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Published online before print August 18, 2006
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* Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Madrid, Spain;
Departamento de Fisiología, Universidad de Barcelona, Barcelona, Spain; and
Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain
3 Correspondence: Servicio de Inmunología, Hospital Universitario de La Princesa,C/ Diego de León, 62, Madrid E-28006, Spain. E-mail: fsanchez.hlpr{at}salud.madrid.org
ABSTRACT
CD69 expression is induced following activation of leukocytes at inflammatory sites and plays a negative regulatory role in the development of collagen-induced arthritis (CIA). To evaluate potential strategies of CD69 targeting in chronic inflammatory diseases, two different anti-CD69 mAbs were generated and their effects on CIA were studied. Administration of the IgG1 anti-CD69 mAb 2.2 to DBA/1 mice with CIA led to an exacerbation of the disease, correlated with down-modulation of CD69 from the cell surface, and reproduced the phenotype of the CD69(–/–) mouse in wild-type animals. In contrast, treatment with the IgG2a anti-CD69 mAb 2.3 was effective in ameliorating CIA when administered in the early or intermediate phases of the disease, causing a decreased production of proinflammatory cytokines in inflammatory foci. Monoclonal antibody 2.3 induces partial depletion of CD69+ cells in vivo. Moreover, adoptive transfer of type-II collagen (CII)-sensitized cells treated with mAb 2.3 to deplete CD69+ cells did not result in arthritis. The attenuation of inflammation correlates with reduced lymphocyte proliferative response in response to CII and with a reduction in the frequency of CII-specific T cells producing IFN-
. We thus conclude that CD69 targeting by mAbs can either enhance or dampen the immune response.
Key Words: antibodies • autoimmunity • T cells • cytokines • chemokines
INTRODUCTION
Collagen-induced arthritis (CIA), a T cell-dependent, Ab-mediated autoimmune condition induced by type II collagen (CII), constitutes a widely accepted experimental model of inflammatory joint disease, particularly rheumatoid arthritis (RA) [1
]. A strong inflammatory cell infiltrate, proliferation of the synovial cell lining (pannus formation), and cartilage and bone destruction are seen in both CIA and RA. Proinflammatory cytokines (IL-1β, TNF-
) and different chemokines are involved in the pathogenesis of the immune-mediated joint damage observed in CIA and RA [2
]. In addition, T cells orchestrate the autoimmune response in CIA, and the generation of CD4-positive CII-specific effector cells in the draining lymph nodes (LN) is critical for its development [3
]. As in other immune-mediated inflammatory conditions, the balance between pro- and anti-inflammatory cytokines is crucial for the outcome of CIA [2
]. Moreover, it is accepted that Th1 cells have a pathogenic role in the development of autoimmune diseases including CIA [4
]. In addition, anti-inflammatory cytokines such as TGF-β1 exert an important immunoregulatory effect in CIA [5
6
7
8
].
CD69 is a homodimeric leukocyte transmembrane protein that is transiently expressed in vitro upon cell activation [9 10 11 12 ] and that is detected in vivo on small subsets of T and B cells in peripheral lymphoid tissues from healthy subjects [13 ]. In addition, CD69 is persistently expressed on leukocyte infiltrates of different chronic inflammatory diseases [14 , 15 ]. After its characterization as an early activation antigen, it was widely proposed that CD69 was involved in the activation and proliferation of different leukocyte subsets [11 , 16 ]. However, recent in vivo data have indicated that this molecule has a different role in the immune system [17 18 19 20 21 ]. Although we have found that hematopoietic cell development, thymocyte positive and negative selection, and T cell maturation are normal in CD69-deficient mice [22 ], these mice show an exacerbated form of (CIA) that correlates with a diminished local synthesis of TGF-β1 [17 ]. Furthermore, CD69-deficient mice challenged with MHC Class Ilo tumors show reduced tumor growth and prolonged survival due to enhanced NK cell activity and reduced TGF-β production [18 ]. Moreover, the in vitro engagement of CD69 induces the production of this immunoregulatory and anti-inflammatory cytokine [17 , 18 ]. Thus, these data strongly suggest that CD69 is a negative regulator of autoimmune reactivity, at least in part through TGF-β synthesis. In contrast, the outcome is different if the control of the adaptive response by T cells is bypassed in the anti-type-II collagen antibody-induced arthritis [21 ].
We have studied the effects on the development of CIA of two different mouse anti-mouse CD69 mAbs administered once the clinical signs have started, following the second injection of CII+CFA. We found that the IgG1 anti-CD69 mAb 2.2, which down-regulates CD69 from the cell surface [23
], reduced TGF-β1 synthesis, enhanced the release of proinflammatory cytokines, and resulted in an exacerbated CIA, thus mimicking the phenotype of the CD69(–/–) mice. In contrast, the IgG2a anti-CD69 mAb 2.3 had a clear-cut beneficial effect on CIA, mainly mediated by the partial depletion of CD69+ proinflammatory cells that leads to the reduction in the frequency of CII-specific cells secreting IFN-. The differential functional effects of these two mAbs on CD69 appeared to be related to their different isotypes and distinct abilities to activate complement. These results thus underscore CD69 as a possible therapeutic target for immune-mediated diseases.
MATERIALS AND METHODS
Mice
Mice were bred at the Centro Nacional de Biotecnologia (Madrid, Spain) under SPF conditions. CD69-deficient mice on a Balb/c background (more than 12 backcrossing generations) were used for the production of mouse anti-mouse CD69 mAb. All experiments involving collagen-induced arthritis were performed using DBA/1j mice that were purchased from Charles River (Barcelona, Spain). All procedures involving animals and their care were performed according to institutional guidelines that are in compliance with international laws and policies.
Induction and assessment of CIA
CFA was prepared by mixing 20 mg heat-killed M. tuberculosis (H37Ra; Difco, Detroit, MI) with 20 ml IFA (Sigma, St. Louis, MO). Chick CII (Sigma, 2 mg/ml) was dissolved overnight at 4°C in 10 mM acetic acid and mixed with an equal volume of CFA. Mice were injected i.d. at the base of the tail and boosted at day 21. Control mice were treated with CFA without CII. Arthritis severity was monitored by direct examination with a digital caliper according to the following scale: grade 0, no swelling; 1, slight swelling and erythema; 2, pronounced inflammation; 3, joint rigidity. Each limb was graded, giving a maximum possible score of 12 per animal.
Monoclonal antibody production
Mouse CD69-specific mAbs were generated by the fusion of NS-1 myeloma cells with spleen cells from a CD69–/– mouse previously immunized three times with mouse 300-19 pre-B cells [18
]. Hybridomas that produced antibodies reacting with murine CD69 were selected and were subcloned twice. Antibody isotypes were determined using a commercial kit (Boehringer Mannheim, Mannheim, Germany). mAb were purified from concentrated supernatants obtained in a INTEGRA CL 350 flask (Integra Biosciences AG, Switzerland) using a protein G column (Pharmacia-Biotech, Uppsala, Sweden). Purified mAb were dialyzed extensively against PBS and stored at –20°C. Purified mAbs were tested for endotoxin using the Limulus amebocyte lysate assay (Cambrex, Bio Science, Walskerville, MD) and endotoxin levels were <0.1 EU/ml
Cytotoxicity assay
Splenocytes from WT mice or CD69–/– mice (negative control) treated with 5 µg/ml Concanavalin A (Con A) for 24 h and then cultured in the presence of IL-2 (100 U/ml) were used as target cells. Recombinant human IL-2 obtained from M. Gately (Hoffmann-LaRoche, Nutley, NJ) was provided by the National Institutes of Health AIDS Research and Reference Reagent program, Division of AIDS. Target cells were loaded with 100 µCi of Na2 51CrO4 (Amersham Pharmacia Biotech) for 2 h at 37°C, washed four times and plated (1x104 cells) in 96-well plates with incomplete culture medium. mAb were added at the indicated final concentration, and the plates were incubated 15 min at 4°C. Then, rabbit complement (Cedarlane, Hornby, Ontario, Canada) at a final dilution of 1/10 was added. Subsequently, the plates were incubated for 45 min at 37°C and centrifuged at 200 g for 5 min. Radioactivity of each supernatant was measured, and the percentage of specific lysis was calculated as follows: % specific lysis = [(sample cpm – spontaneous cpm) / (maximal cpm – spontaneous cpm)] x 100. The spontaneous release of 51Cr was always <10% of maximal cpm, and all experiments were run in triplicate.
In vivo characterization of anti-CD69 mAbs
To determine the in vivo effect of anti-CD69 2.3 mAb, we injected DBA/1 mice with ConA (50 µg per paw per injection) to induce CD69 expression in draining LN. Anti-CD69 2.3 mAb and the isotype-matched control 2.22 mAb were i.v. injected (300 µg), and the possible depletion of activated CD69-expressing T cells was studied by flow cytometry.
As a direct assay to determine the possible role of mAb 2.3 in depletion of CD69-positive cells in vivo, T-lymphoblasts were cultured from spleen cells obtained from C57BL6 mice or CD69–/– mice as described in the previous section. After growing 4 days in IL-2, T-lymphoblasts were collected, washed, resuspended at a concentration of 107 cells per milliliter in RPMI 1640 medium, and labeled with 0.5 µM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) by incubation for 15 min at 37ºC. After incubation, excess CFSE was quenched by adding excess amount of fetal bovine serum and washing in RPMI 1640. After labeling, cells were resuspended in medium at a concentration of 5 x 107 cells per milliliter and 200 µl of the cell suspension were injected per mouse i.v. in the tail vein. Mice were subsequently injected i.p. with 300 µg of anti-CD69 mAb or isotype-matched control. After 48 h, spleen cells from the injected mice were analyzed by flow cytometry for CFSE labeling and CD8 expression, and the proportion of CFSE+ cells was determined.
For treatment of CIA, after boosting immunization with CII (day 21), anti-CD69 mAbs were injected i.p. (500 µg mAb per injection) at indicated times.
Adoptive transfer of arthritis
T cells from draining LN of DBA/1 mice were purified 2 days after boosting with CII+CFA, and treated in vitro with 2.3 mAb or the isotype control 2.22 mAb (IgG2a) in the presence of baby rabbit complement (Serotec, Oxford, UK). Partial depletion of CD69-positive cells was tested by flow cytometry and cells (5x105) were then i.v. transferred to naïve DBA/1 mice. CFA-CII was then injected and CIA development after CII boosting was monitored as indicated above.
Quantitative real-time RT-PCR analyses
Joints or LN were homogenized with a Polytron® (Kinematica, Littau, Switzerland), and total RNA was isolated using the Ultraspec RNA reagent (Biotecx, Houston, Texas). For cDNA synthesis 2 µg of DNaseI-treated RNA were reverse transcribed with MuLV RT (Roche Diagnostics Ltd, Lewes, UK). Real-time PCR was performed in a Lightcycler rapid thermal cycler system (Roche) using primers specific for the different cytokines that generated products of 
200 bp in length. Results for each cytokine were normalized to GAPDH expression and measured in parallel in each sample. The sequences of the primers used were: TGF-β1-F 5'CCG AAG CGG ACT ACT AT3'; TGF-β1-R 5'GTA ACG CCA GGA ATT GT3'; TNF-F 5'TCA TGC ACC ACC ATC AAG GA3'; TNF-R 5'GAG GCA ACC TGA CCA CTC TCC3'; MCP-1-F 5'CAC CAG CAA GAT GAT CC3'; MCP-1-R 5'ATA AAG TTG TAG GTT CTG ATC TC3'; IL1-β-F 5'TCA GGC AGG CAG TAT C3'; IL1-β-R 5'GTC GTT GCT TGG TTC T3'; GAPDH-F 5'TGG GTG TGA ACC ACG A3'; GAPDH-R 5'ACA GCT TTC CAG AGG G3'. IL-4-F 5'CAT CGG CAT TTT GAA3'; IL-4-R 5' CGT TTG GCA CAT CCA TCT CC3'; IFN--F 5' TGG CTC TGC AGG ATT TTC ATG3'; IFN--R 5' TCA AGT GGC ATA GAT GTG GAA GAA3'.
Ex vivo functional assays following in vivo anti-CD69 2.3 mAb treatment
To determine whether in vivo treatment with mAb 2.3 affected the pattern of cytokine secretion by effector T cells, DBA/1 mice were s.c. injected in the footpads with a mixture of CFA+CII prepared as described above, and subsequently injected i.p. with anti-CD69 mAb or isotype-matched control (300 µg per mouse). After 5 days, draining LN from the treated mice were extracted and LN cells were incubated in 96-well plates with boiled CII (100 µg/ml) for 48 h. Then, cells were stimulated with PMA (20 ng/ml) plus ionomycin (500 ng/ml) for an additional 6 h and incubated with 50 µg/ml of brefeldin A (Sigma) during the last 4 h. Cells were stained for CD4, fixed with 2% paraformaldehyde for 10 min, treated with 0.3 saponin for 10 min, stained with PE-conjugated anti-IL-4 and APC-conjugated anti-IFN-, and analyzed on a FACSCalibur for IL-4/IFN- expression on CD4+ cells.
For cell proliferation assays, draining LN were extracted and lymphocytes were cultured in 96-well plates in the presence of different doses of boiled CII, or of an anti-CD3 mAb (10 µg/ml). Cells were incubated for 48 h and then pulsed with 1 µCi per well of [methyl-3H] thymidine (Amersham, Little Chalfont, UK) for the last 12 h of culture before harvesting onto glass fiber filters for determination of [3H] thymidine uptake.
Flow cytometry
Cells (1x106) were incubated with a mixture of anti-CD16/CD32 mAb (BD-PharMingen) to block Fc receptors. Cells were then stained for 30 min on ice with FITC-, PE-, or –Allophycocyanin (APC)-conjugated antibodies or with biotinylated antibodies followed by streptavidin-PE (Molecular Probes), or –APC (BD-PharMingen). The following antibodies were used, anti-CD3 (145-2C11), -CD4 (L3T4), -CD8 (53-6.7),-CD25 (7D4), -IL-4, -IFN-, and -CD69 (H1.2F3) (all from BD-PharMingen). Finally, cells were washed and analyzed on FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA).
RESULTS
In vitro characterization of mouse anti-CD69 mAbs
The immunization of CD69–/– mice with mouse CD69-expressing cells allowed the selection of two mouse anti-mouse CD69 mAbs, 2.2 (IgG1) and 2.3 (IgG2a). We first studied the in vitro capacity of these mAbs to mediate complement-dependent cytotoxicity (CDC) on target cells that expressed CD69. Splenocytes cultured with Con A to induce CD69 expression were incubated with anti-CD69 mAbs plus complement. mAb 2.3 induced cell death in a dose-dependent manner, whereas 2.2 mAb was unable to mediate CDC at any dose tested (Fig. 1A
). We then analyzed the ability of these mAbs to bind to Fc receptors using bone marrow-derived DC from CD69(–/–) mice. We found that 2.3 mAb bound specifically to Fc receptors, since binding was blocked by pretreatment with anti-CD16/32 mAb (Fig. 1B)
. In contrast, 2.2 mAb did not bind to Fc receptors expressed by bone marrow-derived DC. Therefore, anti-CD69 mAbs 2.2 and 2.3 show distinct behaviors in vitro that may have different functional correlates in vivo.
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Effects of CD69 targeting in CIA development
To explore the effects of 2.2 mAb in inflammatory diseases, we injected 2.2 mAb to DBA/1 mice with CIA. We found that 2.2 mAb given during the early onset of disease significantly exacerbated CIA (Fig. 3A
). To determine the possible mechanism of action underlying the effect of 2.2 mAb, mRNA levels for different cytokines in the inflamed paws were analyzed (Fig. 3B)
. Monoclonal antibody administration was associated with reduced expression of TGF-β1 in joints, and an enhanced transcription of IL-1β (Fig. 3B)
. These results indicate that lack of CD69 membrane expression results in an exacerbated inflammation, concurring with our data in CD69-deficient mice [17
].
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, and MCP-1 (Fig. 4C)
, but without a significant effect on TGF-β1 gene expression. Finally, treatment with 2.3 mAb had no effect on the severe inflammatory response at the later stages of disease progression (Fig. 4D)
.
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secretory cells was significantly reduced in T cells from mice treated with 2.3 mAb (Fig. 6B
and 6C)
. This effect of 2.3 mAb can be caused by a selective depletion of T cells that respond in proliferation and cytokine production to collagen, contributing to the observed attenuation of CIA. DISCUSSION
In this paper, we establish the therapeutic potential of CD69 targeting in autoimmune disease by using anti-CD69 mAbs of distinct isotypes to augment or inhibit the severity of CIA and show that such mAbs are also valuable reagents to further analyze CD69 function. Analysis of CD69-deficient mice revealed that CD69 is not essential for the development of the immune system [22 ], but it is in fact involved in the control of both autoimmunity [17 ] and NK cell-mediated tumor rejection [18 ], in part through local regulation of TGF-β secretion. Here, we show that the IgG1 anti-CD69 mAb 2.2 mediates down-regulation of CD69 and that its administration induces a similar phenotype to that described in CD69-deficient mice, with reduction of TGF-β production and increased susceptibility to CIA. These data are fully consistent with the inhibitory effects of mAb 2.2 on the growth and metastasis of Class I-lo tumors in CD69+/+ mice, where enhanced NK cell-dependent tumor killing is observed, exactly as found in CD69–/– animals [23 ].
In contrast, the IgG2a anti-CD69 mAb 2.3 potently inhibits the development of CIA. We have found that the initiation of the immune secondary response, when responsive effector T cells are still in the LN, is the period most sensitive to 2.3 mAb treatment. As CD69 is a marker of activated cells with a net proinflammatory effect on CIA, the depletion of all or some of this cellular subset would prevent the development of CIA. Indeed, we found a reduction in the numbers of CD69+ cells in activated LN of 2.3 mAb-treated animals, suggestive of partial depletion. To evaluate this hypothesis directly, we transferred CFSE-labeled T-lymphoblasts to animals treated with 2.3 mAb or an isotype control and measured cell numbers in spleen two days later. The results show a clear partial depletion of labeled cells by 2.3 mAb. This is further supported by adoptive transfer experiments (Fig. 5) , which clearly demonstrate that the in vitro complement-dependent depletion of activated CD25+CD69+ T cells by 2.3 mAb prevents the subsequent transfer of disease by CII-sensitized T cells.
Because depletion of CD69+ cells by 2.3 mAb is only partial, we evaluated to what extent this partial depletion can explain a reduced reactivity against CII, leading to reduced inflammation. Treatment in vivo with 2.3 mAb reduced the proliferation in response to CII and the frequency of CII-specific IFN--secreting cells. These data show that the partial depletion driven by 2.3 mAb effectively affects the number of effector cells to CII, thus explaining the decreased severity of CIA.
Because both anti-CD69 mAbs 2.2 and 2.3 compete for binding to CD69 (not shown), suggesting that both recognize an overlapping epitope on the molecule, the opposing effects of 2.3 and 2.2 mAbs on CIA are therefore related to their different capacity to bind and activate complement, leading to partial depletion of effector cells expressing CD69 in the case of the 2.3 mAb. However, we cannot rule out that the different binding to Fc receptors can play a complementary role. mAb 2.3 is an IgG2a, an isotype that is able to bind in monomeric form to the high-affinity FcRI receptor on mouse macrophages [24
]. In contrast, the IgG1 isotype of 2.2 mAb requires aggregation or formation of immune complexes to interact with Fc receptors [25
]. It can be hypothesized that 2.3 mAb bound to Fc receptors on DCs and could cross-link CD69 molecules on the cell surface in vivo during cell-cell interactions, generating intracellular signals. It is feasible that cross-linking of CD69 mediates the local production of TGF-β in the LN (22, 23), resulting in inhibition of priming of the Th1 response [26
].
Further studies are required to confirm or refute these possibilities, and other explanations must also be considered. For example, binding of 2.2 or 2.3 mAbs may also have differential effects on down-regulation and signal transduction. In addition, CD69 may play a role in the activation of CD4+ regulatory T cells. In this regard, a subset of CD4+CD69+ cells has been detected in peripheral lymphoid tissues and inflammatory infiltrates in a murine lupus model. These cells are anergic, unable to synthesize proinflammatory cytokines [27 ], and inhibit cytokine synthesis by CD4+CD69- cells. Interestingly, peripheral blood mononuclear cells from lupus patients show an increased expression of CD69 [28 ], and the poor in vitro response of these cells to different stimuli is well known. Likewise, freshly isolated human synovial fluid T cells from RA patients display a profound state of hyporesponsiveness that correlates with the expression of CD69 [29 ]. Therefore, some T lymphocytes bearing CD69 appear to possess the two main characteristics of T regulatory cells, namely anergic behavior and regulatory effects [27 , 30 ].
Both the exacerbation of CIA and the inhibition of tumor growth observed in CD69-deficient mice [17 , 18 ] are also observed in wild-type animals treated with 2.2 mAb, which down-modulates CD69 and thus transiently fully reproduces the CD69–/–phenotype [23 ]. Thus, 2.2 mAb provides us with a tool to study the effect of CD69 deficiency in a way complementary to the genetic approach. The coincident results in both systems reveal CD69 as an immunoregulatory molecule [31 ]. However, results in other systems [21 ], previous results in vitro showing an activating role for CD69 [11 , 16 ], and the speculative function of a putative CD69 ligand expressed on macrophages as a proinflammatory molecule [32 ] all suggest that the effects mediated through this molecule could be complex [31 ]. To summarize, our results with the 2.2 mAb confirm in an independent experimental system that CD69 is an immunoregulatory molecule, as previously found in CD69–/– mice [17 , 18 ]. However, CD69 is also a marker of antigen-specific activated T cells with a net proinflammatory effect. Thus, the depletion, though partial, of this subset of activated cells with anti-CD69 mAb 2.3 is a powerful tool to dampen immune response.
ACKNOWLEDGEMENTS
We thank Dr. Roy R. Lobb and Roberto González-Amaro for helpful discussion. This work was supported by grant BFU 2005-08435/BMC from the Spanish Ministry of Education and Science, and the Ayuda a la Investigación Básica 2002 from Juan March Foundation. D. S. is supported by BEFI 01/9191 from the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo). M. G. is an investigator from "Ministerio de Ciencia y Tecnología Programa Ramon y Cajal.
FOOTNOTES
1 These authors contributed equally to this work. 
2 Current address: Immunobiology Lab., Cancer Research UK, London, UK
Received December 21, 2005; revised April 10, 2006; accepted May 11, 2006.
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) or 2.3 mAb (
) or their isotype controls, 2.8 (
) or 2.22 (
), respectively, were added in the presence of rabbit complement and % specific lysis was determined. Arithmetic means ± SD of 3 independent experiments is depicted. (B) Binding of anti-CD69 Abs to Fc receptors in DCs. Bone marrow cells from CD69–/– mice were differentiated to DCs as indicated in Materials and Methods. Left: Binding to Fc receptors of 2.2 mAb in the absence (bold line) or the presence of anti-CD16/32 (thin line) was analyzed as indicated in Materials and Methods. Right: Binding to Fc receptors of 2.3 mAb in the absence (bold line) or presence (thin line) of anti-CD16/32. One experiment out of three is shown.
) were injected at days 22 and 28 (arrows) following the first immunization. (B) Experiment was performed as in A, but anti-CD69 2.3 mAb (
