responses in HY TCR-tg CD2-/- or 2C TCR-tg CD2-/- mice (28). Moreover, the T cell repertoire was altered in non-tg C57BL/6 CD2-/- with a striking difference in TCR V{alpha} gene usage. To investigate the function of CD2 in CD4 T cells, we compared CD4 T cell function among CD2 wild-type (wt) and CD2 knockout animals bearing the TCR-Cyt–5CC7-I (5CC7) TCR specific for a pigeon cytochrome c (PCC) peptide presented by I-Ek and on a RAG-2-/- background. We observed impaired maturation of CD4 single-positive (SP) thymocytes, as well as defective activation and differentiation of T helper cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

5CC7 TCRtg RAG-2-/- H-2a (5CC7tg CD2+/+) were purchased from Taconic Farms (Germantown, NY). To create the 5CC7 TCRtg RAG-2-/- CD2-/- H-2a (5CC7tg CD2-/-) mice, the 5CC7 TCRtg RAG-2-/- H-2a (5CC7tg CD2+/+) mice were bred with N15 TCRtg RAG-2-/- CD2-/- H-2b which were generated as previously described (27). The heterozygous 5CC7 TCR+/- N15 TCR+/- CD2+/- H-2a/b RAG-2-/- F1 mice were intercrossed and offspring which were 5CC7 TCR+ N15 TCR- CD2-/- RAG-2-/- H-2a were selected (hereafter referred to as 5CC7 tg CD2-/-). The expression of the 5CC7 TCR, H-2Kk, and H-2Ek, as well as lack of RAG-2, N15 TCR, H-2b, or CD2 gene expression was confirmed based on the FACS analysis of peripheral blood cells. To create 5CC7 TCRtg human CD2 tg (5CC7tg hCD2+) mice, 5CC7 TCRtg+/+ RAG-2-/- H-2a (5CC7tg) mice were bred with C57BL/6, hCD2tg+/- heterozygous mice (29, 30) and F1 mice (5CC7 TCR+/- mCD2+/+ hCD2+/- H-2a/b RAG-2+/-) were used in the following experiments. All lines were maintained and bred under sterile barrier conditions at the animal facility of Dana-Farber Cancer Institute (Boston, MA).

Peptide synthesis

PCC wt 17-mer peptide (aa 88–104, KAERADLIAYLKQATAK), PCC wt 14-mer peptide (aa 91–104, RADLIAYLKQATAK), and variants of the PCC wt 14-mer peptide were synthesized by standard solid phase methods on an Applied Biosystems 430A synthesizer (Foster City, CA) at the Biopolymers Laboratory of Massachusetts Institute of Technology (Cambridge, MA). All peptides were purified by reverse phase HPLC (Hewlett Packard HPLC 1100, Palo Alto, CA) with a 2-mm C4 column. Peptides were analyzed for purity and correct molecular composition by electrospray mass spectrometry, amino acid analysis, and HPLC. The PCC 14-mer variant peptides are named to indicate the substituted amino acid and the position in the original PCC sequence (e.g., 97F denotes replacement of tyrosine by phenylalanine at the seventh residue of the PCC wt 14-mer peptide).

Abs and flow cytometric analysis

The following mAbs and staining reagents were used: PE-conjugated anti-CD2 (RM2-5), PE- or CyChrome-conjugated anti-CD4 (RM4-5), FITC- or CyChrome-conjugated anti-CD8{alpha} (53-6.7), FITC-conjugated anti-V{alpha}11.1, 11.2 (RR8-1), FITC-conjugated anti-IFN-{gamma} (XMG1.2), PE-conjugated anti-IL-4 (11B11), PE-conjugated anti-CD25 (PC61), FITC-conjugated anti-CD44 (IM7), FITC-conjugated anti-CD62 ligand (CD62L) (MEL-14), biotin-conjugated anti-CD45RB (16A), biotin-conjugated anti-CD69 (H1.2F3) (BD PharMingen, San Diego, CA), and FITC-conjugated streptavidin (Life Technologies, Grand Island, NY). For flow cytometry, single-cell suspensions of thymocytes, or lymph node cells were double- or triple-stained at 5 x 106 cells/ml in PBS-2% FCS containing the Abs at saturating concentrations according to standard procedures. Phenotypes and proportions of each subpopulation were analyzed on a FACScan (BD Biosciences, Mountain View, CA) using the CellQuest program. Dead cells were excluded from the analysis by forward and side scatter gating.

Proliferation assay

Lymph node cells from 5CC7tg CD2+/+ or 5CC7tg CD2-/- mice (1 x 105/well) were incubated at 37°C with 2 x 104 irradiated B lymphoma cells, CH27, which had been preloaded for 2 h with the indicated doses of PCC and its variant peptides in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% FCS and 50 µM 2-ME. After 48 h incubation, 0.4 µCi per well of [3H]TdR (ICN Biomedicals, Aurora, OH) was added, and after an additional 18 h of culture at 37°C, the cells were harvested and the incorporated radioactivity was measured.

In vitro differentiation of naive T cells and intracellular cytokine staining

To induce differentiation of naive T cells, lymph node cells from 5CC7tg CD2+/+ or 5CC7tg CD2-/- mice (5 x 105/well) were incubated at 37°C with 1 x 105 irradiated CH27 cells, which had been preloaded for 2 h with the indicated doses of PCC and its variant peptides, in RPMI 1640 medium containing 10% FCS and 50 µM 2-ME in the absence or presence of 100 U/ml rIL-2 or 10 ng/ml rIL-4. After 4 days of priming, T cells were harvested and counted. The primed T cells (1 x 106/well) were then restimulated for 5 h at 37°C with 2 x 105 CH27 cells preloaded with 10 µM PCC wt 17-mer peptide in the presence of 1 µl/ml Golgi-plug (BD PharMingen).

To induce differentiation of naive 5CC7tg hCD2+tg T cells, the lymphocytes from lymph nodes of 5CC7tg hCD2+tg or 5CC7tg hCD2-tg littermates were purified using the StemSep Murine CD4+ T cell enrichment kit (StemCell Technologies Vancouver, Canada). Ninety-six percent of the resultant T cells were CD4+/hCD2+. A total of 5 x 105 purified T cells/well were cultured for 5 days with 1.5 x 105 irradiated I-Ek/CD58+ transfected DCEK (DCEK58; originally generated by Dr. R. Germain, National Institute of Health, Bethesda, MD) loaded with indicated concentration of peptide. The same protocol as above was followed for restimulation.

Intracellular staining for cytokines was performed according to BD PharMingen’s protocol. Briefly, T cells were harvested, stained with CyChrome anti-CD4 mAb for 30 min at 4°C, and then fixed/permeabilized with Cytofix/Cytoperm solution (BD PharMingen). Cells were double-stained with FITC-conjugated anti-IFN-{gamma} and PE-conjugated anti-IL-4 mAbs and fixed CD4-positive cells were gated and analyzed on a FACScan using the CellQuest program. Nonstimulated cells (negative control) were <1% positive for IFN-{gamma} and IL-4.

Measurement of IFN-{gamma} production

IFN-{gamma} production was induced under the same culture conditions used for proliferation assays (see above). After 48 h of incubation, supernatants were collected and assayed for IFN-{gamma}, using a mouse IFN-{gamma} ELISA kit (Mouse IFN-{gamma} OptEIA Set, BD PharMingen). The sensitivity of the assay was 31.3–2000 pg/ml for IFN-{gamma}, and results were calculated as the mean of duplicate wells.

RNase protection assay

Five-hour restimulated 5CC7tg hCD2+tg or 5CC7tg hCD2-tg T cells (with CH27) were harvested and RNA was purified using the RNeasy mini kit (Qiagen, Valencia, CA). Four micrograms of RNA was used in RNase protection assays with a RiboQuant kit and mCK-1 template sets according to the manufacturer’s protocol (BD PharMingen). Equivalent RNA loading was monitored by L32 and GAPDH probes within the kit. Differences in cytokine RNA production by RNase protection was quantified visually and by ImageQuant version 1.1 (Molecular Dynamics, Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Defective maturation of thymocytes in 5CC7tg CD2-deficient mice

To analyze the role of CD2 in thymocyte development and mature T cell function in MHC class II-restricted TCR-expressing T cells in vivo, we established 5CC7tg RAG-2-/-CD2-/-H-2a (hereafter referred to as 5CC7tg CD2-/-, for simplicity) mice that express the 5CC7 MHC class II-restricted TCR specific for a PCC peptide bound to I-Ek. T lineage cells from the thymus and lymph nodes of these animals were then directly compared with 5CC7tg RAG-2-/- CD2+/+H-2a (5CC7tg CD2+/+) mice. As expected, CD4+ SP cells from the 5CC7tg CD2-/- lymph nodes lack surface CD2 expression (Fig. 1GoA). However, as defined by the anti-V{alpha}11 (Fig. 1GoA) and anti-V{beta}3 mAbs (data not shown), these lymph node T cells express the same 5CC7 TCR level as lymph node T cells derived from 5CC7tg CD2+/+ mice. Furthermore, the surface density of CD4 coreceptor as enumerated by the anti-CD4 mAb RM4–5 is equivalent.



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  		FIGURE 1. Quantitative and qualitative T lineage abnormalities in 5CC7tg RAG-2-/- CD2-deficient mice. A, CD2 and TCR expression on lymph node T cells of 5CC7tg RAG-2-/- CD2+/+ and 5CC7tg RAG-2-/- CD2-/- H-2a mice. Lymph node cells from 8-wk-old 5CC7tg RAG-2-/- CD2+/+ and 5CC7tg RAG-2-/- CD2-/- mice were triple-stained with FITC-anti-V{alpha}11, PE-anti-CD2, and CyChrome-anti-CD4. The dot-plot profiles of CD2 (x-axis) vs CD4 (y-axis) expression in 10,000 live cells and histograms of V{alpha}11 on CD4+ lymph node cells are shown. The percentage of cells in each subset quadrant is indicated. B, Thymocyte numbers are reduced in 5CC7tg RAG-2-/- CD2-deficient mice. The total thymocyte and CD4+ lymph node cell numbers from 7- to 8-wk-old 5CC7tg RAG-2-/- CD2+/+ and 5CC7tg RAG-2-/- CD2-/- mice are enumerated. {circ}, 5CC7tg RAG-2-/- CD2+/+ mice (n = 9); •, 5CC7tg RAG-2-/- CD2-/- mice (n = 11). {circ} and •, Values of individual mice and bars represent average values for a given group. The thymocyte numbers of 5CC7tg RAG-2-/- CD2-/- mice are significantly lower than those of 5CC7tg RAG-2-/- CD2+/+ mice (p < 0.0005) without significant differences in their respective numbers of CD4+ lymph node T cells (p = 0.32). C, Thymocyte development is impaired by the lack of CD2. The thymocytes from 5CC7tg RAG-2-/- CD2+/+ (left panel) and 5CC7tg RAG-2-/- CD2-/- (right panel) mice at 8 wk of age were double-stained with PE-anti-CD4 and CyChrome-anti-CD8{alpha}. The expression of CD4 (y-axis) and CD8{alpha} (x-axis) on thymocytes was detected by flow cytometry after gating on 10,000 live cells. The percentages of each subset are indicated. Results are representative of 9–11 experiments. D, A reduction in the numbers of DP and CD4+ SP thymocytes in 5CC7tg RAG-2-/- CD2-deficient mice. The numbers of DP, CD4+ SP, CD8+ SP, and DN thymocytes were calculated by quantifying the total numbers of thymocytes from 7- to 8-wk-old 5CytCC7tg RAG-2-/- CD2-/- or CD2+/+ mice and the percentages of each subset as determined by FACS analysis. {circ}, 5CC7tg RAG-2-/- CD2+/+ mice (n = 9); •, 5CC7tg RAG-2-/- CD2-/- mice (n = 11). {circ} and •, Values of individual mice and bars represent average values for a given group. The DP and CD4+ SP thymocyte numbers of 5CC7tg RAG-2-/- CD2-/- mice are significantly lower than those of 5CC7tg RAG-2-/- CD2+/+ mice (DP, p < 0.002; CD4+ SP, p < 0.00005). Statistical analysis involved a one-sided Student t test in all cases.

 
Fig. 1GoB shows that the number of peripheral CD4+ lymph node T cells is comparable in both strains (5CC7tg CD2+/+, 15.6 ± 5.3 x 106 (n = 9) vs 5CC7tg CD2-/-, 13.0 ± 7.0 x 106 (n = 11), p = 0.32). However, comparison of the thymocyte cell number and subset distribution between the 5CC7tg CD2+/+ and 5CC7tg CD2-/- mice reveals substantial differences. At 7–8 wk of age, 5CC7tg CD2-/- mice have a smaller thymus with a significant reduction in the number of thymocytes (5CC7tg CD2+/+, 60.9 ± 17.3 x 106 (n = 9) vs 5CC7tg CD2-/-, 28.4 ± 16.0 x 106 (n = 11), p < 0.0005) (Fig. 1GoB). Furthermore, as shown by the CD4/CD8{alpha} double-staining profiles in Fig. 1GoC, there is a decrease in percentage of CD4+ SP subset and an increase in double-negative (DN) and CD8+ SP subset in 5CC7tg CD2-/- thymocytes, compared with 5CC7tg CD2+/+ mice. Although the total number of 5CC7tg CD2-/- thymocytes is about half of the control 5CC7tg CD2+/+ thymocytes, 5CC7tg CD2-/- mice show no significant differences in the absolute numbers of DN and CD8+ SP thymocytes, but ~2-fold less double-positive (DP) and ~4-fold less CD4+ SP thymocytes than 5CC7tg CD2+/+ mice (Fig. 1GoD). These results indicate that the absence of CD2 influences intrathymic development in MHC class II-restricted 5CC7tg mice, as we have previously shown in the MHC class I-restricted N15tg mice (27).

To further clarify the defect in T cell development in 5CC7tg CD2-deficient mice, we injected the cognate peptide PCC i.v. and examined the phenotypic changes of thymocytes at subsequent time intervals. As shown in Fig. 2GoA, 2 days after i.v. injection with 20 µg of PCC wt 17-mer peptide, the majority of DP thymocytes were deleted by an Ag-dependent cell death mechanism in both 5CC7tg CD2+/+ and 5CC7tg CD2-/- mice, suggesting that CD2-deficiency does not affect the sensitivity of immature DP thymocytes to negative selection induced by the cognate peptide. Moreover, 10 days after PCC injection, 5CC7tg CD2+/+ mice regenerated DP thymocytes efficiently, thereby restoring the total thymic cellularity (Fig. 2Go, A and B). By contrast, 5CC7tg CD2-/- mice exhibited a severe defect in T cell differentiation at the DN stage and remained abnormal in phenotype with a striking reduction of DP and CD4+ SP cells: thymocytes from 5CC7tg CD2-/- mice 10 days after PCC injection are predominantly DN (69%) with 12% CD4+ SP and 9% DP cells (Fig. 2Go, A and B). Collectively, these results clearly show that CD2-deficient mice have a defect in the early T cell development, especially at the transition from DN to DP thymocytes.



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  		FIGURE 2. Aberrant restorative kinetics in thymocytes following peptide-induced negative selection in 5CC7tg RAG-2-/- CD2-deficient mice. Six- to 8-wk-old 5CC7tg RAG-2-/- CD2+/+ and 5CC7tg RAG-2-/- CD2-/- mice were injected i.v. with 20 µg of PCC wt 17-mer peptide in 0.1 ml PBS. Two, 5, or 10 days later, thymocytes were counted and analyzed by flow cytometry for expression of CD4 and CD8{alpha}. A, The profiles of CD4 (y-axis) vs CD8{alpha} (x-axis) expression in 10,000 live cells 2 or 10 days after PCC injection are shown. The percentages of each subset are indicated. The result is representative of five to six independent experiments. B, The total number of thymocytes is shown at different time points. Each point represents mean ± SD of five animals. Two days after injection, there was no difference in the CD4/CD8{alpha} subset distribution of thymocytes from 5CC7tg RAG-2-/- CD2+/+ and 5CC7tg RAG-2-/- CD2-/- mice (p = 0.19). By contrast, 5 and 10 days after PCC treatment, the 5CC7tg RAG-2-/- CD2-/- mice showed a defect in the efficiency of generation of DP and CD4+ SP thymocytes (5 days, p < 0.0005; 10 days, p < 0.02).

 
Impaired Ag-specific activation and proliferation of naive T cells in 5CC7tg CD2-deficient mice

To next examine the role of CD2 in mature CD4+ murine T cell function, we tested the Ag responsiveness of CD2-deficient mice by culturing lymph node T cells with PCC cognate peptide or PCC variants in vitro. Peptide variants of PCC were made by substituting amino acid residues at the dominant (positions 99 and 102) and subdominant (position 97) TCR contact residues; such substitution is known to produce altered T cell responses (31, 32). To first address whether CD2-deficiency affects early phases of naive T cell activation, we assessed expression of the cell surface activation-associated IL-2R{alpha} (CD25) and CD69 molecules which represent reliable indicators of the number of T cells that are activated in tissue culture (33, 34). For this purpose, lymph node T cells from 5CC7tg CD2+/+ and 5CC7tg CD2-/- mice were stimulated in vitro for 18 h with varying molar concentrations of the PCC wt peptides (17-mer and 14-mer) or other altered peptide ligand (APL) variants, 97F, 99R, 102S, and 102A, using irradiated CH27 cells as I-Ek-bearing APC. CD25 and CD69 expression were then assayed by flow cytometry. As shown in Fig. 3GoA, PCC wt 17-mer, wt 14-mer, and 97F peptides were capable of activating the vast majority of T cells present in culture at high peptide concentrations (10-4–10-5 M) in both 5CC7tg CD2+/+ and 5CC7tg CD2-/- mice. However, at lower peptide concentrations (10-7–10-8 M), the percentages of activated T cells as assessed by CD25 and/or CD69 expression induction from 5CC7tg CD2+/+ mice are significantly higher than those from 5CC7tg CD2-/- mice. Similarly, substantial differences are also observed in the activation responses of the 5CC7tg CD2+/+ vs 5CC7tg CD2-/- lymph node T cells after stimulation with other APLs, as assessed by CD25 and CD69 expression. For example, compared with 5CC7tg CD2+/+ lymph node cells, the dose-response curve from 5CC7tg CD2-/- lymph node cells after treatment with 102S peptide shows a reduced sensitivity (i.e., shift to the right) by a factor of 10–100 at concentrations of 10-5–10-7 M. Furthermore, when incubated with CH27 cells prepulsed with the weaker agonists, 99R and 102A, ~25% of 5CC7tg CD2+/+ lymph node cells express CD25 and CD69 using peptide concentrations of 10-4 M. In contrast, 5CC7tg CD2-/- lymph node cells cannot be activated by either 99R or 102A stimulation at any peptide concentration tested (Fig. 3GoA). These findings indicate that in the mature lymphoid compartment, CD2 enables lower molar concentrations of peptide Ags to induce T cell activation as assessed by expression of these cell surface markers.



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  		FIGURE 3. Diminished activation and proliferation upon Ag-triggered stimulation of naive T cells in 5CC7tg RAG-2-/- CD2-deficient mice. A, Lymph node T cells (1 x 105 cells) from 5CC7tg RAG-2-/- CD2+/+ and 5CC7tg RAG-2-/- CD2-/- mice were stimulated in vitro with varying molar concentrations of the PCC wt peptides (17-mer and 14-mer) or APLs, 97F, 99R, 102S, and 102A, using irradiated CH27 cells (2 x 104 cells) as I-Ek-bearing APC. After 18 h of culture, cells were stained with Cychrome-anti-CD4, PE-anti-CD25, and biotin-anti-CD69, followed by FITC-streptavidin. Gated CD4+ cells were analyzed and the percentages of CD25 or CD69 positive cells were determined by flow cytometry. B, Lymph node cells (1 x 105 cells) from 5CC7tg RAG-2-/- CD2+/+ and 5CC7tg RAG-2-/- CD2-/- mice were incubated in vitro with the indicated concentrations of the PCC wt peptides (17-mer or 14-mer), 97F, or 102S, using irradiated CH27 cells (2 x 104 cells) as I-Ek-bearing APC. Forty-eight hours later, [3H]TdR was pulsed for 18 h and T cell proliferation was judged by [3H]TdR incorporation. The mean of duplicate cultures is shown. The result is representative of three to five independent experiments.

 
We next examined Ag-induced proliferative responses of unprimed lymph node T cells from 5CC7tg CD2+/+ and 5CC7tg CD2-/- mice. Although the PCC wt 17-mer peptide stimulated proliferative responses of both 5CC7tg CD2+/+ and 5CC7tg CD2-/- T cells in a dose-dependent manner, the [3H]thymidine incorporation in 5CC7tg CD2-/- lymph node cells is clearly less than in 5CC7tg CD2+/+ lymph node cells at peptide concentrations from 10-5 to 10-7 M (Fig. 3GoB). In addition, compared with 5CC7tg CD2+/+ T cells, 5CC7tg CD2-/- T cells show significantly decreased proliferative responses to stimulation with PCC wt 14-mer or APLs, 97F and 102S. For example, at peptide concentrations of 10-4 and 10-5 M, the responses from 5CC7tg CD2-/- T cells relative to 5CC7 tg CD2+/+ are significantly reduced or eliminated (Fig. 3GoB). Similarly, when we used naive T cells from 5CC7tg hCD2tg mice and I-Ek+/hCD58+ fibroblasts as APC in in vitro proliferation assays, we observed a similar 10- to 100-fold molar peptide concentration shift in hCD2- vs hCD2+ cells (see below and data not shown). Collectively, these results demonstrate that primary responsiveness to antigenic peptides is impaired in the 5CC7tg CD2-/- naive lymph node T cells as determined by Ag-specific induction of CD25/CD69 expression and proliferation in primary culture. CD2 can augment TCR responses to pMHC by a factor of 10–100 fold. Although not shown, addition of exogenous IL-2 fails to restore responsiveness of 5CC7tg CD2-/- T cells to the level observed with 5CC7tg CD2+/+ T cells (data not shown).

Reduced sensitivity of lymph node T cells in 5CC7tg CD2-deficient mice to undergo Th1 and Th2 polarization

To next determine whether CD2 deficiency affects differentiation of CD4+ T cells into Th1- and Th2-like effectors, naive lymph node cells from 5CC7tg CD2+/+ and 5CC7tg CD2-/- mice were stimulated with varying doses of PCC wt peptide or APLs presented on CH27 cells in an initial in vitro culture for 4 days. An equivalent number of surviving T cells from each culture was then restimulated for 5 h with a single dose of PCC wt 17-mer peptide and assayed for the ability to produce Th1 and Th2 cytokines by intracellular IFN-{gamma} and IL-4 double staining. Although the CD4+ T cells from both 5CC7tg CD2+/+ and 5CC7tg CD2-/- mice expressed primarily IFN-{gamma}, but not IL-4, after priming with PCC wt 17-mer peptide, the percentages of IFN-{gamma}-producing cells in 5CC7tg CD2-/- T cells are 3- to 8-fold less than those of 5CC7tg CD2+/+ T cells at peptide concentrations from 10-5–10-8 M (Fig. 4Go, A and B). In the presence of exogenous rIL-2, which is known to promote Th1 responses in CD4+ T cells in in vitro priming culture, both 5CC7tg CD2+/+ and 5CC7tg CD2-/- T cells increased the percentages of IFN-{gamma}-producing cells. Again, compared with 5CC7tg CD2-/- mice, 5CC7tg CD2+/+ produce a greater number of IFN-{gamma}-producing cells, especially at lower peptide concentrations (10-8–10-9 M), (Fig. 4Go, A and B). This result suggests that CD2-deficiency causes a significant defect in the ability of CD4+ T cells to differentiate into Th1 cells.



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  		FIGURE 4. Lymph node CD4+ T cells in 5CC7tg CD2-deficient mice manifest reduced sensitivity to both Th1 and Th2 polarization. Lymph node cells (5 x 105 cells) from 5CC7tg RAG-2-/- CD2+/+ and 5CC7tg RAG-2-/- CD2-/- mice were stimulated in vitro with varying doses of PCC wt 17-mer, wt 14-mer, or 97F peptides presented on CH27 cells (1 x 105 cells) in the presence or absence of 100 U/ml IL-2 or 10 ng/ml IL-4 and cultured for 4 days. An equivalent number of surviving T cells (1 x 106 cells) was restimulated for 5 h with 10 µM PCC wt 17 mer peptide presented on CH27 cells (2 x 105 cells) in the presence of 1 µl/ml Golgi-plug. Cells were fixed/permeabilized and stained with FITC-anti-IFN-{gamma}, PE-anti-IL-4, and CyChrome-anti-CD4. A, The dot-plot profiles of IL-4 (x-axis) vs IFN-{gamma} (y-axis) expression in gated CD4+ cells after in vitro priming with the indicated concentration of PCC wt 17-mer with or without cytokines are shown. The percentages of each subset are indicated. The result is representative of three independent experiments. B, The percentages of IFN-{gamma}- or IL-4-positive cells after in vitro priming with the indicated molar concentration of PCC wt 17-mer in the presence or absence of rIL-2 or rIL-4 are shown. The result is representative of three independent experiments. C, The percentages of IFN-{gamma}-positive cells after in vitro priming with 10 µM PCC wt 14-mer or 97F (left) and IL-4 positive cells after in vitro priming with the indicated doses of PCC wt 14-mer or 97F in the presence of 10 ng/ml rIL-4 (right) are shown. The result is representative of two independent experiments.

 
For purposes of investigating the role of CD2 in Th2 differentiation, CD4+ T cells from 5CC7tg CD2+/+ and 5CC7tg CD2-/- mice were primed with PCC wt 17-mer peptide in the presence of rIL-4 to promote Th2 polarization in in vitro culture (9, 10). Both the 5CC7tg CD2+/+ and 5CC7tg CD2-/- T cells can differentiate into IL-4 producing Th2-like cells in the presence of rIL-4, but the percentages of Th2-polarized cells in 5CC7tg CD2+/+ T cells is significantly higher than those in 5CC7tg CD2-/- T cells, especially at low peptide concentrations (10-8 M) (Fig. 4GoB). Similarly, as shown in Fig. 4GoC, significant differences are observed in both Th1 and Th2 polarization of the 5CC7tg CD2+/+ and 5CC7tg CD2-/- T cells after in vitro priming with PCC wt 14-mer peptide and a weaker agonist, 97F. These findings indicate that CD2 molecules affect differentiation of CD4+ T cells.

Th1 and Th2 differentiation of naive T cells in human CD2/5CC7 double-tg mice

To extend the analysis of the role of CD2 in the differentiation of Th1 and Th2 to the human CD2 molecule, we created 5CC7 TCR-tg human CD2-tg double-tg mice and examined their cytokine production after in vitro differentiation. For this purpose, the fibroblast line DCEK58, which is transfected with class II MHC I-Ek and hCD58 but lacks the expression of other costimulatory molecules such as ICAM-1, VCAM-1, CD48, very late Ag-4, B7-2, Ox-40L, 4-1BBL, LFA-1, and heat-stable Ag (32, 35), was used to stimulate T cells from 5CC7tg hCD2tg+ or 5CC7tg hCD2tg- mice at different concentrations of PCC peptide as indicated in Fig. 5Go. The advantage of using DCEK58 is that the cell line expresses little or no endogenous cytokines while CH27 produces IL-6, IL-10, and to some extent, IL-13 (data not shown). Additionally, there are no costimulatory molecules expressed aside from B7-1 (35). Therefore, DCEK58 is an ideal APC to assess the function of the CD58/CD2 interaction in the absence of other adhesion/costimulatory pairs and without the influence of endogenous cytokines so that the differentiation of T cells to either Th1 or Th2 subsets can be observed based upon TCR signal strength, and CD2-CD58 adhesion and/or costimulation.



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  		FIGURE 5. Differentiation of murine CD4 T cells into Th1 and Th2 effectors in the presence of hCD2. Purified lymphocytes from 5CC7tg hCD2tg+ or 5CC7tg hCD2tg- were cultured with irradiated DCEK58 (I-Ek+, CD58+) pulsed with different concentrations of PCC wt 17-mer peptide for 5 days, and restimulated with a single dose of PCC (10-6 M)-pulsed CH27 cells. A, Intracellular cytokine staining of T cells. The T cells were first surface-stained with cychrome-anti-CD4, then fix/permeabilized and double-stained with PE-anti-IL4 and FITC-anti-IFN-{gamma}. The expression of IL4 and IFN-{gamma} was detected by FACS after gating on CD4+ 10,000 live cells. The percentages of each subset are indicated. B, RNase protection assay analysis of cytokine profiles at transcription level. Four micrograms of total RNA extracted from restimulated T cells was hybridized with 3 x 105 cpm freshly labeled mCK-1 probe sets, according to the manufacture’s protocol (BD PharMingen). Different PCC peptide concentrations employed during the initial stimulation are indicated above each lane.

 
A total of 5x 105 purified T cells were cocultured with 1.5x 105 DCEK58 for 5 days in the presence of PCC wt peptide, but without addition of exogenous cytokines. From these cultures, equal amounts of viable T cells were restimulated with CH27 cells loaded with 10-6 M peptide for another 5 h (T:CH27 cell ratio = 4:1). Subsequently, intracellular cytokine staining or RNase protection assays were performed. CH27 cells offer comparable stimulation to hCD2+ and hCD2- T cells because these cells express many of the key accessory/costimulatory molecules including ICAM-1, CD48, B7, and HSA (data not shown), yet lack hCD58. As shown in Fig. 5GoA, with decreasing molar amounts of PCC peptide, T cells express less IFN-{gamma}, while the amount of IL-4 expression increases as assayed by intracellular cytokine staining. This peptide dose-dependent shift in polarization from Th1 to Th2 is consistent with previous reports (8, 36). The presence or absence of the hCD2 transgene does not alter the T helper polarization. To next investigate whether Th1/Th2 polarization is regulated at the transcriptional level, we employed an RNase protection assay to quantify the expression of cytokine mRNAs. IL-4 and IL-5 mRNA levels are significantly increased upon reduction of the PCC peptide concentration (Fig. 5GoB). In contrast, mRNAs of the Th1 cytokines, IFN-{gamma} and IL-2, increase with increasing peptide concentrations. IL-15 and IL-9 are not expressed while IL-10, IL-13, and IL-6 cannot be assessed due to constitutive expression in CH27 (data not shown). These data demonstrate that there is no difference in murine T cell Th1-Th2 polarization in the presence or absence of the human CD2 transgene.

Abnormality in effector/memory T cell generation after in vivo priming in 5CC7tg CD2-deficient mice

To examine the role of CD2 in Ag-specific T cell responses in vivo, we injected 5CC7tg CD2+/+ and 5CC7tg CD2-/- mice with the cognate PCC wt 17-mer peptide i.v. and examined the phenotypes of in vivo primed lymph node T cells by assessing cytokine production and expression of the cell surface molecules, CD44, CD45RB, and CD62L. Five or 10 days following a single i.v. injection with 20 µg of PCC wt 17-mer peptide, lymph node cells from 5CC7tg CD2+/+ and 5CC7tg CD2-/- mice were restimulated in vitro for 2 days with varying doses of the same peptide presented on CH27 cells, and assayed for cytokine production by ELISA. When incubated for 48 h with PCC prepulsed CH27 cells, both the 5CC7tg CD2+/+ and 5CC7tg CD2-/- lymph node cells obtained 5 days after in vivo PCC injection secrete IFN-{gamma} (Fig. 6GoA), but not IL-4 (data not shown), into the culture supernatant at each peptide concentration tested (10-5–10-7 M). However, the values of IFN-{gamma} secreted from 5CC7tg CD2+/+ lymph node cells are significantly higher than those from 5CC7tg CD2-/- T cells at 10-5–10-7 M PCC. Furthermore, even greater differences are observed in IFN-{gamma} production of the 5CC7tg CD2+/+ and 5CC7tg CD2-/- lymph node T cells when lymph node cells are harvested 10 days after PCC injection at lower concentrations (Fig. 6GoA).



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  		FIGURE 6. Impaired effector/memory CD4 T cell responses after in vivo priming in 5CC7tg CD2-deficient mice. A, Lymph node cells (1 x 105 cells) from 5CC7tg RAG-2-/- CD2+/+ and 5CC7tg RAG-2-/- CD2-/- mice 5 or 10 days after a single 20 µg PCC wt 17-mer peptide i.v. injection were incubated with the indicated concentrations of PCC wt 17-mer peptide and irradiated I-Ek-bearing CH27 cells (2 x 104 cells). Forty-eight hours later, supernatants were assayed for IFN-{gamma} production. Results expressed as mean ± SD from three to four different mice are shown. The production of IFN-{gamma} in 5CC7tg RAG-2-/- CD2-/- T cells is significantly lower than that in 5CC7tg RAG-2-/- CD2+/+ T cells 5 days (10-5 M, p < 0.005; 10-6 M and 10-7 M, p < 0.05) and 10 days (10-5 M, p < 0.0005; 10-6 M and 10-7 M, p < 0.05) after PCC injection. B, Lymph node cells from 5CC7tg RAG-2-/- CD2+/+ and 5CC7tg RAG-2-/- CD2-/- mice 10 days after i.v. injection of 20 µg PCC wt 17-mer peptide were stained with PE-anti-CD4, CyChrome-anti-CD8{alpha}, and FITC-anti-CD44, FITC-anti-CD62L, or biotin-anti-CD45RB followed by FITC-streptavidin. Histograms of CD44, CD45RB, or CD62L of CD4+ SP gated lymph node cells are shown. The result is representative from three independent mice. Percentages of cells in the relevant gates are shown. Open and solid curves represent results from uninjected and injected animals, respectively.

 
Naive T cells express a low surface density of the homing molecule CD44, a high density of the homing molecule CD62L, and a high density of the receptor tyrosine phosphatase CD45 isoform RB. Following an encounter with Ags, the expression pattern of these surface markers reverses, consequently a CD44highCD45RBlowCD62Llow surface phenotype is associated with the differentiation of T cells toward an effector/memory phenotype (37). To determine how T cell differentiation, as defined by the expression of these surface markers, might be influenced by the deficiency of CD2 in vivo, we measured the expression of CD44, CD45RB, and CD62L on lymph node cells from 5CC7tg CD2+/+ and 5CC7tg CD2-/- mice 10 days post-PCC injection. As shown in Fig. 6GoB, while there is no difference in the expression pattern of CD62L after immunization between 5CC7tg CD2+/+ and 5CC7tg CD2-/- lymph node T cells, substantial differences in the expression levels of CD45RB and CD44 after in vivo priming with PCC peptide was observed between 5CC7tg CD2+/+ and 5CC7tg CD2-/- mice. For example, after PCC injection, about half of the 5CC7tg CD2+/+ T cells showed a dramatic reduction of CD45RB expression, which is a typical phenotype of effector/memory cells. By contrast, 5CC7tg CD2-/- mice also showed a reduction in the expression level of CD45RB, but to a much lesser extent (20 vs 40%). Taken together with the data on IFN-{gamma} production, it seems likely that 5CC7tg CD2-/- mice have a defect in the differentiation of T cells into effector/memory cells after in vivo priming.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although it was originally reported that mice deficient in CD2 show no obvious immunological phenotype (24), more recent studies using CD2-deficient animals expressing one of several class I MHC-restricted TCR transgenes (27, 28) demonstrated that CD2 expression influences thymic development. Moreover, quantitative analysis led to the conclusion that the responses of peripheral T cells to specific Ags were impaired in the absence of CD2. Consistent with the latter results, animals harboring double knockouts of CD2 plus LFA-1 or CD28 illustrate how CD2 sets the threshold of T cell signals, thereby augmenting T cell activation (25, 26). Because the role of CD2 in Ag-specific helper T cell function has not been elucidated, we examined the contribution of CD2 to the differentiation and activation of helper T cells in the present study.

CD2 and murine CD4 SP thymocyte maturation

Prior studies showed that CD2 is first expressed on preTCR bearing DN thymocytes and then maintained on all thymocytes at further developmental stages (38). Consistent with an important function for CD2 in development, N15tg CD2-/- mice manifest a reduction in number of DP as well as CD8 SP thymocytes without any alteration of DN thymocytes. Further analysis of CD3-CD4-CD8- thymocytes provided evidence for a blockade at the CD25+CD44- to CD25-CD44- transition, analogous to the block found in mice lacking a functional preTCR complex (39), suggesting that CD2 facilitates the transition from DN to DP thymocytes. That result using a class I-restricted TCR transgene derived from CD8 SP T cells is further extended to a class II-restricted TCR transgene found on T cells of the CD4 subset; a decreased number of DP and CD4 SP thymocytes are observed in 5CC7 CD2-/- mice. This differentiative defect is made more prominent following peptide-induced apoptotic depletion of DP thymocytes, implying that CD2 expression contributes to restoring development, thereby aiding in maintenance of cellular homeostasis. Collectively, these findings show that CD2 function is important in both SP CD8 and SP CD4 differentiation.

CD2 and mature T cell function

Coligation of CD2 and CD58 molecules on opposing human cells redistributes CD2 to the region of cell-cell contact and in doing so, offers an avidity enhancement for adhesive forces to optimize the Ag recognition process (16, 40, 41). Although the affinity of rodent CD2 for rodent CD48 is ~10-fold weaker than that between hCD2 and CD58 (40), the importance of murine CD2-CD48 interaction in T cell activation has been demonstrated in several murine T cell lines (42, 43). Moreover, in N15tg CD2-/- mice, there is a 100- to 1000-fold greater requirement for peptide to induce IFN-{gamma} production or T cell proliferative responses to pMHCI than in N15tg CD2+/+ mice. Interestingly, unlike with CD8 SP T cell cytokine production and proliferation, murine CD2 deficiency did not affect CD8 cytotoxic T cell activity (27). In this context, the present study with 5CC7 CD2-/- indicates that murine CD2 is important in optimizing helper T cell proliferation and cytokine production to pMHCII, both for cognate peptide Ag recognition and stimulation by weak agonist peptide variants. In the latter case, CD2 is essential.

Role of CD2 in Th1/Th2 differentiation

Although strong TCR signaling (i.e., high Ag dose and/or strong T cell ligand affinity) induces the differentiation of naive T cells into Th1 effectors (6, 8), the roles of costimulatory molecules in helper polarization are more variable. Anti-LFA-1 mAb blockade was shown to inhibit Th2 differentiation while leaving unaffected Th1 development (44). In contrast, by using a CD28 knockout line bearing a TCR transgene, it was demonstrated that CD28 promotes Th2 differentiation (45), consistent with the results obtained by CTLA4-Ig blockade (44, 46). The latter result also was confirmed by in vivo experiments (47). ICOS, an inducible costimulator with homology to CD28 expressed on activated T cells, showed an ability to up-regulate IL-4 but not IFN-{gamma} (48). The T cell costimulatory molecule Ox40 was reported to promote either the differentiation of Th2 cells (49, 50) or Th1 cells (51), and a knockout of the Ox40 ligand (Ox40L) expressed on APCs resulted in impaired T cell priming and reduction of both Th1 and Th2 cytokines (52). Other studies suggest that CD40-CD154 interaction drives naive T cells into Th1 or Th2 differentiation (reviewed in Ref. 53). Therefore, it has been suggested that aside from TCR stimulation, the ligation of cell surface accessory/costimulatory receptors additionally influences the differentiation of helper T cells in some complex manner (54).

The differentiation of helper T cells is highly dependent on the cytokine environment in which the T cell finds itself. IL-12 and IL-4 are both sufficient and necessary for Th1 and Th2 polarization, respectively. The in vivo source of IL-12 may be either macrophages or dendritic cells (DC) (55, 56, 57). Different subsets of DC, i.e., lymphoid-related and myeloid-related DC, secrete varying amounts of IL-12 with lymphoid-related DC producing more biologically active IL-12 (56). Therefore, in vivo the lymphoid-related DC population induces high levels of IFN-{gamma} and IL-2, but little or no Th2 cytokines, whereas the myeloid-related subset induces large amounts of the Th2 cytokines IL-4 and IL-10, in addition to IFN-{gamma} and IL-2 (57). Other sources of initial cytokines influencing Th differentiation arise from B cells (58), NK T cells (59), or T cells themselves. Recent progress in the control of CD4 T cell polarization at the transcriptional level defines the molecular mechanisms involved in polarization processes integrating exogenous and endogenous stimuli (60, 61, 62).

Given that CD2 ligation in man and mouse (22, 27) augments IL-12 responsiveness, including IFN-{gamma} production, it was reasonable to assume that CD2 would serve as an important component of Th1 development. Less obvious was the CD2 role in Th2 differentiation. However, when we used lymph node T cells from 5CC7tg CD2+/+ and 5CC7tg CD2-/-, there was a decrease in the production of both IFN-{gamma} and IL-4 from the latter. There are several earlier reports demonstrating a role for CD2 in cytokine production by T cells. CD2-CD58 ligation preferentially augments the production of IFN-{gamma} by super antigen-activated CD8+ human T cells (63), consistent with the results of Wingren et al. (64) that CD2-CD58 ligation (but not the LFA-1/ICAM-1 pathway) is involved in enhanced production of IFN-{gamma} in PHA and IL-2 activated human T cells. However, by stimulating T cells with anti-CD3 and mitogenic anti-CD2 mAbs, others demonstrated that human memory T cells produced more Th2 cytokines including IL-4 and IL-5 as compared with Th1 cytokines (65). Interestingly, more recent studies indicate that CD58 potently induced IL-10 and IFN-{gamma} production from cells which correspond to the T regulatory 1 subset (Tr 1) (66). Besides those results obtained from Ag-independent stimulation, Le Guiner et al. (67) used melanoma-specific CD8+ CTL T cell clones to study the effects of LFA-3 costimulation in cytokine secretion, and demonstrated that LFA-3 systematically enhances cytokine IL-2, IFN-{gamma}, and TNF secretion. Until now, direct evidence of CD2 involvement in cytokine production by helper T cells in an Ag-specific manner has been unavailable.

Our current results demonstrate that without mouse CD2, naive T cells produce less cytokines compared with CD2+/+ T cells under various conditions (Fig. 4Go). However, by using a human CD2 transgene on the 5CC7 background, we also show that the human CD2 transgene does not affect the polarization of CD4+ cell differentiation (Fig. 5Go and data not shown). Additionally, there is no quantitative difference in IFN-{gamma} or IL-4 production between human tg CD2+ or non-tg CD2- T cells. Whether this lack of difference results from the presence of CD58 on APCs only during the inductive polarization culture, the non-RAG-/- background of the double hCD2/5CC7tg mice, or other factors is currently unknown. It is also noteworthy that there are two membrane-proximal cysteines in the murine and rat CD2 tails that are absent from the horse and human homologs (21). Hence, mouse CD2 is possibly palmitoylated so that it is constitutively localized in signal-molecule-enriched lipid rafts while human CD2 is inducibly associated with rafts (21). This difference may offer distinct signals to cells that can affect differentiation of CD4+ T cells to Th1 or Th2. In support of this possibility, it is known that CD2 ligation by CD58 preferentially up-regulates NF-AT nuclear transcription factor (68). Moreover, NF-AT together with other transcription factors, such as AP-1 and NF-{kappa}B, binds to the promotors of many cytokines, for example, IL-2 and IL-4 (69). Therefore, the different lipid rafts association mechanism may confer different roles of CD2 in signal transduction, finally leading to different outcomes in mouse vs human CD2.

For analysis of the mouse CD2 knockout, the primary T cell culture was performed using CH27 as the APC pulsed with different amounts of PCC peptide. CH27 cells express IL-6, IL-10, and IL-13 as assayed by RNase protection (data not shown). Under these conditions, the 5CC7tg/CD2+/+ and CD2-/- T cells differentiated exclusively into Th1 effectors in the absence of exogenous cytokines. One possible explanation for this is that CH27 expresses these and/or other endogenous cytokines which can skew T cell differentiation toward the Th1 pathway. Another possibility is that there are multiple costimulatory ligands, such as ICAM-1, HSA, and B7, expressed on CH27 which may influence the polarization of mouse T cells. To exclude the latter influence, we employed DCEK58 fibroblasts transfected with class II MHC I-Ek and human CD58 as APC during polarization induction. DCEK58 does not express ICAM-1, VCAM-1, CD48, very late Ag-4, B7-2, Ox-40L, 4–1BBL, LFA-1, or heat-stable Ag (32, 35), and does not express any cytokines as assayed by RNase protection using the mCK-1 template set (data not shown). When we used 5CC7tg/hCD2 double-tg T cells, we did not observe a preference of differentiation of naive T cells to either Th1 or Th2 cells at either the protein level (IL-4 and IFN-{gamma} expression) or the RNA level (IL-4, IL-5, IL-2, and IFN-{gamma}), although we observed a dose-dependent shift from Th1 to Th2 polarity with decreasing peptide concentrations (Fig. 5Go). These findings strongly imply that neither human nor mouse CD2 favors preferential polarization of T helper cells. Rather, an important function of CD2 appears to be related to its capacity to optimize Ag recognition processes.


   Acknowledgments
 
We thank Drs. Linda Clayton and Elena Tibaldi for careful review of this manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI21226. Back

2 Address correspondence to Dr. Ellis L. Reinherz, Laboratory of Immunobiology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. E-mail address: ellis_reinherz{at}dfci.harvard.edu Back

3 Abbreviations used in this paper: pMHC, peptide MHC complex; tg, transgenic; PCC, pigeon cytochrome c; RAG-2, recombination activating gene-2; SP, single-positive; DP, double-positive; DN, double-negative; APL, altered peptide ligand; wt, wild type; CD62L, CD62 ligand; DC, dendritic cells. Back

Received for publication September 4, 2001. Accepted for publication November 19, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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