NK Cells Stimulate Proliferation of T and NK Cells through 2B4/CD48 Interactions

Mice

The strains used in this study include adult C57BL/6 (B6), F5 (TCR transgenic (Tg)³ specific for an influenza nucleoprotein epitope, ASNENMDAM, presented on H-2D^b) (26), DO11.10 (TCR-Tg specific for an OVA epitope, ISQAVHAAHAEINEAGR, presented on I-A^d) (27), RAG-1^−/− (28), perforin-deficient (PKOB)^−/−xRAG-1^−/− (29), B10.D2, CD48^−/− (30), and DBA/2. All animals were bred and housed under standard conditions at the Microbiology and Tumor Biology Center, Karolinska Institutet; Department of Pathology, Emory University School of Medicine; and Department of Pathology, Brigham and Women Hospital, Harvard Medical School (Boston, MA). All animal procedures were approved by the local departments as well as by the Committee for Animal Ethics in Stockholm, Sweden.

Cell lines and reagents

P815 tumor cells were transfected with 2B4 (clone P815-2B4.D1 used, designated P815-2B4; see below) and with H-2K^b (designated P815-K^b) and cultured in RPMI 1640, 5% FCS, 100 U/ml penicillin, and 100 U/ml streptomycin. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise specified. OVA-derived peptide (OVA_323–339), ISQAVHAAHAEINEAGR, was purchased from Interactiva (Ulm, Germany), and the influenza virus nucleoprotein-derived peptide (NP_366–374), ASNENMDAM, was purchased from Research Genetics (Huntsville, AL). All Ab used were purchased from BD PharMingen (San Diego, CA), except for FITC anti-human Ig and human Ig (Jackson ImmunoResearch Laboratories, West Grove, PA); anti-murine CD3 (145-2C11) was purified from cell culture medium. All Ab used in proliferation assays were azide free.

Transfection

Tumor cells were grown to a density of 1 × 10⁶/ml, and ∼1 × 10⁷ total cells were used for transfection. Cells were harvested and washed twice with serum and antibiotic-free medium. Approximately 10 μg of plasmid (pBSRaEN with or without 2B4-D1 cDNA inserted) was linearized and used for transfection by electroporation at 960 μF and 250 V. Cells were kept on ice for 15 min following transfection, then placed into complete medium (RPMI 1640, 10% FCS, 100 U/ml penicillin, 100 U/ml streptomycin) overnight at 37°C, 5% CO₂. The following day, cells were harvested and resuspended in complete medium containing 1 mg/ml G418 and plated into a single 96-well flat-bottom plate. Clones were obtained ∼2 wk later and screened for 2B4 expression by flow cytometry.

Flow cytometry

Fc receptors were blocked by incubating the cells in anti-CD16/32 Ab at 10 μg/ml, after which the cells were stained with the specified Ab or its isotype control at 10 μg/ml. For stainings with Ig fusion proteins, splenocytes were stained with polyclonal human Ig (Jackson ImmunoResearch Laboratories) or 2B4 Ig (see below) at 10 μg/ml, washed twice, and stained with FITC-conjugated goat anti-human Ig at 10 μg/ml. The intensity of fluorescence was measured on a FACScan flow cytometer (BD Biosciences, Mountain View, CA) and analyzed using CellQuest computer software (BD Biosciences).

Cell purification

Murine CD4⁺, CD8⁺, and DX5⁺ cells were purified from spleens of mice using the MACS separation system (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s guidelines, and resuspended in complete αMEM (αMEM, 10 mM HEPES, 2 × 10⁻⁵ M 2-ME, 10% FCS, 100 U/ml penicillin, 100 U/ml streptomycin). Unless noted elsewhere, NK cells were purified from RAG-1^−/− mice. Purity of the NK cells was examined by flow cytometry by confirming the expression of NK1.1 and the lack of CD14 and MHC class II.

NK cell activation

For IL-2 activation, freshly purified DX5⁺ cells were cultured in complete αMEM with IL-2 at 1000 U/ml for 6 days. When not else noted, all NK cells used in this study were IL-2 activated.

Proliferation assays

Dendritic cells (DC) were prepared from bone marrow of mice, cultured for 6 days, as previously described (31), pulsed with ASNENMDAM or ISQAVHAAHAEINEAGR peptide for 24 h, and seeded at 2 × 10⁴ cells/well in U-bottom 96-well plates. A total of 3 × 10⁴ naive CD8⁺ or CD4⁺ TCR-Tg T cells and 4 × 10⁴ DX5⁺ IL-2-activated NK cells were added to each well with the indicated Ab. In the DC-free cultures, 3 × 10⁴ naive CD8⁺ or CD4⁺ B6 T cells were added to each well in a flat-bottom 96-well plate precoated with anti-CD3 Ab at 10 μg/ml (if not otherwise noted) at 4°C for 24 h. All T cell proliferation assays were performed for 3 days, except for the IL-2-induced proliferations that lasted for 5 days. [³H]Thymidine (1 μCi/well) was added for the last 18 h of all assays. The plates were harvested on glass fiber filters and analyzed in a beta scintillation counter (Wallac Oy, Turku, Finland). For NK cell proliferation assays, cells were seeded at 4 × 10⁴ cells/well in U-bottom 96-well plates and cultured in IL-2 at 1,000 U/ml with the indicated Ab. The NK cell proliferation assays were performed for 5 days and measured, as above. In proliferations using CFSE, purified B6 CD8⁺, CD4⁺ T, or DBA/2 CD8⁺ T cells were incubated with 1 μM CFSE for 10 min at 37°C, washed twice, and cultured at 1.5 × 10⁵/well with 2 × 10⁵ DX5⁺ IL-2-activated NK cells or irradiated (20,000 rad) P815 cells in a 12-well plate precoated with anti-CD3 Ab. Proliferation was determined by dilution of the CFSE dye by flow cytometry.

Construction of the m2B4-Ig expression vector and production of the 2B4-Ig fusion protein

Total cellular RNA was extracted from DX5⁺ cells using the TRIzol RNA isolation method (Life Technologies, Rockville, MD), followed by cDNA synthesis using the first-strand cDNA synthesis kit (Pharmacia Amersham Biotech, Piscataway, NJ), according to the protocol using pd(N)6 primers. The cDNA was used as template in a PCR with the primers 2B4-forward (5′-GATCAAGCTTCGCACCATGTTGGGGCAAG CTG-3′) and 2B4-reverse (5′-GATCGGATCCGCATTCGAAGGGACACTTTGAC-3′) containing a restriction site for HindIII and BamHI, respectively. The cycling parameters were as follows: 95°C, 3 min; 35 cycles, 95°C, 30 s; 55°C, 45 s; 72°C, 120 s; 72°C, 10 min. The resulting PCR product was digested with HindIII and BamHI and cloned into the plasmid pcDNA1.1/Amp-hIg, 5′ of a DNA fragment encoding for the Fc part of human IgG1. After verifying the construct by sequencing, vectors containing the m2B4-Ig sequence were transfected into COS-1 cells (32). After 14 days, the culture supernatant was collected and applied to a protein G-Sepharose column. Bound m2B4-Ig molecules were eluted with imidazole and dialyzed against PBS. Purity and integrity were analyzed by silver staining and Western blotting.

CD8⁺ T cell stimulation for CD69 expression

A total of 3 × 10⁵ freshly purified CD8⁺ T cells was cultured in anti-CD3-coated (at concentrations indicated) 48-well plates in 500 μl of complete αMEM for 3 h alone or in the presence of 3 × 10⁵ irradiated P815 cells. After culture, the cells were stained with anti-CD69 FITC and anti-CD8α PE and analyzed by flow cytometry.

Statistical analysis

Student’s two-sample t test was used, assuming unequal variances when comparing triplicates from the different treatments with controls within each experiment, or the paired two-sample t test when comparing groups of combined experiments.

NK cells enhance the anti-CD3-induced proliferation of CD8⁺ and CD4⁺ T cells through 2B4/CD48 interactions

To investigate whether NK cells could directly enhance T cell proliferation, we used a system in which CD8⁺ and CD4⁺ T cells were stimulated with immobilized anti-CD3 Ab in the presence or absence of IL-2-activated NK cells. To visualize the T cell proliferation, CD8⁺ and CD4⁺ T cells were labeled with CFSE, which allows the measurement of proliferating cells by flow cytometry. As shown in Fig. 1⇓A, the addition of NK cells to CFSE-labeled CD8⁺ and CD4⁺ T cells significantly increased T cell proliferation. The addition of anti-2B4 Ab to these cultures reduced the proportion of proliferating CD8⁺ and CD4⁺ T cells to background levels. T cells cultured in the absence of NK cells were not affected by the addition of anti-2B4 Ab (Fig. 1⇓A, top two histograms; data not shown). These results suggest that the enhanced proliferation of T cells upon the addition of NK cells was mediated through cell-cell contact. In support of this notion, the NK cell-mediated stimulation of T cell proliferation was abrogated if the NK cells were separated from the T cells by a cell-impermeable porous membrane (data not shown).

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FIGURE 1.

NK cell-mediated enhancement of T cell proliferation in an APC-free system. A, CFSE-labeled B6 CD8⁺ T cells or CD4⁺ T cells cultured in anti-CD3-coated plates (1 μg/ml) in the presence or absence of NK cells and Ab. The fluorescence intensity was determined by flow cytometry. Numbers represent percentage of nondividing cells. B, CD8⁺ T cells, or C, CD4⁺ T cells cultured in anti-CD3-coated plates (1 μg/ml) in the presence or absence of NK cells and Ab: ▪, IgG2b; ▧, anti-2B4; □, anti-CD48. Proliferation was measured by [³H]thymidine incorporation during the last 18 h. Results are expressed as mean cpm ± SD of triplicate cultures. One representative result of three (A) and two (B and C) independent experiments is shown.

NK cell-mediated enhancement of T cell proliferation was also confirmed by measuring [³H]thymidine incorporation. The addition of IL-2-activated NK cells to the T cell cultures clearly enhanced the proliferative response of both CD8⁺ and CD4⁺ T cells (Fig. 1⇑, B and C). Under these conditions, the enhanced proliferation was reversed either by Ab to 2B4 or to its only known ligand CD48, whereas addition of an isotype control Ab or anti-NK1.1 Ab, another activating molecule expressed on NK cells, had no effect (Fig. 1⇑, B and C; data not shown). Additional control experiments revealed that T cell proliferation was not affected by anti-2B4 Ab in the absence of NK cells (Fig. 1⇑, B and C), nor did NK cells proliferate to any major extent when cultured alone (Fig. 1⇑, B and C). However, to exclude that even a minor proliferation of NK cells would give rise to the enhanced thymidine uptake observed when mixed with T cells, NK cells were treated with mitomycin C to inhibit cell division. Mitomycin C-treated NK cells enhanced the T cell proliferation to the same degree as the untreated NK cells (data not shown).

The 2B4/CD48 interactions regulate Ag-driven proliferation of CD8⁺ and CD4⁺ T cells in the presence of NK cells

We next investigated whether 2B4 expression on NK cells could also regulate Ag-specific T cell proliferation. CD8⁺ or CD4⁺ TCR-Tg T cells were cocultured with NK cells and Ag-pulsed DC in the absence or presence of anti-2B4 or anti-CD48 Ab. In these systems, NK cells increased proliferation of T cells, although the degree to which this occurred varied between different experiments. In experiments in which efficient stimulation of T cell proliferation was seen, blocking of either 2B4 or CD48 significantly reduced the proliferative response of both CD8⁺ and CD4⁺ T cells (Fig. 2⇓, A and B). In contrast, no decrease in proliferation was observed upon addition of an isotype control or anti-NK1.1 Ab (Fig. 2⇓, A and B). Importantly, proliferation was not affected by anti-2B4 Ab in the absence of NK cells.

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FIGURE 2.

The 2B4/CD48 interactions regulate Ag-driven proliferation of CD4⁺ and CD8⁺ T cells in the presence of NK cells. TCR-Tg T cells were cocultured with H-2-syngeneic NK cells (B6 or B10.D2) and DC loaded with specific peptide at concentrations indicated and various Ab, as shown. A, F5 TCR-Tg CD8⁺ T cells, or B, DO11.10 TCR-Tg CD4⁺ T cells. C, F5 TCR-Tg CD8⁺ T cells cocultured with DC loaded with 1 μM specific peptide and perforin-deficient (PKOB^−/−) NK cells in the presence or absence of anti-FasL Ab, and IgG2b or anti-2B4 Ab. Proliferation was measured by [³H]thymidine incorporation. Results are expressed as mean cpm ± SD of triplicate cultures. ∗∗, p < 0.01 compared with cultures with IgG2b. One representative result of three (A and B) or two (C) independent experiments is shown.

NK cells can kill autologous DC in vitro in a perforin-dependent manner (33, 34). Because cross-linking of 2B4 is known to enhance NK cell-mediated cytotoxicity, there was a possibility in the present setting that the anti-2B4 Ab triggered NK cells to lyse peptide-loaded DC, which would result in a relatively reduced induction of Ag-specific T cell response (15, 21, 23, 24). Therefore, similar experiments were performed using NK cells from perforin-deficient mice. In addition, anti-Fas ligand (FasL) Ab was added to the cultures to block any possible Fas-FasL-mediated killing of DC. As shown in Fig. 2⇑C, anti-2B4 Ab significantly reduced the CD8⁺ T cell proliferation even in cocultures using perforin-deficient NK cells in combination with anti-FasL Ab. Similar observations were also made using formaldehyde-fixed DC not being susceptible to NK cell-mediated lysis (data not shown). Fab of anti-2B4 Ab were also able to block T cell proliferation in the presense of NK cells, suggesting that the reduced proliferation was not due to cross-linking activation of NK cells by 2B4 cross-linking or Ab-dependent cellular cytotoxicity on the DC (data not shown).

NK cells make up ∼5% of all lymphocytes in the peripheral blood and spleen. NK cells are also detectable in lymph nodes, albeit at low numbers. The relatively lower numbers of NK cells than T cells in peripheral blood and secondary lymphoid organs led us to address how many NK cells were required to enhance CD8⁺ T cell proliferation. As few as 1 NK cell per 16 T cells enhanced the anti-CD3-induced T cell proliferation (Fig. 3⇓). This result suggests that activated NK cells may affect T cell proliferation in organs and tissues in which NK cells are relatively few in numbers.

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FIGURE 3.

Low numbers of NK cells are sufficient to enhance T cell proliferation. B6 CD8⁺ T cells (30,000/well) were cultured in anti-CD3-coated plates with decreasing numbers of NK cells in the presence of Ab. Proliferation was measured by [³H]thymidine incorporation. Results are expressed as mean cpm ± SE of triplicate cultures. ∗∗, p < 0.01; ∗, p < 0.01 compared with cultures without NK cells. Shown is the result of two independent experiments pooled.

P815 tumor cells expressing 2B4 enhance the proliferation of CD8⁺ T cells

To directly investigate whether 2B4 expression alone is sufficient to enhance T cell proliferation, we transfected CD48-negative P815 tumor cells with a mutant form of 2B4 that has a truncated intracellular domain, to obviate the signaling potential of the molecule. CFSE-labeled CD8⁺ T cells from DBA/2 mice were cultured in a high dose of IL-2 alone or with irradiated P815-2B4 cells or P815 control cells for 5 days (Fig. 4⇓, A–H). The proliferative response of T cells cultured alone (Fig. 4⇓A) was equivalent to that of T cells cultured with P815 wild type (wt) (Fig. 4⇓C) or with P815-K^b, used as a control for the transfection (Fig. 4⇓H). In contrast, the addition of P815-2B4 cells to the cultures resulted in a marked increase in cell division (Fig. 4⇓E). Notably, the addition of anti-2B4 Ab reversed the proliferation nearly to background levels (Fig. 4⇓F), whereas there was no effect on the proliferation of T cells cultured alone (Fig. 4⇓B) or with P815 wt (Fig. 4⇓D). Similarly, Fab′ fragments of the anti-2B4 Ab also reduced the 2B4-mediated increase in T cell proliferation (Fig. 4⇓G).

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FIGURE 4.

The 2B4-expressing P815 tumor cells enhance the proliferation of CD8⁺ T cells. CFSE-labeled DBA/2 CD8⁺ T cells cultured alone or with irradiated P815 tumor cells in the presence of IL-2. Proliferation was determined as decreased fluorescence intensity measured by flow cytometry: A, T cells alone + IgG2b; B, T cells alone + anti-2B4; C, T cells with P815 wt + IgG2b; D, T cells with P815 wt + anti-2B4; E, T cells with P815-2B4 + IgG2b; F, T cells with P815-2B4 + anti-2B4; G, T cells with P815-2B4 + anti-2B4 Fab′; H, T cells with P815-K^b + IgG2b. Numbers represent percentage of nonproliferating cells. One representative result of three independent experiments is shown.

The 2B4 binds exclusively to CD48

From the present and previously published reports, it could not formally be excluded that 2B4 bound to molecules other than CD48. To address this, we produced a fusion protein composed of the extracellular domain of murine 2B4 and a human IgG1 tail, which was used to stain B6 and CD48^−/− splenocytes. All B6 lymphocytes stained positively for 2B4-Ig, whereas no binding was detected on CD48^−/− lymphocytes, suggesting that 2B4 binds specifically to CD48 (Fig. 5⇓A). To confirm that the effects of 2B4 were mediated through CD48 on the proliferating T cells, anti-2B4 and anti-CD48 Ab were added together to anti-CD3-stimulated CD8⁺ T cells and NK cells. No additive effect was observed when combining the two Ab, which suggested that the anti-2B4 Ab specifically blocked the interaction between 2B4 and CD48 (Fig. 5⇓B).

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FIGURE 5.

The 2B4 binds no ligand other than CD48. Splenocytes prepared from B6 and CD48^−/− mice were stained with 2B4-Ig (solid line) or human Ig (dashed line) and analyzed by flow cytometry. B, B6 CD8⁺ cells cultured in anti-CD3-coated plates (1 μg/ml) with NK cells in the presence of various Ab, as shown. Results are expressed as mean cpm ± SD of triplicate cultures. C, CD4⁺ T cells, or D, CD8⁺ T cells from B6 and CD48^−/− cultured in anti-CD3-coated plates (1 μg/ml) in the absence or presence of NK cells in the presence of Ab, as shown: ▦, T cells alone + IgG2b; ▪, T cells + NK cells + IgG2b; ▧, T cells + NK cells + anti-2B4; □, T cells + NK cells + anti-CD48. Shown is the mean [³H]thymidine incorporation ± SE of two (C) or three (D) experiments pooled.

When CD4⁺ T cells purified from CD48^−/− mice were cocultured with NK cells, no significant increase in T cell proliferation was observed (Fig. 5⇑C). Nor did anti-2B4 or anti-CD48 Ab affect the T cell responses (Fig. 5⇑C). When CD8⁺ T cells purified from CD48^−/− mice were cocultured with wt NK cells, an increase in T cell proliferation was observed (Fig. 5⇑D). The latter increase could for obvious reasons not be due to 2B4-CD48 interactions, nor was the proliferation decreased by the presence of anti-2B4 or anti-CD48 Ab. The reason for this effect of NK cells on CD48-deficient CD8⁺ T cells is not known, and was not subject for further analysis. It is possible, however, that the CD48-deficient CD8⁺ T cells retain some compensatory mechanisms in the absence of CD48-dependent interactions. These results confirm the specificity of the anti-2B4 and anti-CD48 Ab used in our studies.

The 2B4-expressing cells enhance the activation of CD8⁺ T cells

The expression of CD69, a marker of recent activation, was examined on anti-CD3-stimulated CD8⁺ T cells that were cocultured with or without 2B4-expressing cells. Strikingly, coculture with P815-2B4 cells increased the fraction of CD69⁺ T cells from ∼15 to ∼50% as early as 3 h after anti-CD3 stimulation (Fig. 6⇓). The addition of anti-2B4 Ab reduced the number of CD69⁺ T cells to ∼20%. In contrast, coculture with P815 wt cells had only a minor effect on the CD69 expression of anti-CD3-stimulated CD8⁺ T cells (Fig. 6⇓). Similarly, coculture with NK cells significantly increased the number of CD69⁺ T cells through 2B4/CD48 interactions (data not shown). Thus, costimulation through 2B4/CD48 interactions not only affected proliferation, but also enhanced short-term activation of T cells.

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FIGURE 6.

The 2B4-expressing cells enhance the activation of CD8⁺ T cells. CD8⁺ T cells were cultured in anti-CD3-coated (0.1 μg/ml) plates for 3 h alone or in the presence of P815 wt or P815-2B4 cells and Ab, as shown. After culture, the cells were analyzed for CD69 expression (a marker for early activation) when gated on CD8α⁺ T cells by flow cytometry. A, Histograms showing CD69 expression on CD8α⁺ T cells from the cultures. B, ▦, T cells alone plus IgG2b; □, T cells plus P815 wt and IgG2b; ▪, T cells plus P815-2B4 and IgG2b; ▧, T cells plus P815-2B4 and anti-2B4 Ab. Results are expressed as percentage of CD69⁺ cells of total CD8α⁺ T cells. One representative result of two independent experiments is shown.

Increased NK cell proliferation upon 2B4/CD48 interaction

To examine whether NK cell proliferation could also be regulated by 2B4/CD48 interactions, freshly purified murine splenic NK cells were cultured in a high dose of IL-2 (1000 U/ml) for 5 days in the presence of Ab. In these cultures, NK cell proliferation was reduced upon blocking either 2B4 or CD48 (Fig. 7⇓). No additional blocking effect was observed when combining anti-2B4 and anti-CD48 Ab (Fig. 7⇓). Taken together, these results show that NK cells augment the proliferation of neighboring T cells as well as other NK cells through direct cell-cell contact via 2B4/CD48 interactions.

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FIGURE 7.

NK cell proliferation increases upon 2B4/CD48 interaction. Murine NK cells were stimulated with IL-2 (1000 U/ml) in the presence of Ab, as shown. Proliferation was measured by [³H]thymidine incorporation. Results are expressed as mean cpm ± SD of triplicate cultures. ∗∗, p < 0.01 compared with cultures with IgG2b. NS, no significant difference between the two bars. One representative result of three independent experiments is shown.