Antigen presentation and immune regulatory capacity of immature and mature-enriched antigen presenting (dendritic) cells derived from human bone marrow

Antigen Presentation and Immune Regulatory Capacity of Immature and Mature-Enriched Antigen Presenting (Dendritic) Cells Derived From Human Bone Marrow Yide Jin, Laphalle Fuller, Gaetano Ciancio, George W. Burke III, Andreas G. Tzakis, Camillo Ricordi, Joshua Miller, and Violet Esquenzai ABSTRACT: Several reports including those from this laboratory have demonstrated that bone marrow cells (BMC) downregulate in vitro both mixed leukocyte reaction and cytotoxic T lymphocyte reactions. We consequently hypothesized that a general property of immature cells of hematopoietic organs is their ability to suppress immune reactivity. As one of these suppressive activities, the lack of costimulatory molecules was proposed as a mechanism by which immature antigen presenting cells of the bone marrow might be involved. In the present report, we used two culture environments, each of which would regulate a different maturation pattern of human bone marrow-derived enriched dendritic antigen presenting cells (DC or APC) to determine the respective effects on in vitro immune regulatory function. Human BMC depleted of CD3⫹ cells were cultured with either: interleukin-4 (IL-4) and granulocyte macrophage– colony stimulating factor (GM-CSF), to maintain DC-enriched populations in an immature state (iAPC); or an interferon-␥ (IFN␥), tumor necrosis factor alpha (TNF-␣), GM-CSF, LPS, and IL-6 cocktail to promote the maturation of DC-enriched APC (mAPC). These iAPC and mAPC were, respectively, phenotypically characterized and also tested in vitro for the following: (1) both direct and indirect-antigen presentation functions; (2) immune regulatory functions on the response of autologous and allogeneic peripheral blood lymphocytes (PBL); and (3) Western blot analysis determining the levels of both major histocompatibility complex (MHC) class I related cytoplasmic ABBREVIATIONS APC antigen presenting cells BCA bicinchoninic acid BMC bone marrow cells CTL cytotoxic T lymphocytes DC dendritic cells

From the Departments of Surgery (Y.J., L.F., G.C., G.W.B., A.G.T., J.M., V.E.) and Microbiology/Immunology (L.F., J.M., V.E.), Division of Transplantation, and the Diabetes Research Institute (C.R.), University of Miami School of Medicine, Miami, FL; Miami Veterans Affairs Medical Center (J.M., V.E.), Miami, FL, USA. Human Immunology 65, 93–103 (2004) © American Society for Histocompatibility and Immunogenetics, 2004 Published by Elsevier Inc.

transporter molecules associated with antigen processing (TAP1) and as well as proteasome activator molecules (PA28␣). The iAPC population expressed fewer dendritic cell markers (CD83 and DCsign), and costimulator molecules (CD86 and CD40) than the mAPC, such that there was an approximate threefold increase in expression of CD83, 2.5-fold increase in DCsign, and a threefold increase in CD40 and CD86 on mAPC than on iAPC (p ⫽ 0.005 for CD83; p ⫽ 0.001 for DCsign; p ⫽ 0.001 for CD86; and p ⫽ 0.001 for CD40). In lymphoproliferative assays, indirect and direct alloantigen presentation by iAPC was weaker than by mAPC (p ⫽ 0.05 and 0.04). In addition, iAPC were able to downregulate allogeneic CTL responses. Also, after pulsing with Epstein-Barr virus (EBV) protein antigens, the iAPC were less efficient in their presentation to autologous EBVspecific T-cell lines, and caused an inhibition of EBV-CTL generation. The expression of TAP1 and PA28␣ was reduced in iAPC in comparison to mAPC. These findings support the notion that a maturation state of BMC-derived APC correlates with their capacity to present antigen. The observed in vitro deficiency of this function by immature bone marrow cells may therefore contribute to the immune downregulatory capacity seen in the BMC compartment. Human Immunology 65, 93–103 (2004). © American Society for Histocompatibility and Immunogenetics, 2004. Published by Elsevier Inc. KEYWORDS: bone marrow; APC; antigen pulsing; MHC class I; MHC class II; antigen presentation

EBV iAPC mAPC MLC RdD

Epstein-Barr virus immature APC mature APC mixed lymphocyte culture recipient-derived donor cells

Address reprint requests to: Dr. Jin, Division of Transplantation, Department of Surgery (R-440), University of Miami School of Medicine, PO Box 012440, Miami, FL 33101; Tel: (305) 355-5100; Fax: (305) 355-5134; E-mail: [email protected]. Received October 17, 2003; accepted November 20, 2003. 0198-8859/04/$–see front matter doi:10.1016/j.humimm.2003.11.002

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INTRODUCTION Reports from several laboratories have described the involvement of bone marrow cell (BMC) derived antigen presenting cells (APC) in establishing peripheral tolerance [1– 4]. It has been demonstrated in animal models that self antigens transported from peripheral tissues to draining lymph nodes can be presented by BMC-derived APC in association with major histocompatibility complex (MHC) class I molecules [2]. Antigen presentation that occurs in the absence of a second signal is thought to lead to an unresponsive state [5]. Recently in human studies, Dhodapkar et al. [4] reported that the injection of immature dendritic cells (DC) pulsed with peptides into healthy patients results in inhibition of specific T-cell responses to the injected peptides. Jonuleit et al. [6] demonstrated that the stimulation of alloreactive CD4 cells with immature, but not mature, DC induced an alloreactive CD4 T-cell line that exhibited regulatory properties. The immune regulatory capacity of human BMC has also been studied in our laboratory. Mathew et al. [7, 8] reported that BMC downregulated T-cell responses to alloantigens. In ex vivo studies, in kidney transplant recipients who also received perioperative donor bone marrow infusions, it was found that recipient-derived donor (RdD) bone marrow cells isolated from the chimeric recipient after 1 year had an enhanced inhibitory effect on antidonor cytotoxic T lymphocyte (CTL) and mixed lymphocyte culture (MLC) reactions [9]. Interestingly, iliac crest BMC either from chimeric renal allograft recipients or their non-chimeric living-related donors exhibited an equivalent capacity to inhibit autologous Epstein-Barr virus (EBV) specific CTL generation [10]. This indicated that the recipient chimeric environment was not absolutely required for this more general immune regulatory effect expressed by BMC, although alloimmune regulation appeared to definitely be augmented by the chimeric state. All of this in vitro work supported the concept of the human bone marrow as a natural immunoregulatory organ, which we have previously proposed [11]. Effective antigen presentation by APC requires the expression of costimulatory molecules [12], enhanced by the maturation process. The immature nature of the hematopoietic APC progenitor cells in the human bone marrow compartment may help to explain their immune downregulatory effect on T cells. To further elucidate this, we compared human iliac crest BMCderived “immature” (iAPC) versus “mature” APC (mAPC; after short-term culture with appropriate cytokine cocktails) with respect to their antigen pre-

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sentation capacity and their immune regulatory function. MATERIALS AND METHODS Generation of BMC-Derived Immature and Mature APC Human iliac crest BMC were freshly aspirated from laboratory volunteers, and living-related kidney transplant donors, or BMC were extracted from the vertebral bodies of cadaver donors as previously described [8, 9]. They were then isolated by Ficoll-Hypaque gradients. The BMC then were depleted of CD3⫹ cells by anti-CD3 mAb-coated magnetic beads (Miltenyi Biotech Inc., Sunnyvale, CA). Aliquots of 2 ⫻ 106 cells were placed into six-well plates in culture medium containing 10 ng/ml of granulocyte macrophage– colony stimulating factor (GM-CSF; Biosource, Camarillo, CA) plus 10 ng/ml of IL-4 (PharMingen, San Diego, CA), and cultured for 5 days. Nonadherent cells were harvested, washed once, and 1 ⫻ 106 cell aliquots were then transferred into the wells of additional six-well plates and either were cultured with 10 ng/ml of IL-4 for an additional 3 days resulting in what was designated as an “immature APC” (iAPC) enriched population, or were cultured for an additional 3 days with a “cocktail” of 10 ng/ml of interferon-␥ (IFN-␥), 50 ng/ml of tumor necrosis factor-␣ (TNF-␣), 100 ␮/ml of interleukin-6 (IL-6; all from PharMingen), and 20 ng/ml of LPS (Sigma, St Louis, MO) to promote “APC maturation” (mAPC). There were no detectable CD3⫹ cells, less than 0.5% of CD34⫹, and less than 2% of CD19⫹ cell impurities in either the iAPC or mAPC populations (flow cytometry, vide infra). Phenotypic Analysis of BMC-derived iAPC and mAPC Samples of mAPC or iAPC (1 ⫻ 105 cells) were then incubated with either 20 ␮l of anti-CD40-FITC, antiCD83-FITC, anti-CD86-PE, or anti-DCsign-FITC (all purchased from PharMingen, San Diego, CA), respectively, at room temperature for 30 minutes. After washing to remove excess antibodies, the cells were analyzed by FACScan Immunocytometry (Becton Dickinson, San Jose, CA). The corresponding isotype controls were also included (Ig-FITC as the isotype control for CD40, CD83, and DCsign; Ig-PE for CD86). Lymphoproliferation Assays Direct alloantigen presentation in MLC. T cells were separated from PBL of laboratory volunteers by negative

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selection with the Pan T Kit (Miltenyi Biotech, Inc.; purity ⫽ 92%–95%), and used as responder cells (R). Mitomycin C-treated allogeneic iAPC and mAPC enriched populations were used as stimulators (S). Cell cultures were prepared with 1 ⫻ 105 responder cells/well versus 5 ⫻ 104 cells/well of either iAPC or mAPC stimulators [2:1 responder to stimulator (R/S) ratio], incubated for 5 days at 37 °C in 5% CO2, pulsed with 3 H-thymidine (3H-TdR) for 16 hours, harvested, and radioactivity counted using a beta scintillation counter (Packard Instruments, Chicago, IL). Indirect alloantigen presentation in MLC. Ultraviolet light wave (UV) triggered apoptotic allogeneic PBL were used as the antigen source. As previously described [13] briefly, 4 ⫻ 106 allogeneic PBL in 4 ml of culture medium were irradiated using UVGL25 (UVP, Inc., San Gabriel, CA) for 8 –10 hours at a distance of 13 cm (wavelength of 254 nm) to induce apoptotic cell death. The iAPC and mAPC were allowed to capture the alloantigen from the apoptotic bodies by overnight coculture (at a 2:1 ratio). Indirect presentation was demonstrated by measuring the response of T cells to stimulation by allogeneic cell apoptotic body fed autologous iAPC and mAPC. Lymphoproliferative assays were prepared at a 2:1 R/S ratio as above such that (purified) T lymphocytes isolated from peripheral blood (above) were the responders, and mitomycin C-treated alloantigen captured autologous iAPC or mAPC were the stimulators in each well. After 5 days in culture, cells were labeled with 3H-TdR for an additional 16 hours, then harvested and counted as above. Antigen Priming with EBV Soluble Antigens Exogenous soluble viral antigen presentation by iAPC and mAPC was tested in both proliferative and cytotoxic responses to EBV protein antigens. Short-term EBVspecific T-cell lines were generated as described previously [10]. Autologous iAPC and mAPC were pulsed with 40 ng/ml of EBV capsid antigen and 100 ng/ml of EBNA-1 (ABI; Advanced Biotechnologies, Columbia, MD) in 10% FCS RPMI medium overnight at 37 °C in 5% CO2. The EBV T-cell lines were rested (in the culture medium without antigen) for 7–10 days and then stimulated with the mitomycin C-treated EBV antigenpulsed iAPC or mAPC, respectively, using 4 ⫻ 106 responder cells to 1 ⫻ 106 stimulator cells. This was followed after another 7 days in culture by a cytotoxicity assay (vide infra) against autologous EBV-transformed blast (EBV-B) targets [10]. Additionally, 3H-TdR incorporation proliferation assays were prepared using 1 ⫻ 105 cells/well of the EBV T-cell lines as responders, and 2.5 ⫻ 104 cells/well of autologous antigen pulsed mAPC or iAPC as stimulators. After 3 days, the cells were

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labeled with 3H-TdR for an additional 16 hours, then harvested, and 3H-TdR incorporation was measured as above. Generation of EBV-Specific CTL in the Presence of iAPC or mAPC The procedure for the (primary) generation of EBVspecific CTL also was described in a previous report [10]. Briefly, PBL of EBV IgG positive donors were used as responders and autologous EBV-transformed B cell blasts (EBV-B) as stimulators at a 10:1 responder to stimulator ratio. Autologous iAPC or mAPC were added as thirdparty (modulator) cells (at a 2:1 responder to modulator ratio) to the EBV-specific CTL generating cultures. As a positive control, mitomycin C-treated autologous PBL were added in place of the iAPC and mAPC. After 10 days in culture, cytotoxicity assays were performed using fresh autologous 51Cr-labeled EBV blasts as the targets at effector to target ratio (E:T) of 40:1. The inhibition index was calculated as a percentage with the following equation: % inhibition ⫽ % of 51Cr release of EBV generation culture ⫺ % of 51Cr release of EBV generation culture with either autologous iAPC or mAPC % of 51Cr release of EBV generation culture Autologous and Allogeneic iAPC versus mAPC Effects on a CD3ⴙ Cytotoxic Cell Alloimmune Response Peripheral blood lymphocytes were cocultured with autologous iAPC and mAPC at a ratio of 2:1, respectively, for 7 days. CD3⫹ cells were retrieved with MACS antiCD3 beads (Miltenyi Biotech), designated as TiAPC-auto and TmAPC-auto, respectively. TiAPC-auto and TmAPC-auto were then tested for their capacity to generate an allogeneic CTL response. An allogeneic EBV-B cell line (disparate at least by one DR, one HLA-A and -B locus) treated with mitomycin C, used as stimulator cells, was cultured with responding TiAPC and TmAPC at a ratio of 1:5 stimulator to responding cells for 7 days. A cytotoxicity assay against the allogeneic EBV-B target cells was then performed. Peripheral blood lymphocytes from laboratory volunteers were also cocultured with allogeneic iAPC and mAPC at a ratio of 2:1, respectively, for 7 days. The CD3⫹ cells retrieved from the above cultures were designated TiAPC-allo and TmAPC-allo, respectively, and were also tested using cytotoxicity assays as above. Finally, both TiAPC-auto and TmAPC-auto as well as TiAPC-allo and TmAPC-allo were then tested for their capacity to generate CTL responses to cells from either the allogeneic iAPC/

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TABLE 1 Phenotypic analysis of bone marrow-derived (CD3⫹ cell depleted) iAPC and mAPC Percent of positive cells I

II a

III b

Markers

fresh CD3 negative BMC

iAPC

mAPCb

CD34 (n ⫽ 3) CD40 (n ⫽ 5) CD86 (n ⫽ 4) CD83 (n ⫽ 5) DCsign (n ⫽ 4) p valuesc

3.52 ⫾ 0.65 4.28 ⫾ 0.94 4.81 ⫾ 1.98 3.60 ⫾ 1.03 4.95 ⫾ 1.54

0.42 ⫾ 0.31 17.08 ⫾ 5.39 19.36 ⫾ 2.28 14.89 ⫾ 3.77 21.00 ⫾ 3.38

0.43 ⫾ 0.21 63.16 ⫾ 6.92 60.09 ⫾ 4.60 44.04 ⫾ 8.56 53.68 ⫾ 5.67

BMC depleted of CD3⫹ cells with anti-CD3 beads (see Methods). BMC depleted of CD3⫹ cells and cultured with cytokines (see Methods) to produce iAPC (n ⬎ 10) and mAPC (n ⬎ 10). c I vs. II I vs. III II vs. III p values: a

b

0.046 0.034 0.14 0.025 ⬍0.01 ⬍0.01 0.013 ⬍0.01 ⬍0.01 0.01 ⬍0.01 ⬍0.01 0.01 ⬍0.01 ⬍0.01 Abbreviations: APC ⫽ antigen presenting cells; BMC ⫽ bone marrow cells; iAPC ⫽ immature APC; mAPC ⫽ mature APC.

mAPC donors (ie, specifically) using ConA blast target as previously described [10], or were tested against Con-A blasts that did not share the iAPC/mAPC alloantigens (ie, nonspecifically). MHC Class I Related Transport Molecules Western blot. Protein cell lysates from iAPC and mAPC (2–3 ⫻ 106 cell pellets) were obtained by boiling with two-fold concentrated SDS sample buffer. Protein concentrations were measured by the bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Inc., Rockford, IL). Equivalent amounts of lysate protein were loaded on 10% gels for SDS-PAGE. After electrophoresis, the proteins were transferred to polyvinylidene fluoride membranes, the membranes blocked with 1% nonfat dry milk-Tris, and then incubated with rabbit IgG anti-PA28␣ (Calbiochem, La Jolla, CA) and rabbit IgG anti-TAP1 (Calbiochem) at a 1:1000 dilution overnight at 4 °C. The excess antibody was removed with intensive washing by PBS-T (1⫻ PBS with 0.5% Tween 20) and anti-rabbit IgG horseradish peroxidase-conjugated antibody (Calbiochem) was added as the second antibody at a 1:10,000 dilution with an incubation of 1 hour at room temperature. The membranes were intensively washed with PBS-T, and the bands were detected with the ECL system (Amersham, Piscataway, NJ). Immunoprecipitation. Alternatively, cell lysates were prepared from the mAPC and iAPC with extraction buffer (20-mM Tris, pH 7.5, 150-mM NaCl, 2-mM EDTA, 1% NP-40, 0.02% NaN3, 10-mM NaF, 1-mM sodium ortho-vanadate, 0.25-mM phenylmethylsulfonyl fluo-

ride, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, and 1 ␮g/ml chymostatin; Sigma). Cell lysates with equivalent protein concentrations were precipitated with 10 ␮l of anti-PA28␣ and anti-TAP1 adsorbed with 50 ␮l of protein G beads. The mixtures were rocked at 4 °C overnight. The beads were pelleted, and the adsorbed proteins were solubilized in 24 ␮l of 2⫻ gel sample buffer and placed in a boiling water-bath for 5 minutes. Protein electrophoresis was performed with 10% SDS gels followed by Western blot analysis as described above. Statistical Methods Statistical analysis was done with Microsoft Excel computer software program (Microsoft Corporation, Redmond, WA) paired t-test.

RESULTS Cytokines Promote Maturation and Expression of Costimulatory Molecules on BMC-Derived DC Enriched Populations When compared with fresh BMC depleted of CD3⫹ cells, both iAPC and mAPC revealed an increase in costimulatory molecules (CD40 and CD86) and DC markers (CD83 and DCsign) in single-color flow cytometry analysis. However, mAPC had significantly higher percentage of these epitopes than iAPC (Table 1, Figure 1), such that there was an approximate threefold increase in expression of CD83, 2.5-fold increase in DCsign, and a threefold increase in CD40 and CD86 on mAPC (p ⫽

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0.005 for CD83; p ⫽ 0.001 for DCsign; p ⫽ 0.001 for CD86; and p ⫽ 0.001 for CD40). Indirect and Direct Antigen Presentation of iAPC and mAPC in MLC The iAPC and mAPC, which expressed different levels of costimulator molecules and DC markers, also differed in their capacity to present antigens in lymphoproliferation assays. In studies of direct alloantigen presentation, purified T cells (see Methods) from laboratory volunteers were the responders, and mitomycin C-treated bone marrow-derived allogeneic mAPC and iAPC were used as stimulators (Figure 2A). In indirect antigen presentation, as described in the Methods section, alloantigens from UV-treated apoptotic PBL were captured by mAPC and iAPC, and then presented to autologous T cells (Figure 2B). There was a three- to fourfold increased antigen presenting capacity of mAPC when compared to iAPC in either direct or indirect antigen presentation.

FIGURE 1 Phenotypic analysis of bone marrow derived iAPC and mAPC. The iAPC were BMC-depleted of CD3⫹ cells and cultured in GM-CSF and IL-4; the mAPC were then also cultured in GM-CSF, IFN-␥, TNF␣, LPS, and IL-6. CD83 and DCsign were expressed on most of the mAPC, which also expressed more CD40, and CD86 when compared with iAPC (mAPC, solid fill; iAPC, solid outline; and isotype control, broken outline). The histograms for fresh BMC overlapped the isotype controls, and were not included. Abbreviations: APC ⫽ antigen presenting cells; BMC ⫽ bone marrow cells; DC ⫽ dendritic cell; GM-CSF ⫽ granulocyte macrophage– colony stimulating factor; IFN ⫽ interferon; IL ⫽ interleukin; iAPC ⫽ immature APC; mAPC ⫽ mature APC; TNF ⫽ tumor necrosis factor.

Antigen Presentation by iAPC and mAPC of EBV Proteins in Lymphoproliferative and Cytotoxicity Assays Antigen pulsing experiments with EBV protein antigens were used to provide additional information on the (indirect) antigen presenting pathway by exogenous protein. After establishing short-term EBV-specific T-cell lines, autologous mAPC and iAPC were pulsed with EBV capsid and EBNA-1 antigens, and then used as APC in lymphoproliferative assays. There was an approximate two-fold increase in proliferation induced by mAPC when compared with iAPC (Figure 3A, n ⫽ 3, p ⫽ 0.045). In previous cytotoxicity studies, we found that in order to maintain the antigen specificity of EBV-CTL lines, secondary EBV antigen stimulation, was necessary [10]. After maintaining the EBV-CTL lines in culture medium for 10 days, EBV capsid and EBNA-1-pulsed mAPC and iAPC were used to boost their specific cytotoxicity. The cytotoxic assay against autologous EBV transformed B-cell lines gave results similar to the results obtained in the proliferation assay, i.e., in contrast to antigen-pulsed mAPC, antigen-pulsed iAPC was less efficient in inducing the cytotoxic activity of the EBVCTL lines (Figure 3B, n ⫽ 3; p ⫽ 0.02 at an E:T ratio of 40:1). Decreased Expression of Peptide Transporter and Proteasome Activator Components in iAPC The results of the above EBV indirect antigen presentation experiments measuring cytotoxic activity possibly indicated that there was a defect in the ability of iAPC to present or process antigens associated with MHC class I molecules. We measured two components also attributed to be associated with the MHC class I antigen processing

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FIGURE 2 Determination of direct and indirect alloantigen presenting capacity. Alloantigen presenting capacity of BMC-derived iAPC and mAPC were measured by a proliferation assay (mixed lymphocyte culture). The iAPC were deficient in the presentation of alloantigen either by (A) direct (n ⫽ 4) or (B) indirect presentation (n ⫽ 3). The p value for direct antigen presentation between iAPC and mAPC was 0.04, and for indirect antigen presentation was 0.05. Bg indicates background 3H-thymidine uptake of unstimulated responder cells. These wells had the same number of the responder cells as the experimental wells but no stimulating cells. Error bars indicate standard error of the means. Abbreviations: APC ⫽ antigen presenting cells; BMC ⫽ bone marrow cells; iAPC ⫽ immature APC; mAPC ⫽ mature APC.

FIGURE 3 Soluble EBV protein antigen pulsing of iAPC and mAPC. Short-term EBV-specific T-cell lines were established by stimulating peripheral T cells with autologous EBV transformed B cells (see Methods). Soluble EBV proteins were presented by iAPC and mAPC to these EBV T-cell lines. Both the (A) proliferative (n ⫽ 3) and (B) cytotoxicity assays (n ⫽ 3) were of less magnitude using iAPC pulsed with EBV protein antigens. There was a twofold difference between iAPC and mAPC in the proliferation assay (p ⫽ 0.045). The difference in EBVspecific cytotoxicity after stimulation with EBV protein antigen pulsed iAPC and mAPC was significant at an E/T ratio of 40:1 (p ⫽ 0.02). Bg indicates the background of the responder, ie, same number of the responder cells as the experimental wells but no (stimulator) APC. The results are presented as mean standard error. Abbreviations: APC ⫽ antigen presenting cells; EBV ⫽ EpsteinBarr virus; E/T ⫽ effector to target ratio; iAPC ⫽ immature APC; mAPC ⫽ mature APC.

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FIGURE 4 Detection of PA28␣ and TAP1 expression. Equivalent protein concentrations of cell lysates from iAPC and mAPC were used for electrophoresis. One representative result of three individual experiments is shown. There was weaker expression of both PA28␣ and TAP1 in iAPC either with Western blot (top) or with immunoprecipitation (bottom). Abbreviations: APC ⫽ antigen presenting cells; iAPC ⫽ immature APC; mAPC ⫽ mature APC; TAP1 ⫽ transporter associated with antigen processing.

pathway, i.e., the proteasome activator, PA28␣, and peptide transporter, TAP1, were both measured for their expression in iAPC and mAPC. The iAPC expressed a reduced level of both of these molecules as detected by both Western blot (Figure 4, top) and immunoprecipitation (Figure 4, bottom). Autologous iAPC suppresses the EBV-Specific CTL Response In our previous studies using the EBV-CTL generation system, immune regulatory properties of BMC were observed [10, 12], such that in the presence of autologous fresh BMC as modulators, the cytotoxic response of autologous peripheral T cells to EBV antigens was inhibited in a range of 62%–74%. In the present experiments, iAPC and mAPC were compared for their ability to inhibit the formation of EBV-specific CTL (Figure 5). The iAPC inhibition index (Methods) was 55% compared with 30% for the mAPC (n ⫽ 5; p ⫽ 0.04). Induction of “In Vitro Anergy” in PBL CD3ⴙ Cells After Culturing With Autologous and Allogeneic iAPC vs mAPC The previous experiment suggested that there might be a direct effect of the iAPC vs mAPC to induce T-cell anergy. In order to further explore this question, we precultured PBL with autologous bone marrow-derived mAPC and iAPC, then isolated CD3⫹ cells from these cocultures designated TmAPCauto and TiAPCauto, respec-

tively (n ⫽ 4). Their cytotoxic responses were then determined versus allogeneic EBV-B cell blasts. The TiAPC alloimmune cytotoxic response was almost threefold weaker than TmAPC at the E:T ratio of 40:1 (Figure 6, p ⫽ 0.03). To mimic an analogous in vivo reaction that might occur after allogeneic donor-specific BMC infusion in our organ transplant recipients [9, 11], PBL were cultured with allogeneic bone marrow-derived mAPC and iAPC, and CD3⫹ cells were separated from these cocultures after 7 days. The CD3⫹ cells from PBL cocultured with allogeneic iAPC, TiAPC-allo, exhibited less cytotoxicity than CD3⫹ cells from PBL cocultured with allogeneic mAPC, TmAPC-allo, toward the ConA blasts that were autologous (allospecific) to the iAPC and mAPC (n ⫽ 3; Figure 7A; p values are 0.02, 0.055, 0.14, and 0.1 for E:T ratios of 40:1, 20:1, 10:1, and 5:1, respectively). This study suggested that in vivo recipient PBL stimulated with allogeneic donor BMC (iAPC) might express a reduced cytotoxic response to cells of the organ donor after marrow infusion. However, in these short-term cultures, this response did not appear to demonstrate allospecific inhibition, since, even after subsequently stimulating the TiAPC-allo and TmAPC-allo with thirdparty cells (ConA blasts bearing different MHC alloantigen than iAPC and mAPC), TiAPC-allo still generated less cytotoxicity when compared with TmAPC-allo (n ⫽ 5; Figure 7B; p values for 40:1, 20:1, 10:1, and 5:1 E:T ratios are 0.008, 0.15, 0.38, and 0.30, respectively).

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FIGURE 5 Inhibition of EBV-CTL generation by autologous iAPC. Autologous iAPC and mAPC were added into EBV-CTL generation cultures at a 1:2 ratio to the responders. After 10 days in culture, 51Cr release assay was performed using EBV targets at E/T ratios of 40:1 using the inhibition index or percent (%) inhibition (for calculation see Methods). There is a statistically significant difference in the cytotoxicity seen when iAPC vs. mAPC were used as third-party modulators at the E/T ratio of 40:1 (p ⫽ 0.04, n ⫽ 5). Abbreviations: APC ⫽ antigen presenting cells; CTL ⫽ cytotoxic T lymphocyte; EBV ⫽ Epstein-Barr virus; E/T ⫽ effector to target ratio; iAPC ⫽ immature APC; mAPC ⫽ mature APC.

DISCUSSION We have generated a fairly large body of data in our previous in vitro studies to support the hypothesis of a natural immune regulatory property of the human BMC compartment [7–13]. We questioned whether the immature nature of the APC in the BMC might participate in this regulatory function. We therefore used cytokine cocktails that are known for their ability to differentiate APC (dendritic cells) or to maintain them in an immature state [4]. From the phenotype analysis of the APC (Figure 1A), the expression of CD83, CD40, and CD86 were all decreased in the iAPC population which is in accord with other studies [14 –20], ie, these same molecules were 1.5- to 3-fold higher in mAPC populations cultured in a cocktail that drives DC to maturation (Table 1). It is generally assumed that both direct and indirect (cross) presentation of allogeneic antigens play a role in allograft rejection. Dendritic cells that mediate indirect presentation are thought to contribute predom-

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FIGURE 6 iAPC induce CD3⫹ cells that inhibit the autologous CTL response to alloantigens. Peripheral blood lymphocytes were cocultured with autologous iAPC or mAPC at a ratio 2:1 for 7 days. CD3⫹ cells were then isolated as TiAPCauto and TmAPCauto, respectively, which were then stimulated with allogeneic EBV B cells (n ⫽ 4). 51Cr release assays were performed after 7 days at different E/T ratios (p values for 40:1, 20:1, 10:1, and 5:1 E/T ratios are 0.03, 0.07, 0.06, and 0.1, respectively). The results are again presented as means ⫾ standard error. Abbreviations: APC ⫽ antigen presenting cells; CTL ⫽ cytotoxic T lymphocyte; EBV ⫽ Epstein-Barr virus; E/T ⫽ effector to target ratio; iAPC ⫽ immature APC; mAPC ⫽ mature APC.

inantly to chronic rejection [21]. In the present report, efficient antigen presentation was mainly a property of bone marrow derived mAPC. The poorer antigen presentation by iAPC, either in indirect presentation of alloantigens from apoptotic cells or after being pulsed with soluble EBV antigens (illustrated in Figures 2B and 3), again suggested as in our previous observations [9, 22, 23] that in vivo the infusion of donor BMC in kidney allograft recipients might not induce a strong allogeneic response. The freshly isolated donor BMC used for infusion might at least in the APC be equivalent to the derived in vitro subset of iAPC, since, in this study, maturation of these cells to mAPC required culturing with a concentrated cocktail of cytokines questionably present in the bone marrow compartment environment. Moreover, fresh CD3-depleted BMC, even in the absence of derived iAPC culture conditions expressed reduced levels of co-stimulatory molecules and DC markers (Table 1). We have previously demonstrated that whole BMC inhibited the generation of EBV-specific CTL, and with this present report this is extended to reveal an effect of iAPC and mAPC on EBV CTL generation. Although mAPC indicated some inhibition compared with the non-marrow control, the difference in the inhibition between iAPC and mAPC appeared significant (E:T ratio of 40:1 [p ⫽ 0.04], Figure 5)

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FIGURE 7 Immature APC induce CD3⫹ cells that inhibit CTL response to allogeneic antigen. Allogeneic PBL were cocultured with bone marrow-derived mAPC and iAPC, and then CD3⫹ cells were respectively isolated from the above cocultures. (A) CD3⫹ cells from allogeneic PBL cocultured with iAPC (TiAPC-allo) exhibited less cytotoxicity than CD3⫹ cells from allogeneic PBL cocultured with mAPC (TmAPC-allo) toward the ConA blasts that were autologous (allospecific) to the iAPC and mAPC (n ⫽ 3). The p values are 0.02, 0.055, 0.14, and 0.1 for E/T ratios of 40:1, 20:1, 10:1, and 5:1,

respectively. (B) After stimulating with third-party cells, TiAPC-allo still generated less cytotoxicity when compared with TmAPC-allo (n ⫽ 5). The p values for 40:1, 20:1, 10:1, and 5:1 E/T ratios are 0.008, 0.15, 0.38, and 0.30, respectively. The results are again presented as means ⫾ standard error. Abbreviations: APC ⫽ antigen presenting cells; CTL ⫽ cytotoxic T lymphocyte; E/T ⫽ effector to target ratio; iAPC ⫽ immature APC; mAPC ⫽ mature APC; PBL ⫽ peripheral blood lymphocytes.

The lack of costimulatory molecule expression on iAPC could be responsible for the weak allogeneic response induced by the direct alloantigen presentation pathway (Figure 2A). However, the failure of iAPC pulsed with the soluble EBV antigen (Figures 3A and 3B) could also indicate an inefficiency in antigen presentation. It has been reported that the protein uptake capacity of iAPC from apoptotic bodies is actually greater than that of mAPC [24]. Therefore, the insufficient indirect antigen presentation of iAPC (Figure 2A) could have possibly resulted from subsequent steps involving the poor antigen processing and the defective antigen transport [25, 26]. The proteasome is a major proteolytic system that enzymatically degrades most cytosolic proteins into peptides for MHC class I presentation [27]. A two-subunit complex, PA28, has been identified as a proteasome activator, and one of these, PA28␣, has been demonstrated to facilitate the transfer of peptides into the endoplasmic reticulum [28 –30]. The well defined two peptide transporters TAP1 and TAP2 have been described to be required for cross presenting exogenous antigen by MHC class I molecules [20, 31, 32]. The reduced expression of PA28␣ and TAP1 in iAPC (Figure 4) may not only be responsible for reduced (en-

dogenous) antigens presented by MHC class I molecules but also for reduced cross-presented antigens by these MHC molecules as well. We have previously demonstrated that peripheral T cells were anergized in cellular immune assays after encountering autologous BMC, with a characteristic of reduced response to alloantigens [13]. In this present study an attenuated cytotoxic response to alloantigens was observed after T cells were co-cultured with autologous iAPC (Figure 6), which indicates a direct immune regulatory effect of these bone marrow-derived APC on T cells. The results presented in Figure 7A suggest a possible in vivo effect that could occur after allogeneic fresh BMC (iAPC) from the donor-infused BMC contacts the recipient immune competent cells. These in vitro studies indicate that allogeneic iAPC from (donor) BMC exhibited a capacity to inhibit the T-cell response to the donor. This downregulation was not only limited to autologous donor antigens, but also to indifferent alloantigens (Figure 7B). This suggests that priming of recipient PBL T cells with donor BMC (iAPC) reduced the ability of these recipient primed donor T cells (TiAPC-allo and TmAPC-allo) to generate CTL to both donor and indifferent cells, ie, is not restricted by (known) MHC-antigen expression. We

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therefore propose that T cells are anergized both due to weak signaling through TCR, and/or to the absence of costimulation, at least in these short-term cultures, a situation that might be shifted toward more allospecific regulation that we have observed in longer-term cultures [9, 33] and in vivo in the long-term chimeric condition seen in the marrows of donor bone marrow-infused kidney transplant recipients [22]. Taken together with our previous studies, we conclude that there is further support that human bone marrow cells possess an immune regulatory property [11]. The maturation of APC derived from BMC correlates with their antigen presenting capacity. The deficiency of immature bone marrow in antigen presentation may be a cogent factor in contributing to this immune downregulation. The benefit of donor bone marrow cell infusion may partially result from this downregulation of T-cell responses due to insufficient allogeneic antigen presentation by MHC class I (or MHC class II) pathways. REFERENCES 1. Xiao BG, Huang YM, Yang JS, Xu LY, Link H: Bone marrow-derived dendritic cells from experimental allergic encephalomyelitis induce immune tolerance to EAE in Lewis rats. Clin Exp Immunol 125:300, 2001. 2. Adler AJ, Marsh DW, Yochum GS, Guzzo JL, Nigam A, Nelson WG, Pardoll DM: CD4⫹ T cell tolerance to parenchymal self-antigens requires presentation by bone marrow-derived antigen presenting cells. J Exp Med 187: 1555, 1998. 3. Finkelman FD, Lees A, Birnbaum R, Gause WC, Morris SC: Dendritic cells can present antigen in vivo in a tolerogenic or immunogenic fashion. J Immunol 157:1406, 1996. 4. Dhodapkar MV, Steinman RM, Krasovsky J, Munz C, Bhardwaj N: Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med 193:233, 2001. 5. Steinman RM, Turley S, Mellman I, Inaba K: The induction of tolerance by dendritic cells that have captured apoptotic cells. J Exp Med 191:411, 2000. 6. Jonuleit H, Schmitt E, Schuler G, Knop J, Enk AH: Induction of interleukin 10-producing, nonproliferating CD4⫹ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med 192:1213, 2000. 7. Mathew JM, Carreno M, Fuller L, Ricordi C, Tzakis A, Esquenazi V, Miller J: Modulatory effects of human donor bone marrow cells on allogeneic cellular immune responses. Transplantation 63:686, 1997. 8. Mathew JM, Carreno M, Zucker K, Fuller L, Kenyon N, Esquenazi V, Ricordi C, Tzakis AG, Miller J: Cellular immune responses of human cadaver donor bone marrow cells and their susceptibility to commonly used immuno-

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