CD4+CD25+ T cells have immunoregulatory and suppressive functions and are responsible for suppressing self-reactive cells and maintaining self-tolerance. In addition to CD4+CD25+ T cells, there is some evidence that a fraction of CD4+CD25− T cells exhibit suppressive activity in vitro or in vivo. We have shown, using aged mice, that aging not only leads to a decline in the ability to mount CD4+CD25− T cell responses, but, at the same time, renders aged CD4+CD25− T cells suppressive. In this study we report two newly established mAbs that could abrogate the suppressive function of aged CD4+CD25− T cells. These mAbs recognized the same protein, the transmembrane phosphatase CD45. Cross-linking of CD45 on aged CD4+CD25− T cells was required for the disruption of their suppressive activity. Surprisingly, these mAbs also abrogated the suppressive action of CD4+CD25+ T cells in vitro. Our results demonstrate an unexpected function of CD45 as a negative regulator neutralizing the suppressive activity of aged CD4+CD25− and young CD4+CD25+ T cells.
Naturally occurring CD4+CD25+ T cells plays an important role in the maintenance of self-tolerance in vivo and in vitro (1, 2, 3). In fact, depletion of CD4+CD25+ T cells in vivo leads to the development of various autoimmune diseases. In vitro analyses revealed that CD4+CD25+ T cells could suppress the activation of other CD4 and CD8 T cells (4, 5). Recently, it was reported that CD28, CTLA-4, and glucocorticoid-induced TNFR family-related gene (GITR)4 are involved in the functional regulation of these suppressive/regulatory CD4+CD25+ T cells (4, 5, 6, 7, 8, 9). For example, stimulation of CD4+CD25+ T cells through GITR led to the abrogation of their suppressive activity. Furthermore, it was shown that the transcription factor Foxp3 (forkhead/winged helix transcription factor gene) is a critical regulator of CD4+CD25+ T cell development and function (10, 11, 12).
Along with these reports of CD4+CD25+ T cells, evidence is emerging that CD4+CD25− T cells contain some regulatory cell population/activity (13, 14, 15, 16, 17, 18, 19). Previously, comparing young and aged mice, we showed that in addition to the well-characterized suppressive/regulatory CD4+CD25+ T cells, hyporesponsive CD4+CD25− T cells in aged (24 mo old) mice, especially after in vitro prestimulation, exhibit a suppressive effect on the activation of normal CD4 T cells (20, 21). This suppression was not mediated by humoral factors and was dependent on contact between the CD4+CD25− T cells from aged mice and the responder CD4 T cells. Stimulation of aged CD4+CD25− T cells through GITR also broke their hyporesponsive and suppressive status.
Little is known, however, about the molecular regulation of suppressive activities by these aged CD4+CD25− T cells. Using an in vitro culture system, we show that CD45, receptor-like protein tyrosine phosphatase, is involved in the functional regulation of suppressive CD4+CD25− T cells from aged mice. Furthermore, our in vitro analyses revealed that CD45 is involved in abrogating the suppressive action of CD4+CD25+ T cells.
Materials and Methods
Male C57BL/6 (B6) mice and Wistar rats (2 mo old) were purchased from Japan SLC. All young (2 mo old) and aged (24 mo old) mice used in our experiments were maintained in a specific pathogen-free animal facility and treated in accordance with institutional guidelines for animal care. Aged mice with evidence of gross pathology were excluded from the study.
PE-conjugated, FITC-conjugated, or unconjugated Abs against CD4, CD25, and pan-CD45 (30-F11) were purchased from BD Pharmingen. Hybridomas producing Ab against pan-CD45 (M1/9.3.4.HL.2), CD45RA (14.8), CD45RB (MB4B4 and MB23G2), and CD45RC (I/24.D6) were obtained from American Type Culture Collection. After staining, cells were analyzed with an EPICS ALTRA (Beckman Coulter). The Fab of Ab was prepared using an ImmunoPure Fab Preparation kit (Pierce).
Preparation of mAbs
Wistar rats were i.p. immunized three times every 2 wk with a CD4+CD25− T cell line prepared from aged mice, then i.v. injected with an aged CD4+CD25− T cell line 1 mo later. Spleen cells were fused with P3X63Ag8.653 myeloma cells (from American Type Culture Collection) 3 days after the final immunization. For details of screening of the mAbs, see Results.
Splenic cells were incubated at 5 × 107/ml for 45 min at 37°C with the culture supernatant (SN) of the hybridoma cells secreting anti-CD25 Ab (7D4), and rabbit complement diluted to a final concentration of 1/10 (Cedarlane Laboratories). The treatment was repeated twice, and the resulting CD25+ cell-depleted cell fraction was used for the preparation of CD4+CD25− T cells using magnetic beads conjugated with anti-CD4 (GK1.5) Ab and a magnetic column (Miltenyi Biotec). In all experiments, the efficiency of cell depletion and the purity of cells were analyzed by flow cytometry after incubation with anti-CD25 (PC61) Ab plus FITC-anti-rat IgG (Caltag Laboratories), or PE-anti-CD4 (H129.19) Ab. The purity of the final CD4+CD25− T cell preparation from young or aged mice was typically >98% CD4+ and <0.2% CD25+, or >96% CD4+ and <0.1% CD25+, respectively. Preparations of CD4+CD25+ T cells have been described previously (20). In some experiments, T cells or APCs were precultured with 59.32 Ab at 100 μg/ml for 60 min at 37°C. After three washes, these cells were used as pretreated cells.
CD4+CD25− T cells (1 × 104/well), as responder cells prepared from young mice, were stimulated with 5% SN of anti-CD3 Ab (145-2C11), which induces maximum proliferation, in the presence of mitomycin C-treated spleen cells (1 × 104/well) as APCs, in 96-well, round-bottom plates in DMEM containing 10% heat-inactivated FCS, penicillin (100 U/ml), streptomycin (100 μg/ml), 2 mM l-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 50 μM 2-ME. In coculture experiments, young and aged CD4+CD25− T cells were cultured at a ratio of 1:1. The proliferation of T cells (triplicate cultures) was determined by measuring the incorporation of [3H]TdR (37 kBq/well) for the final 4 h of the 2-day culture. For prestimulation of CD4+CD25− T cells, in addition to anti-CD3 Ab and APC, murine rIL-2 (10 U/ml; donated by Shionogi) was added to the culture. Seven to 10 days later, cells were harvested, washed, and used as cell lines for immunization and proliferation assays. In some experiments, rat whole spleen cells (1 × 104/well) were stimulated with Con A (1.5 μg/ml) in the presence or the absence of suppressive mouse CD4 T cells.
Cells were washed and lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 50 mM iodoacetamide, 1 mM PMSF, 5 mM EDTA, and 10 μg/ml trypsin inhibitor. After a 20-min incubation on ice and following centrifugation, the cell lysates were recovered and incubated with Ab-coupled Sepharose 4FF (Amersham Biosciences) for 100 min at 4°C. After a wash with lysis buffer, bound proteins were subjected to SDS-PAGE (5% gel), blotted onto a membrane, and immunoblotted. In the Coomassie brilliant blue (CBB) staining of gels, a T cell hybridoma established from young CD4+CD25− T cells was used to prepare large numbers of cells.
The plasmid expression vectors for CD45 were constructed with total RNA from young CD4+CD25− T cells. First-strand cDNA primed with an oligo(dT) primer was synthesized using SuperScript II reverse transcriptase (Invitrogen Life Technologies). The extracellular domain and transmembrane region of CD45 were amplified by RT-PCR with the following primers: CD45 sense, 5′-GTCCGGAATTCCTGATCTCCAGATATGACCATGGGTTTG-3′; and CD45 antisense, 5′-ATTGCTCGAGTCATCTCAATTGCTGGATCTTTTCTTG-3′. The primers used for FLAG-containing truncated CD45RO were: antisense, 5′-AGGCCGCGGCCTTGTCGTCATCGTCTTTGTAGTCACCATCACTGGGTGTAGGTGTTTGC-3′; and sense, 5′-TCCCCGCGGCCGCCAGCCTCACAACTCTTACACCAT-3′ (CD45RO), 5′-TCCCCGCGGCCTGTGCTGCCATGTTTGGGAACATTAC-3′ (Δ3), and 5′-TCCCCGCGGCCAACATTACTGTGAATTACACCTATG-3′ (Δ3.1).
The PCR-amplified fragments digested with EcoRI and SacII or with SacII and XhoI were subcloned into the EcoRI/XhoI site of the mammalian expression vector pME18S and introduced into a normal rat kidney cell line (NRK; provided by Dr. K. Miyake, University of Tokyo, Tokyo, Japan) along with the neo-containing vector using FuGene6 (Roche) according to the manufacturer’s instructions. Cells were cultured in the presence of G418, harvested 10 days later, and stained with various Abs and appropriate secondary Ab.
59.32 and I.11 mAbs abrogate the suppressive activity of aged CD4+CD25− T cells
To examine the molecules involved in the functional regulation of suppressive aged CD4+CD25− T cells, we tried to establish mAbs that can abrogate the suppression. Rats were immunized with prestimulated CD4+CD25− T cells of aged (24 mo old) B6 mice, which gain a stronger suppressive activity through a prestimulation culture (21), and the immunized spleen cells were fused with P3X63Ag8.653 myeloma cells. Supernatants from the resulting hybridomas were screened for their ability to neutralize the suppression caused by aged CD4+CD25− T cells. Selected hybridomas in terms of stable neutralizing activity were subjected to subsequent cloning. Finally, two clones (59.32 (rat IgM) and I.11 (rat IgG2c)) were established. Both mAbs were able to abrogate the suppressive activity of aged CD4+CD25− T cells in a dose-dependent manner and had almost no effect on the proliferation of responder or suppressive aged CD4+CD25− T cells alone under these culture conditions, i.e., 5% anti-CD3 Ab (Fig. 1, A and B). Even with 400 μg/ml 59.32 or I.11 Ab, the proliferation of aged CD4+CD25− T cells cultured along with APC plus anti-CD3 Ab was not augmented. (In a representative experiment, the proliferation of aged CD4+CD25− T cells was 5284 cpm without Ab, 5854 cpm with 400 μg/ml 59.32 Ab, and 3501 cpm with 400 μg/ml I.11 Ab.) The pentameric mAb 59.32 was more effective than the monomeric mAb I.11 in abrogating suppression (for example, □ at 0.1 μg/ml mAbs in Fig. 1, A and B). The 59.32 and I.11 mAbs themselves did not stimulate responder cells in the absence of anti-CD3 Ab even in the presence of APC (194 and 327 cpm, respectively, in a representative experiment).
Next, to investigate the distribution of Ags recognized by the 59.32 or I.11 Ab, whole spleen cells prepared from young (2 mo old) or aged (24 mo old) B6 mice were stained with either Ab. Surprisingly, the staining experiments revealed that both molecules recognized by these Abs were expressed on almost all splenic cells (CD4 T cells and non-CD4 T cells) regardless of age (Fig. 1 C).
Both 59.32 and I.11 mAbs recognize the extracellular domain of CD45
To identify molecules recognized by these mAbs, lysates prepared from young CD4+CD25− T cells were immunoprecipitated with 59.32 or I.11 Ab, subjected to SDS-PAGE under nonreducing conditions, transferred onto a membrane, and blotted with either Ab (Fig. 2,A). Unexpectedly, it was suggested that the two mAbs recognize the same molecules with Mr of ∼190 and 170 kDa. Although both Abs lost the ability to interact with these proteins under reducing conditions, CBB staining of the gel run under reducing conditions revealed two bands with slightly higher Mr than those obtained under nonreducing conditions (data not shown). These results suggest that both (190 and 170 kDa) proteins are monomers. Then the two bands in the CBB-stained gel run under nonreducing conditions were excised, treated with trypsin, and subjected to MALDI-TOF/mass spectrometry analysis using a Shimadzu Biotech AXIMA-CFR mass spectrometer. A subsequent database search revealed that these proteins, although derived from two different bands, are identical; they are the transmembrane protein tyrosine phosphatase CD45 (22, 23). To confirm these results, cDNA was prepared from total RNA extracted from young CD4+CD25− T cells and amplified by PCR with primers that cover all CD45 isoforms, which are produced by the alternative splicing of three exons (exons 4–6) in the extracellular domain. Amplified products were subjected to electrophoresis, and the two major PCR products were cloned into an expression vector, pME18S, and sequenced. One of them was identical with CD45RB (expressing exon 5, but not exons 4 and 6), and the other was identical with CD45RO (lacking exons 4–6). NRK cells transfected with the CD45RO-containing vector along with the Neo-containing vector were cultured in the presence of G418. The remaining cells were stained with anti-CD45 mAbs (30-F11 and M1/9.3.4.HL.2), 59.32 Ab, and I.11 Ab (Fig. 2 B). All four mAbs stained the CD45RO transfectant with a different staining intensity. The CD45RB transfectant was also stained with these four mAbs with similar results (data not shown). These results indicate that both 59.32 and I.11 mAbs recognize the common extracellular region in all CD45 isoforms independent of exons 4–6.
Comparison of 59.32 and I.11 mAbs with other anti-CD45 mAbs
In the next experiment we examined whether other anti-CD45 Abs could abrogate the suppressive activity of aged CD4+CD25− T cells. We used six different anti-CD45 Abs, including Abs reactive to all isoforms of CD45 (M1/9.3.4.HL.2 and 30-F11). First, we stained young CD4+CD25− T cells with various anti-CD45 Abs after pretreatment with the various Abs as a competitive Ab. As shown in Fig. 3 A, staining with the 30-F11 or M1/9.3.4.HL.2 Ab was inhibited in the presence of the 59.32 or I.11 Ab. Also, staining with the 59.32 or I.11 Ab was inhibited by prestaining using the 30-F11 or M1/9.3.4.HL.2 Ab, with a somewhat different competitive efficiency. In contrast, staining with other anti-CD45 Abs (14.8, I/24.D6, MB4B4, and MB23G2) examined in our experiments was not influenced by the 59.32 or I.11 Ab (data not shown). These results indicated that the 59.32, I.11, 30-F11, and M1/9.3.4.HL.2 Abs recognize close determinants on CD45.
The extracellular domain of CD45 contains a cysteine-rich region, followed by three fibronectin type III repeats (24). To examine the epitope recognized by the 59.32, I.11, 30-F11, and M1/9.3.4.HL.2 Abs, we established various CD45RO transfectants with a truncated region. Of 12 truncated CD45RO transfectants we established, the results using three transfectants (CD45RO full length, Δ3, and Δ3.1) are shown in Fig. 3 B. The truncated transfectant Δ3 or Δ3.1 has a deletion in the cysteine-rich region: as position 9–38 or 9–44, respectively (accession no. NM_011210). Although the Δ3 CD45 deletion mutant appears to be expressed at lower levels than wild-type CD45RO, its staining intensity was the same for all four anti-CD45 mAbs examined. The Δ3.1 CD45 deletion mutant was expressed at similar levels, as shown by staining with either anti-CD45 mAb, 30-F11 or M1/9.3.4.HL.2. In contrast to these results, the Δ3.1 mutant was not recognized by either of the suppression-abrogative anti-CD45 mAbs (59.32 or I.11). These results demonstrate that the suppression-abrogative Abs (59.32 and I.11) and other anti-CD45 Abs (30-F11 and M1/9.3.4.HL.2) recognize different epitopes on CD45.
Next, we examined whether the 30-F11 and M1/9.3.4.HL.2 Abs as well as the 59.32 and I.11 Abs can abrogate the suppressive activity of aged CD4+CD25− T cells. 30-F11 and M1/9.3.4.HL.2 Abs had no significant abrogative activity (Fig. 3,C). None of the other four anti-CD45 mAbs examined (see Materials and Methods) showed suppression-disrupting activity (data not shown). These results raise the possibility that the 59.32 and I.11 Abs recognize some molecule other than CD45 that is critical in abrogating the suppressive activity, and merely cross-react with CD45. To address this possibility, young and aged CD4+CD25− T cells were stimulated in the presence of the 59.32 or I.11 Ab with or without a competitive Ab (M1/9.3.4.HL.2) or a noncompetitive Ab (MB4B4; Fig. 3,D). The M1/9.3.4.HL.2 Ab was competitive not only in the staining (Fig. 3,A), but also in the culture experiments (Fig. 3,D); that is, the 59.32 and I.11 Abs could not abrogate the suppression by aged T cells in the presence of the M1/9.3.4.HL.2 Ab. Another competitive Ab (30-F11) showed the same competitive pattern with M1/9.3.4.HL.2 Ab (data not shown). In contrast, noncompetitive Ab (MB4B4 (Fig. 3 D, ○) and MB23G2 (data not shown)) had no effect in these culture experiments. Collectively, these results confirm that the extracellular domains of CD45 are involved in regulating the suppressive activity of aged CD4+CD25− T cells. Furthermore, it is suggested that the 59.32 and I.11 Abs recognize a unique extracellular epitope of the CD45 molecule regardless of certain exons (exons 4–6), which is critical in abrogating the suppressive activity of aged CD4+CD25− T cells.
Cross-linking of CD45 on aged CD4+CD25− T cells is required to abrogate their suppressive activity
CD45 is expressed on all hemopoietic cells (22, 23). To examine which cells, i.e., responder T cells, suppressive CD4+CD25− T cells from aged mice, or APCs, are critical targets of the 59.32 or I.11 Ab in removing suppression in our in vitro culture system, the following experiments were performed. First, along with the titrated amount of anti-CD3 Ab, the responder cells were stimulated in the presence or the absence of a fixed amount of the 59.32 Ab. As shown in Fig. 4,A, the 59.32 Ab had a costimulatory effect on responder T cell stimulation at a suboptimal dose (<5%) of anti-CD3 Ab, demonstrating that the 59.32 Ab can act on responder T cells. The I.11 Ab had similar costimulatory activity (data not shown). Second, we used suppressive CD4+CD25− T cells from aged mice with or without pretreatment with the 59.32 Ab. As shown in Fig. 4,B, we observed a significant (p < 0.002) neutralization of the suppressive activity of aged CD4+CD25− T cells after the pretreatment of aged CD4+CD25− T cells with the 59.32 Ab. In contrast, even with pretreated responder T cells or APCs, aged CD4+CD25− T cells exhibited suppressive activity (Fig. 4,C). Third, the 59.32 Ab was added to a stimulation culture of rat spleen cells and aged mouse CD4+CD25− T cells. The 59.32 Ab can only act against the aged CD4+CD25− T cells because it has no reactivity for rat cells (data not shown). In accordance with the previous report (8), as shown in Fig. 4 D, aged CD4+CD25− T cells suppressed the response of rat T cells. However, in the presence of the 59.32 Ab, the suppression was partially (∼60%) abrogated. Taken together, these results demonstrate that the binding of the 59.32 Ab to aged CD4+CD25− T cells is critical to the neutralization of their suppressive activity.
Next, we used Fabs of the I.11 Ab, instead of the whole Ab, to investigate whether the anti-CD45 Ab acts as a cross-linker or merely a blocker in breaking the suppression. Fig. 4,E shows that the Fabs had markedly lost their ability to neutralize the suppression. The Fab binds less efficiently, as shown by decreased fluorescence at lower concentrations (data not shown). However, even at 10 μg/ml Fab, which is 10 times the dose used in staining-saturation, the suppression was not broken. Therefore, this is not likely to explain the lack of efficacy. These results demonstrate that the cross-linking of CD45 molecules is important, rather than the mere binding of these Abs to CD45. In addition, it is plausible that the cross-linking of CD45 by the 59.32 or I.11 Ab leads to unique signaling, because other anti-CD45 Abs had no effect on the suppressive activity of aged CD4+CD25− T cells (Fig. 3). At present, we do not know how the cross-linking caused by the 59.32 or I.11 Ab differs from that caused by other anti-CD45 Abs that have no neutralizing effect.
59.32 and I.11 Abs have costimulatory functions for young and aged CD4+CD25− T cells
As shown in Fig. 4,A, the 59.32 Ab had a costimulatory function on responder T cell stimulation at a suboptimal dose of anti-CD3 Ab. Furthermore, even with an optimal dose of anti-CD3 Ab that can induce the maximum T cell response, the 59.32 and I.11 Abs exhibited costimulatory function against young CD4+CD25− T cells; that is, long-lasting T cell proliferation was observed in the presence of 59.32 or I.11 Ab, but not 30-F11 Ab (Fig. 5,A). The 59.32 and I.11 Abs also exhibited costimulatory function against aged CD4+CD25− T cells (Fig. 5,B). Aged CD4+CD25− T cells showed poor proliferation compared with young cells on culture days 2–3. However, in the presence of IL-2, aged CD4+CD25− T cells showed comparable proliferation with young cells (data not shown). Furthermore, as shown in Fig. 5,B, the 59.32 and I.11 Abs, but not 30-F11 Ab, induced augmented proliferation in the presence of IL-2. It is notable that these augmented and long-lasting proliferations of young and aged CD4+CD25− T cells were induced in the presence of 59.32 or I.11 Ab, but not 30-F11. These results suggest a reason why 59.32 and I.11 Abs, but not other anti-CD45 Abs, can break the suppression. One possibility is that the suppression-breakable Abs induce elevated IL-2 production from young CD4+CD25− T cells at the same time that IL-2 acts on aged CD4+CD25− T cells. Furthermore, 59.32 and I.11 Abs would augment the susceptibility of aged CD4+CD25− T cells to IL-2 (Fig. 5 B). We have previously shown that IL-2 can break the suppression by aged CD4+CD25− T cells (20). Therefore, IL-2 derived from young CD4+CD25− T cells and 59.32 or I.11 Abs themselves act on aged CD4+CD25− T cells, leading to the abrogation of suppression. Currently, these possibilities are being investigated.
We also examined whether 59.32 and I.11 Ab induced apoptosis of aged cells and resulted in suppression-breakage. As shown in Fig. 5 C, there was no significant induction of apoptosis in aged CD4+CD25− T cells among no Ab and 30-F11-, 59.32-, and I.11 Ab-treated groups. 59.32 Ab slightly (up to 20%) induced cell death of young responder CD4+CD25− cells. I.11 Ab caused a variable increase in apoptosis in the young cells that was not statistically significant. Therefore, it is unlikely that 59.32 and I.11 Ab induce cell death of suppressive aged CD4+CD25− T cells and result in the breakage of their suppressive function (for young CD4+CD25+ cells, see below).
59.32 and I.11 Abs are able to abrogate suppression by regulatory CD4+CD25+ T cells
It has been shown that the stimulation of GITR by anti-GITR agonistic Ab abrogated both suppression by the well-characterized regulatory/suppressive CD4+CD25+ T cells and the suppressive effect of aged CD4+CD25− T cells (8, 20). Next, we examined whether CD45 participates in regulating the suppression by CD4+CD25+ T cells. Interestingly, both 59.32 and I.11 Abs were able to inhibit the suppression by CD4+CD25+ T cells (Fig. 6,A). We did not observe any significant induction of apoptosis of CD4+CD25+ T cell by 59.32 or I.11 Ab in annexin V staining (Fig. 5,C; up to 20%; statistically not significant compared with CD4+CD25− responder cells). Moreover, in the coculture of rat spleen cells and mouse CD4+CD25+ T cells with Con A (polyclonal T cell stimuli), CD4+CD25+ T cells suppressed the activation of rat cells, and this effect was partially (∼50%) abrogated by addition of the 59.32 Ab, which is active against CD4+CD25+ T cells, but not rat cells (Fig. 6 B). These results demonstrate that the 59.32 Ab affects CD4+CD25+ T cells, and that CD45 molecules also play a role in regulating the function of suppressive CD4+CD25+ T cells, raising the possibility that CD45 is important for adjusting the suppressive effect of T cells, that is, CD4+CD25+ T cells and aged CD4+CD25− T cells.
CD45 is a receptor-like protein tyrosine phosphatase, is expressed on all nucleated hemopoietic cells, and exists as multiple isoforms due to alternative splicing of three exons (exons 4–6) in the extracellular domain (22, 23). Studies of CD45-deficient cell lines and CD45-deficient mice initially demonstrated an essential role for CD45 in TCR-mediated signal transduction as a positive regulator (25, 26). However, recently, an unexpected function of CD45 has been reported: CD45 could negatively regulate cytokine receptor signaling (27, 28). In our studies of the aging-dependent generation of suppressive CD4 T cells, we established mAbs (59.32 and I.11) capable of breaking the suppressive activity of these CD4 T cells. Surprisingly, these mAbs recognized CD45. Additional in vitro analysis demonstrated that the cross-linkage of CD45 on suppressive CD4 T cells, importantly through a particular portion of CD45 molecules, leads to abrogation of their suppressive activity. These results demonstrate another functional aspect of CD45, that is, the involvement of CD45 in the functional regulation of suppressive/regulatory CD4 T cells. Although the experimental model is very different, it was previously demonstrated that treatment of Con A-induced suppressor cells with anti-CD45RA Ab could block the suppressor function of such cells in vitro assays using human cells (29). Therefore, these phenomena might depend on the common functional aspect of CD45 molecules.
The transcription factor Foxp3 is specifically expressed in CD4+CD25+ regulatory T cells and is required for their development (10, 11, 12). Furthermore, ectopic expression of Foxp3 confers a suppressive function on young CD4+CD25− T cells. As shown in Fig. 6, the 59.32 and I.11 Abs could break the suppression by CD4+CD25+ regulatory T cells. One possibility is that these Abs influence Foxp3 expression, that is, cause a decrease in Foxp3 in CD4+CD25+ T cells, resulting in loss of the suppressive function. We performed a real-time PCR experiment to evaluate the amount of Foxp3 in CD4+CD25+ regulatory T cells before and after 59.32 Ab treatment. The treatment induced no change in the amount of Foxp3. For example, CD4+CD25+ T cells were cultured along with APC, anti-CD3 Ab, and IL-2 with or without 59.32 Ab (100 μg/ml) for 2 days, then relative Foxp3 expression (Foxp3/hypoxanthine phosphoribosyltransferase) was measured. In a representative experiment, Foxp3 expression was 6.39 for uncultured CD4+CD25+ T cells, 2.17 for cultured CD4+CD25+ T cells without Ab, and 2.42 for those with Ab. Therefore, it is unlikely that these Abs break the suppressive activity by diminishing the expression of Foxp3 in the cells.
We showed that Fabs of I.11 mAb lost their ability to break the suppression (Fig. 4), suggesting that the cross-linking of CD45 is required to disrupt the suppression through CD45. One possible means to cross-link CD45 is via CD45 ligand binding. The search for a CD45 ligand continues (23). We also tried to identify a ligand for CD45 with the use of CD45-Ig fusion protein, in which CD45 consists of the truncated extracellular domain, but maintains the 59.32 and I.11 Ab-binding portion. However, we have not yet observed the binding of CD45-Ig protein to T or B cells before and after stimulation. Furthermore, CD45-Ig did not immunoprecipitate any proteins in supernatants prepared from T cell-stimulated cultures. We also added CD45-Ig proteins to a mixed culture of responder CD4 T cells and suppressive CD4 T cells. However, the proteins had no influence on the suppression by CD4 T cells (data not shown). Taken together, these results suggest another mechanism in the cross-linking of CD45 not involving CD45 ligand.
CD45 exists in both monomeric and dimeric forms (30, 31). Several lines of evidence suggest that dimerization of CD45 down-regulates its function and that the CD45 ectodomain regulates this dimerization (30, 31, 32). Furthermore, genetic inactivation of CD45 in cells enhanced cytokine-triggered proliferation (27). Thus, one possible explanation for the 59.32 and I.11 Ab effects is that, first, dimerization of CD45 molecules through the unique epitopes recognized by the 59.32 and I.11 Abs would occur. Second, this dimerization results in the inactivation of CD45 phosphatase activity and leads to enhanced susceptibility to cytokine signaling (27). In accordance with this hypothesis, aged CD4+CD25− T cells could proliferate for a long culture period in the presence of exogenous IL-2 and 59.32 or I.11 Ab, but not in the presence of IL-2 alone (Fig. 5 B). Importantly, another anti-CD45 Ab (30-F11; suppression-nonbreakable anti-CD45 Ab) was not able to induce any augmented proliferation. These results and hypothesis may explain the reason why the 59.32 and I.11 Abs have unique functions compared with other anti-CD45 Abs. Currently, this hypothesis is under investigation.
Our in vitro analyses revealed that cross-linkage of CD45 on suppressive CD4 T cells is required to abrogate their suppressive activity (Fig. 4, B and E). In addition, this suppression breakage was not influenced by the presence of anti-CD45RB Abs (MB4B4 or MB23G2; Fig. 3 D). These anti-CD45RB Abs, used as competitive Abs, would form the cross-link with CD45RB molecules. Even under this condition, the 59.32 and I.11 (anti-CD45RO) Abs could break the suppression. Therefore, these results suggest that the cross-linkage of CD45RO molecules, but not other CD45 isoforms, is critical to the abrogation of suppression.
The anti-CD45 Abs (59.32 and I.11) reported in this study are unique in terms of their costimulatory activity for CD4 T cell activation. Both Abs were costimulatory for CD4 T cell activation (Fig. 4,A and Fig. 5, A and B). In contrast, none of the other anti-CD45 Abs examined had costimulatory activity at 50% of culture SN of hybridoma; rather, some (I/24.D6, MB4B4, 14.8, and MB23G2) were inhibitory of CD4 T cell activation (51.8, 64.3, 25.0, and 44.6% of CD4 T cell response compared with that without any anti-CD45 Ab, respectively), suggesting multifunctional roles of CD45 in CD4 T cell activation. As shown previously, anti-GITR Ab, which can abrogate the suppressive activity of young CD4+CD25+ T cells and aged CD4+CD25− T cells (20), also provides a potent costimulatory signal to CD4 T cells (8, 33). Therefore, this property might be important to suppression-breaking Abs.
The properties of 59.32 and I.11 Abs revealed from in vitro analyses, that is, the ability to abrogate the suppressive activity of CD4 T cells and the costimulatory activity for other responding CD4 T cells, might be useful in the rescue of aged CD4 T cells. In our preliminary experiments, we immunized young or aged B6 mice with OVA in CFA. Administration of the 59.32 or I.11 Ab on day 0 resulted in markedly reduced cell recovery (21.2 ± 14.7% (n = 7) and 17.9 ± 10.0% (n = 6) recoveries, respectively) from draining lymph nodes on day 7 compared with that from immunized mice not inoculated with Ab. These results demonstrate that the 59.32 and I.11 Abs are depletive in vivo. Previously it was shown that defective T cell priming associated with aging could be rescued by signaling through CD137 with the use of an agonistic anti-CD137 Ab in vivo (34). Therefore, by focusing on CD45, establishing other in vivo agonistic/nondepletive anti-CD45 Abs, and inoculating them in vivo, it might be possible to improve immune responses in aged hosts.
The authors have no financial conflict of interest.
We thank Drs. Osami Kanagawa, Tatsuo Katagiri, and Atsushi Kosugi for fruitful discussion, and Toshifusa Toda (Proteomics Collaboration Research Group, Tokyo Metropolitan Institute of Gerontology) for facilities and advice regarding mass spectrometry analysis.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by a Grant-in-Aid for Scientific Research (B) from JSPS, the Naito Foundation, the Japan Health Foundation, the Uehara Memorial Foundation, the Mitsubishi Pharma Research Foundation, the Mitsubishi Foundation, a Research Grant for Longevity Sciences (16A-2; to J.S.), and a Grant-in-Aid for Scientific Research on Priority Areas from MEXT (to J.S. and Y.I.).
Abbreviations used in this paper: GITR, glucocorticoid-induced TNFR family-related gene; CBB, Coomassie brilliant blue; Foxp3, forkhead/winged helix transcription factor gene; SN, supernatant.