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HLA-B2702 (77–83/83–77) Peptide Binds to ß-Tubulin on Human NK Cells and Blocks Their Cytotoxic Capacity¹

Raquel Tarazona^*, Guillermo López-Lluch^*, Maria-Dolores Galiani^*, Enrique Aguado^*, Fernando Barahona, Rafael Solana^* and José Peña^2,*

^* Department of Immunology, Faculty of Medicine, Hospital Reina Sofía, University of Córdoba, Córdoba, Spain; and Centro de Biología Molecular, University Autónoma of Madrid, Madrid, Spain

	Abstract

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It has been described that peptides derived from a highly conserved region of the 1 helix of the first domain of HLA class I Ags exhibit immunomodulatory capacity blocking both T and NK cell cytotoxicity. In vivo treatment with these peptides prolongs survival of MHC-mismatched allografts. However, the molecular bases of these effects are still unclear. In this study, we further analyze the mechanisms by which the dimeric peptide HLA-B2702 (77–83/83–77) induces suppression of NK cell cytotoxicity. This peptide inhibits natural and redirected lysis mediated by NK cells without significantly affecting effector-target cell binding. We have also isolated and sequenced a protein that binds this inhibitory peptide, which structurally corresponds to ß-tubulin. Tubulin is the major protein of microtubules and is involved in target cell killing. Furthermore, B2702 peptide promotes GTP-independent tubulin assembly, producing aggregates that cannot be depolymerized by cold. Treatment of NK cells with Taxol or demecolcine, which interfere with microtubule organization, also prevents NK cell cytotoxicity. Taken together, these results support the hypothesis that the peptide B2702 (77–83/83–77) exerts its inhibitory effect on NK cell cytotoxicity by inducing polymerization of microtubules and interfering with their normal assembly/disassembly dynamics.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
It has been described that peptides derived from a human highly conserved region of the 1 helix of the first domain of HLA class I Ags exhibit immunomodulatory capacity (1), and that some of these peptides also have the property of significantly prolonging allograft survival in mice (2, 3, 4, 5). In particular, the peptide derived from HLA-B2702 (2702.75–84) significantly prolongs allograft survival, inhibits the differentiation of T lymphocytes (1), and blocks both CTL and NK cell-mediated cytotoxicity (5, 6, 7). These effects seem to be independent of MHC Ags expressed on target or effector cells, and although it was originally speculated that TCR- and HLA-specific killer inhibitory receptors were participating, these possibilities have been formally excluded (5). When the structures responsible for mediating the immunomodulatory effect of this peptide were analyzed, contradictory results have been published. It was originally suggested that HLA-derived peptides that inhibit both CTL and NK cell functions bind two members of the heat shock protein (hsp)³ 70 family, constitutively expressed heat shock cognate (hsc) 70, and heat-inducible hsp70 (6). It was later demonstrated that some of the peptides binding hsc70 and peptides mediating inhibition of cytotoxicity do not correlate, suggesting that the peptide immunomodulatory activity is independent of binding to hsc70 (7). Finally, it has been demonstrated that peptide D2702.75–84 inhibited heme oxygenase-1 (HO-1) activity in vitro in a dose-dependent manner (8, 9). This effect is similar to what has been observed with other inhibitors of HO-1, and more important, other peptides derived from B2702.75–84 with similar immunomodulatory activity displayed similar effects inhibiting HO-1 activity (8). These results suggest that HO-1, which is also known as hsp32, could be responsible for the immunomodulatory effect of the peptides HLA-2702.75–84 (7). However, no definitive results have been reported showing the involvement of HO-1 as directly responsible for the impairment of the immune function observed after peptide treatment both in vivo and in vitro. Thus, the molecular basis of the inhibitory effect of HLA-B2702.75–84 on both CTL and NK cell functions is still unresolved and controversial.

The aim of this work was to study the effect of peptide HLA-B2702, 77–83/83–77 (NLRIALR/RLAIRLN) on NK cell-mediated cytotoxicity and to further analyze the mechanism of action mediating its effect on NK cells. Our results show a significant inhibitory effect of this peptide on NK cell activity and that it interacts with a protein of 50 kDa. Sequencing of this protein demonstrated that it corresponds to ß-tubulin. The addition of B2702 peptide to tubulin induced tubulin polymerization in the absence of GTP, producing microtubules that were not depolymerized by cold. These results support the hypothesis that the binding of B2702 peptide to ß-tubulin is involved in inhibition of NK cell cytotoxicity by inducing tubulin polymerization, and interfering with cytoskeleton organization and tubulin chaperone-like activity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
HLA-derived peptides

Peptides with the sequences summarized in Table I were synthesized using an automated peptide synthesizer (431 Peptide Synthesizer; Applied Biosystems, Foster City, CA), purified by preparative reverse-phase HPLC, and shown to be >90% homogeneous by analytic reverse-phase HPLC. For biochemical analyses, a biotin group was attached to the NH₂-terminal amino acid (Chiron Technologies, Clayton Victoria, Australia). Peptides were dissolved in PBS and used for the different assays, as indicated.

View this table: [in this window] [in a new window]   Table I. Amino acid sequences of HLA class I-derived peptides corresponding to residues 77–83/83–77 of the 1 helix of respective HLA class I molecules

K562, 721.221, P815, and NKL cell lines were used. K562 cells derive from a myeloid tumor cell line from a patient with chronic myelogenous leukemia in a blast phase. 721.221 is a HLA class I (A, B, C)-deficient EBV-transformed human B cell line (10). Both cell lines are susceptible to NK-mediated killing. P815 is a NK-resistant murine mastocytoma cell line that expresses FcR. These cells were grown in RPMI 1640 supplemented with 10% FCS and used as target cells in the cytotoxic assays. The NK cell line NKL derives from an aggressive human NK cell leukemia and was grown in the presence of 200 U/ml of rIL-2 (11), as previously described (12). NKL cells are known to express the complex CD94/NKG2A and the inhibitory receptor ILT2 (13).

Cell purification

PBMC from healthy volunteers were separated on density gradient (Lymphodex, Fresenius, Germany). Polyclonal NK (pNK) cells were isolated from PBMC after depleting monocytes and B cells by plastic adherence and passage through nylon wool; for T cell depletion, nonadherent cells were incubated with anti-CD3 (anti-Leu-4; Becton Dickinson, Mountain View, CA) for 30 min at 4°C. After two PBS washes to remove unbound Abs, the cells were bound to goat anti-mouse Ig-coupled magnetic beads (Dynal, Oslo, Norway) for 30 min at 4°C, and then, a magnetic particle concentrator was used (MPC1; Dynal). The resulting population (negative fraction) consisted of 75–90% CD3^-CD56⁺CD16⁺. Positive fraction contained >90% of T lymphocytes, as shown by flow cytometric analysis.

Fluorescence analysis

For flow cytometric analysis, PBMC were stained for 30 min at 4°C in the presence of 1% BSA with the following mAbs: PE-conjugated anti-CD3 (Leu-4), and anti-CD14; FITC-conjugated anti-CD19, anti-CD16 (Leu-11a), and anti-CD56 (Leu-19), from Becton Dickinson. Fluorescence analysis was performed on a FACSort cytometer (Becton Dickinson) after acquisition of 10⁴ events. Viable cells were selected using forward and side scatter characteristics. Resulting profiles were analyzed using CellQuest software (Becton Dickinson).

Cytotoxicity assay

⁵¹Cr release assays were performed as described elsewhere (14). Purified NK cells from different donors and the NK cell line NKL were used as effectors. Target cells were incubated for 90 min with 50 µCi of ⁵¹Cr sodium chromate (CIS Biointernational, Cedex, France) before being mixed with effector cells. An E:T cell ratio of 20:1 was chosen based on results of experiments using serial dilutions of NKL or pNK cells. Assays were performed in triplicate in round-bottom microtiter plates. After 4 h at 37°C, 25 µl of supernatant was collected from each well and spotted onto glass fiber Spot-on filter mats (Wallac, Turku, Finland). Filter mats were analyzed on a 1205 Betaplate counter (Wallac). Peptides were used at different concentrations ranging from 25 to 200 µg/ml, and were preincubated with the effector cells during 30 min before the addition of the target cells. When indicated, peptides were incubated overnight. Cell viability was not affected by incubation with peptides at the doses and times used in our experimental procedures. The percentage of specific cytotoxicity was calculated as follows: ((cpm experimental - cpm spontaneous)/(cpm maximum - cpm spontaneous)) x 100, in which spontaneous release was that obtained from target cells incubated with medium alone, and maximum release was that obtained from target cells incubated with 5% Triton X-100. Spontaneous lysis was always <10% of maximum release. Percentage of inhibition was calculated as ((percentage of specific lysis in the absence of peptides - percentage of specific lysis in the presence of peptides)/(percentage of specific lysis in the absence of peptides)) x 100. In some experiments, the 5:1, 10:1, and 20:1 E:T cell ratios were used and the LU per 10⁶ cells were also calculated. One lytic unit was defined as the number of effector cells required to lyse 20% (LU_20%) of a population of 5 x 10³ target cells. For redirected lysis of the P815 cell line by pNK cells, anti-CD16 mAb (3G8; Immunotech, Marseille, France) was added to the assay at a final concentration of 10 µg/ml. The killing of P815 by pNK cells in the absence of anti-CD16 mAb was always below 2%.

Conjugate formation

Effector-target cell binding was analyzed as previously described (15). pNK cells purified from healthy donors, as described above, were preincubated overnight with B44 or B2702 peptides or with demecolcine (Molecular Probes, Leiden, The Netherlands) and used as effectors. The 721.221 NK-sensitive cells were used as targets. Effector-target cell mixtures (10:1) were centrifuged (900 rpm, 1 min) and incubated at 37°C in a CO₂ chamber. After 1 h, the cells were gently resuspended and conjugates were counted. This incubation time was chosen based on the results of preliminary experiments at different times (not shown), and represents the maximum percentage of conjugate formation. Blind-coded slides were analyzed by two individuals using a phase-contrast microscope (Zeiss, Oberkochen, Germany). Percentage of effector cells involved in conjugates was calculated after counting a minimum of 500 cells.

Precipitation of peptide-binding proteins

NKL cells, pNK cells, and T lymphocytes (50 x 10⁶/ml) were centrifuged, and cell pellets were lysed with (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)/PBS (pH 7.4) containing protease inhibitors, pepstatin (1 µg/ml), leupeptin (10 µg/ml), aprotinin (10 µg/ml), and PMSF (0.2 mM) on ice for 30 min. Cell debris were spun out by microcentrifugation for 15 min at 4°C. Cell lysates were incubated with biotinylated peptides (B2702 or B44) overnight at 4°C. Peptide-binding proteins were precipitated using Ultralink immobilized streptavidin (Pierce, Rockford, IL) for 1 h at 4°C with gentle mixing. After incubation, proteins were pelleted from the lysates by microcentrifugation and washed five times with immunoprecipitation buffer (20 mM sodium phosphate buffer (pH 7.5), 500 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.02% sodium azide).

Electrophoresis and Western blot analysis

Peptide-binding proteins were separated by reducing SDS-PAGE. After electrophoresis, gels were stained with Coomassie blue stain. For Western blot assays, proteins were transferred to polyvinyldifluoride membranes (Amersham Life Science, Little Chalfont, U.K.). The membranes were probed with anti-ß-tubulin (Sigma, St. Louis, MO), followed by HRP-conjugated anti-mouse Ab (Transduction Laboratories, Lexington, KY). Immunodetection using ECL (Amersham Life Science) was performed according to the manufacturer’s instructions. After ECL detection, the membranes were stripped of bound Abs by incubating them in a solution of 100 mM 2-ME/2% SDS/65 mM Tris-HCl (pH 6.7) for 20 min at 60°C. Then the membranes were reprobed using anti-hsp70 (StressGen Biotechnologies, Victoria, British Columbia, Canada), followed by HRP-conjugated anti-mouse Ab, and revealed with ECL as before.

Protein sequencing

Amino acid sequencing of the 50-kDa protein was performed using an automated system sequencer (Beckman LF3000) at the Protein Sequencing Laboratory, Institute of Fundamental Biology "Vicent Villar Palasí," University Autónoma (Barcelona, Spain). The sequence was compared with known sequences from the Swiss-Prot database.

Effect of Taxol and demecolcine treatment on NK cell cytotoxicity

NKL cells were incubated overnight with different doses of Taxol or demecolcine (Molecular Probes) from 10 to 100 µM. After two washes, the cells were counted and their viability was established. Then NKL cells were used as effector cells in a standard cytotoxic assay as before. Viability of NKL cells was not affected at the doses of Taxol and demecolcine used in the experiments.

In vitro tubulin polymerization assay

Tubulin polymerization was analyzed as previously described (16). Purified bovine tubulin was suspended in assembly buffer containing 0.1 M MES (pH 6.5), 1 mM EGTA, 0.5 mM MgCl₂, 0.1 mM EDTA, and 2.5 M glycerol (all reagents from Sigma, St. Louis, MO). The mixture was transferred to a quartz cuvette equilibrated at 37°C. Assembly was conducted at 37°C and monitored spectrophotometrically at 350 nm by following the change in turbidity that is representative of microtubule formation. Changes in the OD were monitored every 3 min in a Spectronics Genesys 8 spectrophotometer. As a control, GTP (1 mM) was used to induce tubulin polymerization. Stock solution (40 mg/ml) of B2702 and B44 peptides was prepared in DMSO and dissolved in assembly buffer to a final concentration of 200 µg/ml. The presence of DMSO at the concentration present in the assay (0.5%) did not affect tubulin polymerization. The depolymerization induced by cold was also studied by switching the assembly temperature to 4°C, after 40 min of incubation at 37° with GTP, Taxol (23.4 µM), and B2702 peptide (36 µg/ml).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
NK cell-mediated cytotoxicity is specifically blocked by B2702 peptide

The effect of B2702 peptide in the killing of K562 by the NK cell line NKL and by pNK cells obtained from donors with different HLA haplotypes was studied. The results show that B2702 peptide strongly inhibited NKL-mediated killing in a dose-dependent manner, whereas other related peptides used as controls did not significantly affect cytotoxicity (Fig. 1A). When the effect of B2702 peptide was studied using pNK cells, we found that B2702 peptide inhibited the NK cell cytotoxicity, but not the other peptides used (Fig. 1B). The results of two experiments, in which the cytotoxicity data were calculated as LU_20%/10⁶ cells, showed that the number of LU_20%/10⁶ cells was reduced upon B2702 peptide treatment (100 µg/ml) from 20 to 13, and from 24 to 13 when NKL were used as effector cells. Cytotoxicity by pNK was also reduced after B2702 peptide treatment from 13.5 to 9.5, and from 14.5 to 6 LU_20%/10⁶ cells. No reduction in the number of LU was observed when the other peptides were used. No relationship was found between the inhibitory effect of the B2702 peptide and the haplotype of the effector cells used. To identify which population, effector or target, is involved in the decreased lysis due to B2702 peptide, we treated both target and effector cells with this HLA-derived peptide. Effector or target cells were incubated separately with the peptide overnight, then washed and used in a standard cytotoxicity assay. Inhibition was only observed when effector cells were incubated with B2702 peptide and no effect was detected after treatment of target cells with the peptide (Table II). The influence of the B2702 peptide on the binding of target and effector cells was also studied. The results in Fig. 2 show that the conjugate formation of pNK and 721.221 cells was not significantly affected either by B2702 or B44 peptides.

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Effect of HLA-derived peptides on the lysis of K562 target cells by NKL (A) and pNK (B) cells. An E:T ratio of 20:1 was used. Peptides were added to the assays at the indicated concentration 30 min before the addition of target cells. Each point represents the mean of three independent experiments. The mean ± SD values of specific lysis are 33 ± 15 for NKL and 26 ± 3 for pNK cells. Percentage of specific lysis and percentage of inhibition were calculated as described in Materials and Methods. No variations were observed between pNK cells from different donors.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. B2702 peptide does not significantly affect conjugation of pNK cells with 721.221 cells. Effector pNK cells were used untreated or after overnight treatment with B2702 and B44 peptides at 100 and 200 µg/ml or with demecolcine (demec.) at 10 and 50 µM. The 721.221 NK-susceptible cell line was used as target. E:T ratio was 10:1. Conjugate formation was analyzed after 1 h of incubation at 37°C. Percentage of conjugated effector cells was calculated after counting a minimum of 500 cells. Results represent the mean of three independent experiments.

The P815 cell line was efficiently killed by pNK cells in the presence of anti-CD16 mAb, which binds the FcR on target cells and the triggering receptor CD16 on effector cells, whereas killing of P815 in the absence of anti-CD16 mAb was always below 2%. When the B2702 peptide was added to the assay, redirected lysis was blocked, but at a lesser extent than that observed in direct NK cell-mediated lysis (Fig. 3). B44 peptide did not produce any effect on lysis. Analysis of the cytotoxicity data based on calculation of LU_20%/10⁶ cells showed similar levels of inhibition by the B2702 peptide. Thus, in an additional independent experiment, the cytotoxicity of pNK cells against P815 + anti-CD16 mAb and 721.221 target cells was 17.5 and 21.5 LU_20%/10⁶ cells, respectively; after B2702 peptide treatment (200 µg/ml) was 10 and 5.5 LU_20%/10⁶ cells; and after B44 peptide treatment (200 µg/ml) was 19.5 and 23.5 LU_20%/10⁶ cells.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. Effect of HLA-derived peptides on anti-CD16-redirected cytotoxicity against P815 cells compared with natural cytotoxicity of 721.221 cells. Lysis of the NK cell-resistant P815 cell line by pNK cells occurred only in the presence of anti-CD16 mAb at 20 µg/ml. The inhibitory B2702 peptide and B44 control peptide were used at 200 µg/ml. Three representative experiments are shown. Specific lysis values (mean ± SD) of these three experiments are 41 ± 6 for P815 and 55 ± 12 for 721.221 cells. Percentage of specific lysis and percentage of inhibition were calculated as described in Materials and Methods.

To further elucidate the mechanism of action of the B2702 peptide, specific peptide-binding proteins were isolated from NKL and pNK cells. Peptide-binding proteins isolated from T lymphocytes were also used as control. Cell lysates were treated with biotinylated peptides and precipitated with Ultralink immobilized streptavidin, and peptide-protein interactions were determined by SDS-PAGE. A protein of molecular mass of 50 kDa that binds to the inhibitory peptide B2702, but not to the noninhibitory peptide B44, was found (Fig. 4). As previously described (6), the B2702 peptide binds to an additional protein of 70 kDa on T cells (Fig. 4).

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Coomassie staining of peptide-binding proteins. Peptide-binding proteins were precipitated using B2702 and B44 peptides and identified on 10% SDS-PAGE. The inhibitory B2702 peptide binds specifically to a 50-kDa protein on the NK cell line, NKL, pNK cells, and T lymphocytes. An additional protein of 70 kDa is also observed on T lymphocytes, but not in NKL cells and pNK cells. Control peptide (B44) and Ultralink immobilized streptavidin alone (ctrl) did not precipitate any protein.

Amino acid sequencing of 14 N-terminal residues of the protein identified in the 50-kDa band obtained from NKL lysates indicates that this protein corresponded to the ß-tubulin, isotypes I and III (Table III). The presence of ß-tubulin was further confirmed by Western blot using specific Abs against this protein. Thus, ß-tubulin was coprecipitated with the B2702 peptide in all of the cells tested, including NKL and T cells (Fig. 5A). The p50 band on the Western blot corresponded to the single band observed in the SDS-PAGE gel of NKL lysates precipitated with B2702 peptide. It is of interest to note that, although this p50 band was not detected on Coomassie staining when the noninhibitory peptide B44 was used, binding of this peptide to ß-tubulin was detected by Western blot, indicating that B44 peptide can also interact with tubulin. The amount of tubulin precipitated with the B44 peptide is apparently lower than the amount precipitated with the B2702 peptide. Although these differences could be interpreted by different binding affinities of peptides to tubulin, the data presented do not exclude other possibilities. Thus, although the B44 peptide might interfere with the binding of the anti-tubulin mAb to tubulin, the fact that no precipitation band was detected by Coomassie staining when this B44 peptide was used does not support this possibility. Furthermore, the results presented below, indicating that B2702 but not B44 peptide induces polymerization of tubulin, suggest that the different amount of protein precipitated by these peptides could be due to the induction of tubulin polymerization by the B2702 peptide during the experimental procedure that will carry increased amounts of tubulin subunits in the precipitate, but not when B44 peptide was used. Ultralink immobilized streptavidin alone did not precipitate tubulin (Fig. 5A).

View this table: [in this window] [in a new window]   Table III. N-terminal amino acid sequence of the eluted 50-kDa band compared to human ß- and -tubulins

View larger version (53K): [in this window] [in a new window]   FIGURE 5. Identification of peptide-binding proteins by Western blot. Peptide-binding proteins were precipitated, separated on 10% SDS-PAGE, blotted, and then probed with anti-ß-tubulin Ab. A, A band of 50 kDa corresponding to ß-tubulin was identified in the NK cell line, NKL, and T lymphocytes after precipitation with B2702 peptide. B, After stripping of bound Abs, membranes were revealed with anti-hsp70 Ab. A band of 70 kDa corresponding to hsp70 was detected in NKL lysates and T cell lysates when precipitated with B2702 peptide. Control peptide (B44) and Ultralink immobilized streptavidin alone (ctrl) did not precipitate either ß-tubulin or hsp70.

Effect of Taxol and demecolcine on NK cytotoxicity and conjugate formation

When NKL cells were treated with Taxol and demecolcine, which have the capacity to interfere with microtubule organization, we found that cytotoxic activity decreased (Fig. 6). As shown in Fig. 2, demecolcine did not significantly affect binding of pNK to 721.221 target cells. This demonstrates that correct ß-tubulin polymerization is required to develop normal cytolytic activity, whereas it is not required for conjugate formation (15).

View larger version (30K): [in this window] [in a new window]   FIGURE 6. Effect of Taxol and demecolcine treatment on NKL cytotoxicity. Inhibition of cytotoxicity after treatment of the NK cell line, NKL, with Taxol or demecolcine at different doses. Cells were incubated overnight with the drugs and used in a conventional lysis assay against K562 cells. Each point represents the mean of three different experiments. Percentage of specific lysis was calculated as described in Materials and Methods.

To study whether the B2702 peptide has a direct effect on tubulin function, in vitro tubulin polymerization in the presence of peptides was analyzed. Fig. 7A shows that the addition of GTP at 37°C induced tubulin polymerization to microtubules, evidenced by a rapid increase in OD. When the assembly temperature was switched to 4°C, a gradual depolymerization of microtubules was observed (Fig. 7B). The addition of B2702 peptide induced polymerization of tubulin to microtubules at 37°C in the absence of GTP (Fig. 7A), and stabilized them against depolymerization by cold (Fig. 7B). A similar effect promoting polymerization and stabilization of microtubles was observed when Taxol was used (Fig. 7B). In contrast to the B2702 peptide, B44 peptide failed to induce tubulin polymerization (Fig. 7A).

View larger version (20K): [in this window] [in a new window]   FIGURE 7. B2702 peptide induces polymerization of tubulin in the absence of GTP and stabilization of microtubules. A, Tubulin polymerization: assembly of microtubules with assembly buffer alone (control), GTP (1 mM), B2702 (200 µg/ml), and B44 peptide (200 µg/ml). Additions were made at time 0. The assembly temperature was 37°C. DMSO was contained at a final concentration of 0.5%. B, Microtubule stabilization by B2702 peptide: switch of the assembly temperature to 4°C (arrowhead) induces depolymerization of GTP (1 mM)-induced microtubules, but did not induce depolymerization of B2702 (36 µg/ml) or Taxol (23.4 µM)-induced microtubules. Turbidity, as a measure of polymerization, was followed at 350 nm every 3 min. Results are represented as increments of absorbances. Two representative experiments are presented.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

To identify the mechanism mediating the inhibitory effect of B2702 peptide on NK cells, we have isolated the proteins that bind this peptide. Our results demonstrate that B2702 peptide strongly binds to ß-tubulin in the NKL cell line, pNK cells, and T lymphocytes. Binding of B2702 peptide to hsp70 was also demonstrated in T lymphocytes and in the NKL cell line. Tubulin is the major protein of microtubules, and dimers of the and ß isoforms are the core of microtubules (17, 18, 19, 20). It has hydrophobic domains located at the surface of the protein and hydrophilic tails especially rich in acidic residues. Tubulin polymerization to form microtubules is a reversible process that requires the presence of GTP. Microtubules form diverse structures that are used for many different functions, including the determination of cell polarity, cellular locomotion, and granules secretion. Our results show that whereas GTP induces tubulin polymerization to form microtubules that depolymerize when temperature is switched to 4°C, B2702 peptide induces not only tubulin polymerization in the absence of GTP, but also stabilization of microtubules against depolymerization by cold. This effect is similar to the effect of Taxol, a well-known antimitotic drug that induces tubulin polymerization in the absence of GTP stabilizing microtubules irreversibly (21, 22, 23, 24). Thus, B2702 peptide has a direct effect on tubulin polymerization that results in the disruption of tubulin assembly/disassembly dynamics, strongly supporting that the inhibition of NK cell cytotoxicity mediated by B2702 peptide is related to its capacity to bind tubulin and to interfere with tubulin polymerization and function.

The possible involvement of tubulin in NK cell lysis machinery has been previously shown by several authors. Thus, it has been reported that rearrangements of tubulin-dependent cytoskeleton are produced in both effector and target cells (25, 26). Microtubules provide a polarized scaffold along which cytotoxic granules bind and move (27), although inhibition of granule exocytosis that results in reduced NK spontaneous and Ab-dependent cytotoxicity may happen without affecting rearrangement of tubulin (15). It has also been shown that inhibition of cell microtubule assembly in vivo by colchicine prevents rejection of renal allografts in rats promoting graft long-term survival (28). Sirianni et al. (29) have demonstrated that a defect in tubulin polymerization is involved in the impairment of the NK cell function in HIV-1-infected individuals. Moreover, although microtubule assembly is not required for effector-target cell binding, it is essential for further processes involved either in target cell killing (29) or in the inhibition of growth of Cryptococcus neoformans mediated by NK cells (30). Our results using Taxol or demecolcine, another microtubule disruptor that inhibits tubulin polymerization (25), also show that the integrity of microtubule dynamics is required for NK cell cytotoxicity. Demecolcine, as B2702 peptide, did not significantly affect conjugate formation, indicating that microtubules are not essential for effector-target cell binding (15) and that inhibition of NK cytotoxicity by B2702 peptide or demecolcine is a postbinding event.

A weak binding of the B44 peptide to tubulin was detected by Western blot. Furthermore, biochemical studies have demonstrated that B44 peptide and other HLA 1 helix-related peptides bind with different affinities to the hydrophilic tails of - and ß-tubulins (T. Sarkar, T. Manna, R. Mahapatra, A. Poddar, B. Bhattacharyya, R. Solana, R. Tarazona, and J. Pena, manuscript in preparation). However, the B44 peptide did not significantly affect NK cytotoxicity or induce tubulin polymerization, supporting the association between cytotoxicity and microtubule dynamics. The causes why B44 peptide did not block NK cell cytotoxicity or induce tubulin polymerization in our experimental systems could be related to different binding sites of B2702 and B44 peptides.

Although the main function of tubulin is the maintenance of microtubule organization, Guha et al. (31) have demonstrated that tubulin also has the ability to suppress the thermally and chemically induced aggregation of other proteins, indicating that tubulin has some functions and features similar to many known molecular chaperones. Molecular chaperones are defined as a class of unrelated proteins whose function is to assist in the correct folding of other proteins in the cells (32). Furthermore, the binding of other HLA 1 helix-related peptides to - and ß-tubulins inhibits their chaperone-like activity (T. Sarkar, T. Manna, R. Mahapatra, A. Poddar, B. Bhattacharyya, R. Solana, R. Tarazona, and J. Pena, manuscript in preparation). It is of interest to note that B2702-related peptides bind to hsp70 and HO-1, both molecules with chaperone activity, in human T lymphocytes (6, 8). However, some CTL-inhibitory peptides did not bind hsp70 family chaperones (8). Our results confirming that B2702 peptide binds hsp70 on T lymphocytes and to a lesser extent on NK cells suggest that modulation of chaperone-like activity of both tubulin and hsp70 by B2702 peptide may contribute to its immunosuppressive effect. In addition, it has been described that other immunosuppressant molecules, such as deoxyspergualin (33) and mizoribine (34), specifically interact with hsc70 and hsp60, respectively, supporting the possible role of hsp on cell cytotoxicity.

In conclusion, our results support that B2702 peptide inhibits NK cell cytotoxicity by strongly binding ß-tubulin and inducing tubulin polymerization, which could affect not only cytoskeleton organization, but also chaperone-like activity of tubulin.

	Acknowledgments

 
The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health: human rIL-2 from Dr. Maurice Gately, Hoffmann-LaRoche (Nutley, NJ).

	Footnotes

 
¹ This work was supported by Grants SAF 96-0259 (to J.P.), FIS98-1051 (to J.P.), FIS98/1052 (to R.S.), and SAS 1889 (to J.P.). R.T. was supported by a research contract from the Spanish Ministry of Education (until May 1999) and from Fondo de Investigaciones Sanitarias (98/3082, from May 1999). M.D.G. is a research fellow from the CajaSur Foundation.

² Address correspondence and reprint requests to Dr. José Peña, Departamento de Inmunología, Hospital Universitario Reina Sofía./. Menendez Pidal s/n, 14004 Córdoba, Spain.

³ Abbreviations used in this paper: hsp, heat shock protein; HO-1, heme oxygenase-1; hsc, heat shock cognate; pNK, polyclonal NK.

Received for publication January 18, 2000. Accepted for publication September 15, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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