Human CD26 expression in transgenic mice affects murine T-cell populations and modifies their subset distribution

Human CD26 Expression in Transgenic Mice Affects Murine T-Cell Populations and Modifies Their Subset Distribution Luca Simeoni, Alessandro Rufini, Tiziana Moretti, Pietro Forte, Alessandro Aiuti, and Antonio Fantoni ABSTRACT: CD26 is a type II transmembrane glycoprotein with dipeptidyl peptidase (DPPIV) activity, constitutively expressed in different cell types and contributing to T-cell activation by acting as costimulatory molecule. Although data suggest an important role for CD26 within the immune system, the physiologic function of this molecule is still unknown. To investigate the role of CD26 in vivo we have produced transgenic mice expressing the human molecule in T cells. Human CD26 (huCD26) is constitutively expressed in all thymocytes and peripheral T lymphocytes of these transgenic mice and is endowed with an enhanced DPPIV activity. CD26 transgene expression induces major phenotypic changes to T-cell populations within the thymus and in peripheral blood. After the onset of sexual maturity, huCD26 expression induces an age-related overreduction of thymus ABBREVIATIONS DPPIV dipeptidyl peptidase IV huCD26 human CD26 TCR T-cell receptor ITAM immunoreceptor tyrosine-based activation motif LAT linker for activation of T cells TRIM T-cell receptor interacting molecule

cellularity accompanied by a relative impairment of thymocyte proliferation following lectin stimulation. Also the peripheral blood T-cell pool is reduced in huCD26 transgenic mice and this is accompanied by an increase of the apoptotic rate of CD4⫹ and CD8⫹ subpopulations. Taken together these data suggest that CD26 interferes with transduction pathway(s) needed for the maturation of T cells and plays an important role in T lymphocyte homeostasis in peripheral blood. Human Immunology 63, 719 –730 (2002). © American Society for Histocompatibility and Immunogenetics, 2002. Published by Elsevier Science Inc. KEYWORDS: CD26; thymus; T-cell homeostasis; transgenic mice; proliferation

DN DP SP GH IGF-I GHRH

double negative double positive single positive growth hormone insulinlike growth factor I growth hormone-releasing factor

INTRODUCTION CD26 is a 110 kDa, highly pleiotropic receptor endowed with different biologic activities [1– 6]. It is constitutively expressed in endothelial cells, in hepatocytes and on kidney brush border membranes where it acts as a From the Sezione di Genetica Molecolare (L.S., A.R., P.F., A.F.) and the Sezione di Biochimica Clinica (T.M.), Dipartimento di Biotecnologie Cellulari ed Ematologia, Universita´ di Roma, Rome, Italy; and the San Raffaele Telethon Institute for Gene Therapy (A.A.), Milan, Italy. Address reprint requests to: Dr. Antonio Fantoni, Sezione di Genetica Molecolare, Dipartimento di Biotecnologi Cellulari ed Ematolgia, Universita´ di Roma “La Sapienza,” Viale Regina Elena 324, 00161 Rome, Italy; Tel: ⫹39 (6) 4454302; Fax: ⫹39 (6) 4462891; E-mail: [email protected]. Received May 7, 2002; revised June 14, 2002; accepted June 19, 2002. Human Immunology 63, 719 –730 (2002) © American Society for Histocompatibility and Immunogenetics, 2002 Published by Elsevier Science Inc.

proteolytic enzyme with dipeptidyl peptidase IV activity (DPPIV) [7], important for renal transport and intestinal digestion [8]. By its peptidase activity it cleaves to several chemokines in vitro [9 –12] whose immune functions are yet not fully characterized. The expression of CD26 is regulated in T-lymphoid cells, where it acts as a costimulatory molecule for T-cell activation [13–18]. In cord blood T lymphocytes, as well as in thymocytes, CD26 signaling requires the T-cell receptor (TCR)/CD3 complex [13, 14, 17, 19, 20]. The activation of peripheral T lymphocytes by CD26 may also require the CD2 pathway [14, 18]. CD26 induces a series of molecular events that lead to 0198-8859/02/$–see front matter PII S0198-8859(02)00433-0

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the phosphorylation of a subset of proteins similar to those triggered through TCR [21]. To exert cell activation at least one functional immunoreceptor tyrosinebased activation motif (ITAM) of the CD3 zeta chain has to be tyrosine phosphorylated [22, 23]. The nature of the T-cell activation triggered by CD26 is unclear because it is mediated neither by the recently discovered TCR associated adaptor protein Linker for activation of T cells (LAT) nor by T-cell receptor interacting molecule (TRIM) [24], and also because the CD26 cytoplasmic tail has only six amino acid residues, devoid of any common signalling motifs [17]. CD26 interacts with other signalling molecules, such as tyrosine phosphatase CD45 [25, 26], adenosine deaminase [27, 28], and the insulin like growth factor II receptor [29], which may have a so far unexplored role in this activation process. This study is aimed at exploring the role of human CD26 (huCD26) in driving in the mouse T lymphocyte maturation from thymus to periphery. Bone marrowderived precursors of T lymphocytes undergo in the thymus a complex series of selection events leading to the production of mature T lymphocytes expressing either CD4 or CD8 coreceptors. Thymocytes can be attributed to three developmental stages, on the basis of CD4 and CD8 expression: (a) most immature cells expressing neither CD4 nor CD8 molecules (double negative, DN); (b) immature cells expressing both CD4 and CD8 coreceptors (double positive, DP); and (c) mature cells expressing either CD4 or CD8 (single positive [SP]). Several data suggest that CD26 could play a crucial role in thymus development that is, CD26 expression is tightly regulated in both human [14] and murine thymus [13]; mature thymocytes in the medulla of human thymus display the highest surface level of CD26 [14, 30]; and the level of DPPIV activity increases during thymocyte development, reaching the highest level in mature single positive CD4 or CD8 cells in human [30] as well as in the mouse [31]. In addition, CD26 has a specific signal transduction property in thymocytes [14]. In fact, modulation of CD26 by monoclonal antibodies in these cells induces an enhanced rise of intracellular calcium, and a proliferative response after anti-CD3 treatment. We have studied different aspects of T-lymphocyte biology in transgenic mice constitutively expressing huCD26 in lymphoid cells. We built the CD26 transgene under the regulatory sequences of human CD2 that confer high level, T-cell specific, position-independent gene expression in mouse cells [32–35]. Human CD26 is correctly exposed on murine T lymphocytes, where it displays an intense dipeptidyl peptidase activity. The transgene constitutive expression adds to murine endogenous, regulated CD26 expression and brings to a CD26 activity markedly increased on T cells from these ani-

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mals. We report that CD26 overexpression affects cellularity of thymus and peripheral blood T cells in aging transgenic mice, interfering with their proliferative and apoptotic activities. MATERIALS AND METHODS General Restriction endonuclease and T4 DNA ligase were purchased from Promega (Promega Co., Madison, WI, USA) and New England Biolabs (New England Biolabs Inc., Beverly, MA, USA). All enzymes were used according to the manufacturers’ instruction. Radionucleotides were from NEN Dupont (NEN, Boston, MA, USA). The plasmids p5⬘CD2/coding-CD2 and p3⬘CD2 were provided by Dr. D. Kioussis of the National Institute for Medical Research (London, United Kingdom). The plasmid pKG5-CD26 was provided by Dr. B. Fleischer of the Bernhard Nocht-Institute for Tropical Medicine (Hamburg, Germany). huCD26 DNA Construct and Generation of Transgenic Mice The plasmid p5⬘CD2/coding-CD2, containing the cDNA coding for CD2 under the control of the 5⬘ CD2 promoter [33], was linearized with Bam HI and Xba I and ligated to a 5.5 Kb Bam HI/Xba I fragment comprising the 3⬘ regulatory region of CD2 gene. The latter fragment was excised by digestion with Xba I and Bam HI from the plasmid p3⬘CD2. In the resulting construct [34, 35], the cDNA coding for CD2 is located between the 5⬘ and 3⬘ regulatory regions of CD2 gene. Subsequently, the coding CD2 DNA excised from the construct with Eco RI and Bam HI was replaced by the 4.1 Kb Kpn I/Bam HI fragment containing the VP1 intron of SV40 and the coding region for huCD26 to give the construct 5⬘-CD2.VP1.CD26-C.CD2-3⬘. The 4.1-Kb fragment was ligated to the Eco RI/Bam HI sites by the use of a 50 bp Eco RI/Kpn I linker. The 4.1 Kb fragment Kpn I/Bam HI was excised from the construct pSVL.VP1.CD26 by a digestion with Kpn I and Bam HI. The construct pSVL.VP1.CD26 was obtained by cloning in the plasmid pSVL both the 2.9 Kb Xho I/Xho I region coding for CD26 and the 1.2 Kb Kpn I/Xho I fragment containing the VP1 intron of SV40. The cDNA coding region for huCD26 was obtained from plasmid pKG5 by digestion with the restriction endonuclease Xho I. The construct was excised from the vector pBS with Kpn I and Not I and separated from vector sequences by electroelution after electrophoresis on 0.6% agarose gel; the remaining construct was microinjected in (C56BL/6J X DBA/2) F1 fertilized mouse eggs, according to the method of Hogan and coworkers [36]. Founders were identified by Southern transfer anal-

Characterization of huCD26 Transgenic Mice

ysis using the 2.9-Kb Xho I/Xho I cDNA fragment as a probe. Four different transgenic founder mice were produced in which the presence of huCD26 was at high levels. Cell Preparations Thymus was obtained by dissection from 3- to 45-weekold mice. Spleen, pooled mesenteric and inguinal lymph nodes, and femoral bone marrow were obtained from 1to 8-month-old mice. In all cases, tissues were mechanically disrupted and passed through 70-␮m nylon cell strainer (Becton Dickinson, Mountain View, CA, USA) to obtain single-cell suspensions under sterile conditions. Cells were placed in ice-cold RPMI 1640 medium supplemented with 10% fetal bovine serum (GIBCO/BRL, Life Technologies, Paisley, Scotland), 2 ⫻ 10⫺4 M Lglutamine, 5 ⫻ 10⫺5 M 2-mercaptoethanol, 100-␮g/ml penicillin/streptomycin, and 10-mM HEPES (all from Sigma, St. Louis, MO, USA). Peripheral blood was collected from the retro-orbital sinus. Human peripheral blood lymphocytes were isolated by Ficoll-Hypaque (Pharmacia LKB, Uppsala, Sweden) discontinuous gradient centrifugation. Recovered cells were then washed twice and resuspended at a concentration of 106/ml in Ca⫹⫹ and Mg⫹⫹ free phosphate buffered saline supplemented with 0.1% of bovine serum albumin for immunofluorescent staining. Total cell numbers were determined by microscopic observation using a Burker hemocytometer. Complete mouse blood counts were performed on a CellDyn (Abbot Laboratories, North Chicago, IL, USA) using the veterinary package. Flow Cytometry The following fluorescin (FITC), or phycoerythrin (PE) conjugated monoclonal antibodies were used: anti-human CD26 1F7 FITC (mouse IgG1) [37]; anti-mouse CD4 FITC clone H129.19 (rat IgG2a), anti-mouse CD8 PE clone 53-6.7 (rat IgG2a), anti-mouse CD3 PE clone 29B (rat IgG2b), anti-mouse CD45R (B220; specific for a glycosylation epitope on B cells) FITC clone RA3-6B2 (rat IgG2a) all from GIBCO/BRL. Optimal concentrations of all reagents were determined in preliminary experiments to assure saturation of all antigenic sites. For single or dual fluorescence analysis, single cells suspensions (1 ⫻ 106 cells in 100 ␮l) of lymphoid organs or blood samples (100 ␮l) were incubated with the appropriate monoclonal antibodies for 30 minutes at ⫹4°C. Red blood cells were depleted by incubation in FACSTM lysing solution (Becton Dickinson). Cells were washed, and then resuspended in medium suitable for flow cytometry. Fluorescences were scored from 10.000 viable cells on a FACScan (Becton Dickinson).

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DPPIV Enzymatic Activity Dipeptidyl peptidase IV activity in thymocytes was measured as previously reported [38]. Briefly, single cell suspension of thymocytes were resuspended at 1 ⫻ 105 cells in 96-well plates (Costar, Cambridge, MA, USA) in the appropriate buffer containing 2 mM of the chromogenic substrate Gly-pro-pnitroanilide (Gly-pro-pNA) with or without 1.5 mM of the DPPIV inhibitor DiprotinA. Enzymatic activity was measured after incubation at the indicated time at 37°C and 5% CO2 atmosphere, and the absorbance was determined at 405 nM. Cell Proliferation Splenocytes were cultured at a density of 2 ⫻ 105/0.2 ml in RPMI medium supplemented with 10% fetal bovine serum (GIBCO-BRL), 2 ⫻ 10⫺4 M L-glutamine, 5 ⫻ 10⫺5 M 2-mercaptoethanol, 100-␮g/ml penicillin/streptomycin, 10-mM HEPES, 1-mM sodium pyruvate, 1-mM nonessential amino acids (all from Sigma) in a U-bottomed 96-well microplate (Costar). After 48-hour incubations at 37°C and 5% CO2 atmosphere, the cells were labeled with 0.5 ␮Ci of [3H]-thymidine (185 GBq/ mmol; Amersham Life Science International PLC, Buckinghamshire, United Kingdom) for 18 hours and harvested onto glass fiber filter (Wallac Oy, Turku, Finland). Radioactivity was measured by standard scintillation counting. All assays were performed in triplicate. Thymocytes were cultured as described for splenocytes. For stimulation, anti-mouse CD3 monoclonal antibody clone 145-2C11 (2.5 ␮g/well) (Pharmingen, San Diego, CA, USA), 200 ng/ml ionomycin plus 1 ng/ml phorbol 12-myristate 13-acetate (PMA), or 5 ␮g/ml concanavalin A (ConA), or 2.5 ␮g/ml pokeweed mitogen (PWM) (all from Sigma) was added into the culture. For anti-CD3 stimulation, wells were coated with antibody 145-2C11 for 2 hours at 37°C in 50 ␮l phosphate buffered saline (PBS) and then washed with ice-cold PBS prior to use. After 72-hour incubations at 37°C and 5% CO2 atmosphere, the cells were labeled with 1 ␮Ci of [3H]-thymidine (185 GBq/mmol) (Amersham) for 12 hours and harvested onto glass fiber filter (Wallac). Radioactivity was measured by standard scintillation counting. All assays were performed in triplicate. Apoptosis To evaluate apoptosis, single cell suspension (1 ⫻ 106) of freshly isolated thymocytes were stained with Annexin V-FITC and Propidium Iodide (Bender MedSystem, Boheringer Ingelheim, Germany) according to the manufacturer’s instructions. For peripheral blood apoptosis, 100 ␮l of whole peripheral blood was simultaneously stained with Annexin V-FITC and CD4-PE or CD8-PE.

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Apoptotic cells were following analyzed by flow cytometry.

RESULTS Generation of huCD26 Transgenic Mice In order to generate transgenic mice for human CD26 we prepared a DNA construct where the CD26 cDNA was under the control of the regulatory regions of human CD2 [32, 33]. Genetic crosses of founder mice revealed that the transgene behaves as a single Mendelian trait. As previously reported by our laboratory, these sequences conferred high level, T-cell specific, position-independent gene expression in mouse cells [34, 35]. The tissue specific expression of the CD26 transgene was studied by northern transfer of total RNA extracted from various organs. CD26 expression appeared to be restricted to T cells (data not shown), consistent with the expression of other CD2 driven human transgenes, such as CD4 [34] and HLA-Cw4 [35]. The presence of the CD26 transgene had no apparent effect on the survival of mice, as far as the average size of litters was identical to controls and no evident pathology was observed up to 16 months old. huCD26 Gene Expression and Activity in Mouse Lymphoid Tissue In order to analyze the distribution and the correct exposition of huCD26 on the cell surface, we utilized flow cytometry experiments with two anti-human CD26 monoclonal antibodies, Ta1 [39] and 1F7 [37], with different epitope specificity. We observed that the thymocyte population from line #13 homozygous mice displayed a CD26 membrane density virtually identical to that of PHA activated human PBMCs [Figure 1] and that both 1F7 [Figure 1] and Ta1 (not shown) were able to recognize human CD26 on cell surface, thus suggesting the correct conformation of the human protein on the murine T-cell membrane. The level of DPPIV activity for the Gly-Pro-pNA chromogenic substrate was two- to threefold higher in transgenic thymocytes (Figure 2); therefore, demonstrating the overexpression of the enzymatic activity in mouse cells. Immune fluorescence analysis of thymus and spleen did not reveal alterations in the intensity profiles of markers such as CD3, CD4, CD8, CD45R, and endogenous CD26 (not shown). Data reported so far indicate that the huCD26 transgene is expressed with high density on mouse thymocytes and T lymphocytes, it has a correct conformation, and it fully retains its enzymatic function.

FIGURE 1 Immunofluorescence analysis of human CD26 surface expression from thymocytes of normal and transgenic mice and mononuclear cells of human peripheral blood. In panels (a) and (b), thymic cells were isolated from adult mice and stained with anti-CD26 monoclonal antibody (1F7). Panel (c) illustrates that heparin-treated human peripheral blood was collected and mononuclear cells were isolated by Ficoll density centrifugation. Purified human peripheral mononuclear cells were cultured at 2 ⫻ 106 cells per ml and were activated for 72 hours with 10 ␮g/ml of phytohemaglutinin in RPMI 1640 supplemented with 10% of fetal bovine serum. For each staining 1 ⫻ 106 cells were used.

huCD26 Transgenic Mice Exhibit an Age-Related Decrease in the Number of T Cells The CD26 molecule has two important biologic functions: signal transduction and the capacity to process growth factors and other regulatory proteins. On this basis, overexpression of huCD26 should lead to abnormalities in mouse T-cell populations. To explore this hypothesis, we analyzed in transgenic mice the cellularity of lymphoid organs. Transgenic thymuses revealed (Figure 3a) an evident overall decrease of cell number. This reduction became detectable only 6 weeks after birth, and was completely evident at 8 weeks (32% reduction), 15 weeks (48%), and 45 weeks (51%), when last tested. To confirm that CD26 overexpression affects also peripheral T-cell populations, the T lymphocyte cellularity was measured in the peripheral blood, obtaining similar

Characterization of huCD26 Transgenic Mice

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TABLE 1 Percentage of peripheral blood lymphocyte subpopulations Lymphocyte subsets Total T lymphocytes CD3⫹ Helper T lymphocytes CD4⫹ Cytotoxic T lymphocytes CD8⫹ CD4/CD8 ratio Total B lymphocytes B220⫹

hu-CD26 mice

Normal mice

33.7 ⫾ 3.5 18.6 ⫾ 3.5 16.5 ⫾ 2.5 1.1 53.2 ⫾ 3.3

43.2 ⫾ 2.4 26.7 ⫾ 0.7 13.0 ⫾ 1.4 2.0 45.2 ⫾ 2.2

As described in Materials and Methods, blood samples were simultaneously stained with anti-CD3 (PE) and anti-B220 (FITC), or with anti-CD8 (PE) and anti-CD4 (FITC). Values were expressed as percentage ⫾ standard deviation. At least three mice per group were used.

FIGURE 2 Kinetics of DPPIV enzymatic activity. Thymocytes from huCD26 transgenic and control mice were incubated at 37°C in peptidase buffer containing 2 mM Gly-PropNA chromogenic substrate with or without 1.5 mM of DPPIV inhibitor Diprotin A.

results. As illustrated in Figure 3b, the size of the leukocyte circulating population was significantly lower in transgenic mice (mean 3.4 ⫾ 1.5; n ⫽ 9) than in wild type control mice (mean 5.5 ⫾ 1.6; n ⫽ 5). This decrease affected particularly the leukocyte subset of lymphocytes with almost a 40% reduction of cell number (mean 2.4 ⫾ 1.2; n ⫽ 9 for transgenic mice, versus 4.1 ⫾ 1.5; n ⫽ 5; for wild type mice). This effect was evident for CD3⫹, PB T lymphocytes

(Table 1), thus confirming that the selective decrease of T cell number is consistent with CD26 enforced expression. In agreement with this observation, the proportion of B lymphocytes was only slightly increased when compared with controls (Table 1). When related to age, it became evident that the overexpression of CD26 started to affect the circulating T-cell population after 4 months old, giving raise to a severe decrease in 6- to 8-month-old mice. Other T-cell populations may behave differently, responding to strategies typical of different lymphoid organs. Indeed, single-cell suspensions from spleen, lymph nodes, and bone marrow from transgenic mice did not

FIGURE 3 Analysis of cellularity in murine thymus and peripheral blood. (a) Total thymocytes were isolated from mice at different weeks after birth and counted as described in Material and Methods. At least four mice per group were included. Statistical analysis were performed using Student’s t-test. Differences between hu-CD26 transgenic and wild type mice were statistically significant at 8 (p ⫽ 0.03), 15 (p ⫽ 0.0007), and 45 (p ⫽ 0.003) weeks after birth. (b) Peripheral blood was collected, by EDTA-treated capillary from the retro-orbital sinus. Complete blood counts were performed on a CellDyn 2500 using the veterinary package. A number of nine transgenic and five control mice were analyzed. In the figure means values ⫾ standard error of the mean are reported. Asterisks indicate the statistically significance differences relative to the cpm levels **p ⫽ 0.02, *p ⫽ 0.02.

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TABLE 2 Number of lymphoid cells and blood leukocytes in hu-CD26 transgenic and normal mice Total number of lymphoid cells (⫻106/organ) Lymphoid organ

hu-CD26 mice

Normal mice

Spleen Lymph node Bone marrow Blood leukocytesa

129 ⫾ 52 5.2 ⫾ 2.9 3.0 ⫾ 0.4 3.4 ⫾ 1.5

134 ⫾ 41 3.5 ⫾ 3.4 3.3 ⫾ 0.3 5.5 ⫾ 1.6

Data are expressed as arithmetic mean ⫾ standard deviation. A minimum of five mice were included in each group. a Note that the number of leukocytes is ⫻106 per ml.

demonstrate any obvious difference when compared with wild type mice (Table 2). huCD26 Expression Unbalances the CD4 and CD8 Populations of Transgenic Mice CD26 overexpression may differentially affect various T-cell subpopulations. To characterize the reduction of thymus and peripheral blood cellularity in huCD26 transgenic mice, we tested thymocytes and peripheral blood T cells for CD4 and CD8 expression. As illustrated in Figure 4, no significant difference were detected in the proportion of CD4⫺/CD8⫺ double negative (DN) and CD4⫹/CD8⫹ double positive (DP) thymocytes. However, single positive (SP) mature T populations resulted differentially affected by CD26 overexpression. In fact, while the proportion of CD8⫹ thymocytes was markedly increased, mature single pos-

itive CD4⫹ cells resulted unaffected, resulting in the alteration of the CD4/CD8 ratio. These data suggest that terminal differentiation of T cells is altered in huCD26 transgenic mice. This increased percentage of CD8⫹ thymocytes is not age related as it was observed from 3 to 45 weeks of age, when last tested. This suggests that two different phenomena occur in the thymus of transgenic mice: (a) the reduction of total cellularity after the onset of sexual maturity, and (b) the increase in the proportion of CD8 SP thymocytes. We have reported that T-lymphocyte cellularity is reduced also in the peripheral blood of huCD26 transgenic mice. The modification of the proportion of terminally mature CD8 single positive thymocytes raised the question of whether the CD4/CD8 ratio in the periphery would be maintained. Again, as reported for peripheral blood cellularity, no modification was detected in 1- to 4-month-old mice for the percentage of CD4⫹ and CD8⫹ T lymphocytes. Conversely, we detected in older mice (6 – 8 months) a decrease of both CD4⫹ and CD8⫹ T lymphocyte number in the peripheral blood, this decrease being more marked for the CD4⫹ population (Table 1). T-Cell Apoptosis in the Thymus and Peripheral Blood The finding that thymus and peripheral blood cellularity was decreased in transgenic mice raised the question of whether an increase in the apoptotic rate occurred in these organs. This question was answered by enumerating apoptotic cells by cytofluorimetric analysis of freshly

FIGURE 4 Immunocytometric analysis of CD4⫹ and CD8⫹ T-cell subsets in thymus of huCD26 transgenic and wild type mice. Thymocytes from 4-week-old mice were double stained with anti-CD4 (FITC) and anti-CD8 (PE) and 10.000 viable cells were analyzed on a FACScan. The percentages of the relevant gated cells in the regions marked on each panel are illustrated. One representative mouse of 16 is depicted.

Characterization of huCD26 Transgenic Mice

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FIGURE 5 Analysis of Annexin V⫹ T lymphocytes in peripheral blood. Peripheral blood was simultaneously stained with Annexin V-FITC and anti-CD4-PE (upper panels) or anti-CD8-PE (lower panels) and 10.000 viable cells were analyzed on a FACScan. One of three independent experiments is illustrated.

isolated cells stained with propidium iodide (PI) and FITC-labeled Annexin V. No differences were obtained in thymus of transgenic mice when compared with normal mice (data not shown), which was interpreted as due to the rapid elimination of thymic apoptotic cells by phagocytic cells [40, 41]. However, in the peripheral blood a significant increase (at least twofold) in the number of Annexin V⫹ lymphocytes was observed in both CD4⫹ and CD8⫹ subpopulations (Figure 5). Thus, apoptosis of peripheral blood T cells resulted increased in huCD26 transgenic mice, possibly giving raise to the reduction of this cell pool. Thymocytes From huCD26 Transgenic Mice Display a Defective Proliferative Response It is generally held that pathways that regulate proliferation, survival, development, and homeostasis of T cells are tightly linked. In view of this and having demonstrated that huCD26 expression in transgenic mice perturbate both T lymphocyte cellularity and peripheral blood apoptosis, we checked if thymocyte proliferation was also adversely affected. Initially proliferative activity was assessed by [3H]-

thymidine incorporation in response to lectins Concanavalin A (ConA) and pokeweed mitogen (PWM). These mitogens interact with TCR and activate thymocyte proliferation mostly by mediating surface signalling events. Proliferation resulted markedly and significantly lower in huCD26 transgenic mice when compared with wild type mice (Figure 6a). CD26 inhibition of T-cell proliferation might have been exerted at several levels of the activation pathway, in response to different selected mitogens. In order to explore this, we tested the effect of mitogens with different specificities, among these were the ones that could compensate for the observed decrease of proliferation. The inhibition of proliferation of ConA-stimulated transgenic thymocytes was reversed by the addition of a phorbol ester (PMA), which causes a direct activation of PKC, thus bypassing earlier signaling events (Figure 6b). This result indicates that CD26 overexpression exerts an inhibitory effect at levels preceding PKC activation and was confirmed by the finding that CD26-induced inhibition was absent when thymocytes were activated by PMA plus the calcium ionophore ionomycin (Figure 6a). T-cell activation by lectins is indicative of a broad

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range of effects, among which the direct and specific stimulation of the TCR-CD3 complex and also other T-cell costimulatory processes. To study the action of CD26 transgene uniquely and directly on TCR, we measured thymocyte proliferation in response to anti-CD3 monoclonal antibodies 145-2C11 and found an identical stimulation of thymocytes from control as well as from huCD26 mice (Figure 6a), thus suggesting that CD26 may act on some costimulatory functions needed for T-cell proliferation. Because most T cells present in the thymus pertain to early steps of maturation, our results could be referred to CD26 inhibition exerted specifically on immature T-cell populations. This hypothesis was tested by proliferation experiments on splenocytes that comprise mostly mature T cells. All combinations of stimulatory molecules elicited the effective and equal activation of both normal and CD26 transgenic splenocytes, indicating that the inhibitory effect of CD26 overexpression on T-cell proliferation is limited to immature T cells in the thymus (Figure 6c).

FIGURE 6 Proliferative response in huCD26 transgenic and control mice. Thymocytes (a, b) or splenocytes (c) from 4-week-old hu-CD26 transgenic and wild type mice (2 ⫻ 105 cells per well) were stimulated with anti-CD3 (2.5 ␮g per well), ConA (5 ␮g/ml) and PWM (2.5 ␮g/ml) for 72 hours. Cells were stimulated with PMA (1 ng/ml) and ionomycin (200 ng/ml) as a positive control (P⫹I). The decreased proliferation to ConA stimulation is reversed by the addition of PMA (b). Cultures were pulsed with 1 ␮Ci of [3H]-thymidine for the last 12 hours and processed for standard scintillation counting. Results from three transgenic and three control mice are depicted as means counts per minute (Cpm) of incorporated [3H]-thymidine ⫾ standard error of the mean of triplicate cultures. One of four independent experiments is illustrated. Asterisks indicate the statistically significance differences (p ⫽ 0.001) relative to the counts per minute levels.

DISCUSSION The features of the CD26 molecule, although extensively studied in this last decade, remains obscure for several important aspects. CD26 seems involved in a large variety of biologic functions, including metabolic processes, cell adhesion, chemotaxis, HIV infection, and malignant transformation [1– 6]. T-cell immune function is the most promising research area concerning CD26 because CD26 expression is regulated in the course of T-cell development and during acquisition of the activation status. CD26 triggering results in a series of events, such as phosphorylation of different proteins including the TCR/CD3 ␨ chain, IL-2 production, and T-cell proliferation [21–23]. In turn, CD26 is capable to deliver activation signals to T cells, possibly by modulating the phosphatase activity of CD45 [25, 26]. Therefore, it seems likely that CD26 plays a critical role in T-cell development and signalling, although the in vivo role of CD26 in the immune system is unknown at present. As a first approach to this problem, we have investigated the role of CD26 in the function and distribution of murine T-cell populations both in the thymus and in lymphoid peripheral organs. To achieve this goal we produced transgenic mice constitutively expressing the human gene. The DNA construct was based on a cassette containing the transgene under the control of extended regulatory sequences of human CD2. This human promoter, contrary to mouse CD2 that is normally expressed both in T and B lymphocytes [42], drives high transgene expression almost

Characterization of huCD26 Transgenic Mice

exclusively in the whole thymocyte subset and in peripheral T lymphocytes. The huCD26 gene shares most of its sequences with the mouse equivalent [19] and is also capable of delivering signals in mouse cells [22], thus indicating that the human molecule fully interacts with the mouse cell environment. In huCD26 mice resting thymocytes displayed a very high level of constitutive huCD26 expression, as high as the one observed for human peripheral mononuclear cells activated by phytohemaglutinin. This constitutive expression of huCD26 added to the regulated endogenous mouse CD26, thus leading to a general overexpression of the CD26 molecule on the surface of mouse T cell lineages. The human protein on the murine T-cell surface was recognized by two different anti-CD26 monoclonal antibodies 1F7 and Ta1, thus demonstrating the correct conformation and transmembrane positioning of the transgene huCD26 product. We have also reported that huCD26 displayed an intense DPPIV. Therefore, this initial characterization of transgenic mice allowed us to conclude that the human transgene product is constitutively expressed, fully preserves its structure and functionality in the environment of mice cells, and has no negative effect on the survival and the reproductive capacity of mice. Although HUCD26 expression induced several phenotypic changes to our transgenic mice, that is mainly a marked decrease of the total thymocyte cell number, the increased presence of CD8⫹ single positive cells in the thymus and the overall reduction of the peripheral blood T-cell pool. Thymus ontogeny proceeded normally in transgenic mice, at least in the earlier stages of development. After the sixth week of age, thymuses from huCD26 transgenic mice revealed, in comparison with wild type mice, an evident decrease in overall thymocyte number. Interestingly, this accelerated reduction of thymocyte number started at a specific developmental stage of adult mice, that is the onset of sexual maturity (at the first month of age), concomitantly with the exponential decline of thymic cellularity (thymic involution) observed in wild type mice [43– 45]. Therefore, this process resulted anticipated in transgenic mice when compared with normal mice. Although thymic involution is poorly understood, the regulation of thymus homeostasis is known to be due to several hormonal and neuroendocrine factors, such as glucocorticoids, growth hormones, insulin, peptidic hormones, and neuropeptides [43, 46 –51]. Indeed, replacement of growth factors or hormones results in inhibition of either thymic involution or T-cell senescence with ageing [52–56]. On the basis of these observations we

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speculate that huCD26, in transgenic mice, by virtue of its overexpression and increased enzymatic activity, alters the function of growth factors or hormones, thus affecting thymus homeostasis. In this regard, one of the major candidates could be the growth hormone (GH). GH exerts a pleiotropic action on thymus by regulating thymulin levels [57, 58], which controls thymic epithelial cells proliferation in vitro [59] and the expression of receptors and ligands of extracellular matrix [60]. Moreover, GH has direct effects on thymocytes by positively regulating thymocyte number [61] and ConA-induced proliferative response and cytokines production [58]. The molecular mechanisms of these effects are poorly understood at present, except for the observation that GH action is mediated by the insulinlike growth factor I (IGF-I) [58 – 60, 62– 64]. CD26 inactivates the growth hormone-releasing factor (GHRH) [65, 66], thus decreasing GH secretion [67, 68]. The enzymatic activity of CD26 could disregulate the neuroimmune mechanism involving GHRH, GH, IGF-I, and lead to the alterations observed in huCD26 transgenic mice. Thymuses from huCD26 transgenic mice reveal an increase of the proportion of CD8 SP thymocytes. This observation reflects the possibility that CD26 is involved in the commitment of DP thymocytes to the CD8 SP lineage. Although further investigations are needed to address this issue, it is well established that both CD26 expression and DPPIV enzymatic activity are increased during thymocyte maturation and that CD26 is an important signalling molecule in thymocytes. We suggest that in our huCD26 transgenic model CD26 overexpression interferes with thymocyte signaling. This hypothesis is consistent with the observation that thymocytes from huCD26 transgenic mice demonstrated a reduced proliferative response to ConA and PWM. Though in our experimental condition no differences were observed following TCR stimulation, unknown pathways specific for T cells were perturbated in huCD26 transgenic mice. We report that reduction of lectin-induced proliferation occurs at earlier step of activation because the addition of the PKC activator PMA fully restored proliferative response after ConA stimulation. It has been suggested that CD45 and LFA-1 molecules play important roles in lectin-induced activation. In fact, it has been reported that lectin receptors associate with CD45 after lectin activation [69], and that antiLFA-1 antibodies inhibited lectin-induced T-cell aggregation [70]. Interestingly, CD26 coaggregates with CD45 into lipid rafts, thus facilitating colocalization of CD45 to T-cell receptor signaling molecules p56(Lck), ZAP-70, and TCRzeta [26]. Indeed, CD45-mediated signaling pathway is the major candidate to further investigate molecular mechanisms responsible for the

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reduced proliferation, following lectin stimulation, observed in transgenic mice. In addition to disregulation of thymocyte homeostasis and proliferation, huCD26 transgenic mice demonstrate alterations in the homeostasis of T lymphocytes in the peripheral blood. The number of peripheral T lymphocytes is reduced in 6- to 8-month-old huCD26 transgenic mice by a factor of 2 when compared with wild type animals. Among T subsets, the CD4⫹ helper subpopulation is the most affected. On the contrary, both the total cellularity and T-cell subsets observed in other lymphoid organs such as spleen, bone marrow, and lymph nodes resulted normal in transgenic mice. The survival of mice is not affected in our animal housing conditions. Moreover, transgenic mice survive the inoculation with a nonlethal dose of M. tuberculosis, or with the nonpathogenic M. bovis in a manner comparable with wild type mice (Fantoni et al., unpublished results). This finding reveals that the efficiency of immune functions is not affected in transgenic mice. The reduced number of mature T cells in the peripheral blood of huCD26 transgenic mice can be explained, in part, by the reduction in the number of total thymus cellularity, which could potentially correspond a reduction of thymic output. An alternative or additional explanation for the reduced number of peripheral blood T-cell pool observed in huCD26 transgenic mice is the finding that the percentage of Annexin V⫹ cells is increased in both CD4⫹ and CD8⫹ subpopulations. This increase in the number of apoptotic cells is already observed in 3- to 4-monthold mice. Thus, the reduction of the peripheral T-lymphocyte number observed in 6- to 8-month-old mice results from the modification of a homeostatic mechanism occurring early in mice. The reason for this increased apoptotic rate in peripheral blood T lymphocytes is unknown at present. In conclusion, we suggest that CD26 overexpression may affect a common mechanism needed for differentiation, proliferation, and survival of both SP thymocytes and peripheral T lymphocytes. The perturbation of this mechanism could be responsible for the different cell fate observed in T lymphocytes from huCD26 transgenic mice. We believe that the huCD26 mouse model is a useful tool to further investigate the role of huCD26 in controlling T-lymphocyte homeostasis, and to identify the cellular and molecular mechanisms underlying the effects of huCD26 overexpression on T-cell murine populations.

ACKNOWLEDGMENTS

We are indebted to Prof. Paola Verani and Dr. Filippo Berardelli (Istituto Superiore di Sanita`, Roma) for support and

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helpful discussions. We thank Franco Varano and Massimo Spada for competent animal care.

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