- Email: [email protected]
Campath-1H Does Not Alter Bone Marrow Cell Regulatory Function
Campath-1H Does Not Alter Bone Marrow Cell Regulatory Function Yide Jin, Laphalle Fuller, Anne Rosen, Gaetano Ciancio, George W. Burke III, Camillo Ricordi, Andreas G. Tzakis, Joshua Miller, and Violet Esquenazi ABSTRACT: We have previously reported in laboratory volunteers (in vitro) and renal transplant recipients (ex vivo) that bone marrow cells (BMC) are potent downregulators of the immune response. Also, the use of alemtuzumab (Campath-1H, C1H) for immunodepletion is associated with the most potent lasting effects yet seen on T-cell immunity in renal transplantation. We questioned whether the administration of C1H to kidney allograft recipients of donor bone marrow cell (DBMC) infusions would lead to stronger or weaker immunoregulatory effects. Human BMC depleted of T cells (nT-BMC) were either untreated or treated with C1H and rabbit complement and compared for their ability to downregulate autologous or allogeneic T-cell responses and to generate T regulatory (T reg) cells. The proliferative responses to anti-CD3 monoclonal antibody of T cells derived from cocultures with C1H-treated or untreated autologous nT-BMC were equally suppressed, i.e., an equivalent alteration in CD3 complex signaling, not regained by the addition of interleukin 2. Adenosine triphosphate levels were also markedly reduced in T cells both from C1HABBREVIATIONS ATP adenosine triphosphate BMC bone marrow cells C1H Campath-1H CTL cytotoxic lymphocytes DBMC donor bone marrow cells
From the Lillian Jean Kaplan Renal Transplant Center of the Division of Transplantation of the Department of Surgery (Y.J., L.F., A.R., G.C., G.W.B., C.R., A.G.T., J.M., V.E.), the Department of Microbiology/ Immunology (L.F., C.R., J.M., V.E.), and the Diabetes Research Institute (C.R.) at the University of Miami, Leonard M. Miller School of Medicine; and the Miami Veterans Affairs Medical Center (J.M., V.E.), Miami, FL, USA. Address reprint requests to: Dr. Y. Jin, Department of Surgery, Division of Transplantation, University of Miami School of Medicine, P.O. Box 012440 (R-440), Miami, FL 33101; Tel: (305) 355-5100; Fax: (305) 3555134; E-mail: [email protected]. Received February 2, 2005. Human Immunology 66, 637– 643 (2005) © American Society for Histocompatibility and Immunogenetics, 2005 Published by Elsevier Inc.
treated and untreated nT-BMC cocultures. The ability of C1H-treated or untreated nT-BMC to suppress autologous T-cell cytotoxic function was also equivalent, with a marked, but equivalent, capacity to induce CD4/CD25high T regs from CD3⫹ cells, which effectively downregulated cytotoxic T cells. To mimic the clinical infusion of DBMC into (allogeneic) recipients, peripheral blood mononuclear cells were also cultured with allogeneic C1H-treated and untreated nT-BMC. T cells derived from these cultures secondarily stimulated with the same-donor mature antigen-presenting cells exhibited suppressed cytotoxicity by 85% and 54%, respectively. These in vitro studies suggest that C1H does not abrogate BMC immunoregulation and thus may allow its lympho-depleting effect to be synergistic. Human Immunology 66, 637– 643 (2005). © American Society for Histocompatibility and Immunogenetics, 2005. Published by Elsevier Inc. KEYWORDS: Campath-1H; bone marrow antigenpresenting cell; regulatory function
EBV nT-BMC PBL T regs TCR
Epstein-Barr virus human BMC depleted of T cells peripheral blood mononuclear cells T regulatory cells T-cell receptors
INTRODUCTION Alemtuzumab (Campath-1H, or C1H; Ilex Oncology, San Antonio, TX) is a humanized monoclonal antibody that recognizes the CD52 epitope, a small glycosylphosphatidylinositol glycoprotein expressed at a high level on T and B lymphocytes and to a lesser extent on other mononuclear cells [1– 4]. It appears that the CD52 epitope is close to the cell membrane, so that antigenantibody binding will easily lead to cell lysis through the complement or antibody-dependent cell-mediated cytotoxicity pathways [5, 6]. On this basis, C1H has been used in the past to treat lymphoid malignancies and rheumatoid arthritis [3, 4]. CD52 has also been reported 0198-8859/05/$–see front matter doi:10.1016/j.humimm.2005.03.005
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to be expressed on human dendritic cells (DC), vis-à-vis administering C1H to help eliminate host antigen-presenting DC so as to reduce graft-versus-host disease after bone marrow transplantation has also been considered as an ameliorating mechanism [5], in addition to host immunodepletion to facilitate engraftment [6]. Recently, C1H was also found to be a very effective immunosuppressive agent in organ transplantation [7–10]. We and others have previously reported on the effects of donor bone marrow cells (DBMC) infusions in kidney and liver transplant recipients [11–15]. Several in vitro and ex vivo studies1 demonstrated that bone marrow cells (BMC) downregulate immune responses [16 –18]. We questioned whether C1H would have any impact on this BMC immune regulatory function. As such, would DBMC infusion, combined with C1H administration, have additive benefit if used clinically, or would the C1H-depleting effect somehow cancel tolerogenic mechanisms engendered by DBMC? In the present report, C1H-treated BMC were therefore compared with untreated BMC to assess immune downregulatory effects on either autologous or allogeneic T-cell activation and on inducing T regulatory cells (T regs) (infectious tolerance). MATERIALS AND METHODS Induction of Modulatory Effects on the CD3ⴙ Cells of Peripheral Blood Mononuclear Cells by Autologous C1H-Treated or Untreated BMC in Short-Term Culture Human BMC or peripheral blood mononuclear cells (PBL) were depleted of CD3⫹ cells (nT-PBL or human BMC depleted of T cells [nT-BMC]) and placed in tissue culture as previously described [17] in the following experimental protocols: One group of nT-BMC had been cultured in the presence of 20 g/ml of C1H and 15% rabbit complement for 4 days. On day 4, an additional 20 g/ml of C1H with 15% rabbit complement was added with fresh medium, and the cells were further cultured for 2 days. On day 6, the cells were washed three times with RPMI-1640. The (untreated) nT-PBL and (untreated) nT-BMC were cultured under similar conditions without C1H and complement. Three groups of autologous mixed cultures were then prepared: PBL ⫹ nT-PBL (control), PBL ⫹ nT-BMC, and PBL ⫹ nTBMC treated with C1H (the latter as described above). The ratio of PBL/nT-PBL (control) or PBL/nT-BMC was 2:1 for all cultures. The cultures were then incubated in RPMI-1640 containing 10% FCS in 5% CO2 at 37°C for an additional 7 days. Cells that were CD3⫹ were then 1
In vitro indicates cells tested from volunteers (nondonor or recipient specific); ex vivo indicates cells derived from specific clinical organ transplant donors and recipients.
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isolated from each of the three groups of mixed cultures by negative selection with the Pan-T kit (Miltenyi Biotec, Auburn, CA) as per the manufacturer’s instructions. These three different CD3⫹ cell populations (⬎90% purity) were tested for their immune function. Determination of Proliferative Cytotoxic Response of CD3ⴙ Cells Derived From Autologous Cocultures of PBL Stimulated With and Without C1H-Treated nT-BMC Proliferative response to immobilized anti-CD3 monoclonal antibody. CD3⫹ T cells were retrieved from the above three (autologous) cocultures and their proliferative response via signaling through the CD3 complex was tested. Concentrations of 105 cells/well from each group were prepared in triplicate in flat-bottom 96-well plates coated or uncoated with OKT3 (Ortho Biotech Inc, Raritan, NJ); the wells without OKT3 coating were considered background after 3H-thymidine treatment and processing, as previously described [17, 19]. In addition, 100 U/well of exogenous interleukin 2 (IL-2; Cetus, Emeryville, CA) was added to determine whether this was able to restore the response. Cytotoxic T-cell response to alloantigens. CD3⫹ T cells retrieved from the above (three) autologous cocultures were tested for their cytotoxic response to alloantigenic stimulation. Mitomycin C–treated allogeneic Epstein-Barr virus (EBV) blasts were used as the stimulator cells to generate alloantigen-specific cytotoxic lymphocytes (CTL) at a ratio of 5:1 (responder vs. stimulator). After 7 days in the culture, 4-hour 51Cr release assays were performed against the specific targets (the same EBVtransformed cells used as the stimulators) as previously described [19] at an effector-to-target (E/T) cell ratio of 40:1. Generation of T reg by Multiple Stimulation With C1H-Treated or Untreated nT-BMC in LongTerm Culture CD3⫹ cells were isolated from PBL by negative selection with the Pan-T kit (Miltenyi Biotec, Auburn, CA). The CD3⫹ cells were cultured with mitomycin C–treated autologous C1H-treated or untreated nT-BMC at a ratio of 5:1 in the presence of 20 U/ml of exogenous IL-2. Autologous Ficoll-Hypaque gradient isolated peripheral blood mononuclear cells cultured simultaneously without BMC were similarly treated with 20 U/ml of IL-2 and served as controls (PBL controls). The experimental (CD3⫹ cells mixed with C1H-treated or untreated nTBMC) and control (PBL) cells were cultured for 5 days, then maintained in newly replenished medium for an additional 5 days. The CD3⫹ cell cultures were then restimulated with the two (experimental) respective au-
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tologous mitomycin C C1H-treated or untreated nTBMC for a total of three such cycles. The PBL controls were subjected to the same cycles without additional cell supplementation, and finally after the total culture period, CD3⫹ (control) cells were isolated by negative selection. The CD3⫹ cells, cultured with C1H-treated or untreated BMC, and the CD3⫹ control cells were analyzed for phenotypic markers by flow cytometry. The CD3/CD4/CD25high phenotype of the putative T reg was determined with three-color immunocytometry analysis with CD3-FITC, CD25-PE, and CD4-PerCp (BD Biosciences, San Jose, CA). In functional studies, the effect of T reg generated with C1H-treated or untreated nT-BMC on the primed (effector) phase of the CTL response was determined as follows. Primed CTL against allogeneic EBV-transformed B cell blasts were established as previously described [19]. Briefly, PBL autologous to the T reg were stimulated with allogeneic EBV B cell blasts at a 5:1 ratio for 7 days. The resulting PBL primed (cytotoxic) to the allogeneic EBV cells were added to 96-well round-bottom plates. The T reg cells derived from culture with C1H-treated or untreated BMC were added at a 1:1 effector to T reg ratio 1 day before the addition of the 51Cr-labeled EBV target cells. Control cultures contained the added PBL cultured for the same three cycles as the putative CD3⫹ T reg cells. Cytotoxic Response of CD3ⴙ Cells Derived From Cocultures of PBL With Allogeneic C1H-Treated or Untreated nT-BMC Cocultures of PBL stimulated with (1-DR mismatched) allogeneic C1H-treated or untreated nT-BMC were performed, with PBL versus nT autologous PBL again serving as the responder control. After 7 days of coculture, CD3⫹ cells were isolated by negative selection (with Pan-T Kit, Miltenyi Biotec, Auburn, CA). These were then restimulated with mature dendritic cell (mDC)enriched populations (generated as previously described [20]) from the same (allogeneic) BMC donor at a 5:1 responder to stimulator ratio for 7 days. Cytotoxic activity against the EBV target cells again from the same donor was tested in 4-hour 51Cr release assays. Measurement of Adenosine Triphosphate Levels of CD3ⴙ Cells Derived From Cocultures With Autologous PBL Versus C1H-Treated or Untreated nT-BMC Adenosine triphosphate (ATP) levels of the T cells retrieved from these cocultures were measured as described by the ImmuKnow kit manufacturer (Cylex, Columbia, MD). Briefly, 3 days after incubation with immobilized anti-CD3 monoclonal antibody (vide supra), 105 CD3⫹ cells were collected, pelleted, and treated with 100 l of lysis reagent to release ATP. A total of 50 l of cell
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FIGURE 1 C1H treatment does not alter BMC capacity to anergize T cells. T cells retrieved from cocultures of PBL/nTBMC or PBL/C1H-treated nT-BMC exhibited an equivalent reduction in proliferation to immobilized anti-CD3, compared with T cells retrieved from controls (PBL/nT-PBL). Exogenous IL-2 exhibited a slight rescue effect, perhaps to a lesser degree on T cells from PBL/C1H-treated nT-BMC (n ⫽ 4). In the absence of IL-2, p ⫽ 0.003 (PBL/nT-PBL vs. PBL/nT-BMC) and p ⫽ 0.002 (PBL/nT-PBL vs. PBL/C1H-treated nT-BMC). In the presence of IL-2, p ⫽ 0.039 (PBL/nT-PBL vs. PBL/nTBMC) and p ⫽ 0.019 (PBL/nT-PBL vs. PBL/C1H-treated nT-BMC). Data are expressed as means ⫾ SE.
lysates were transferred into a measurement plate, and 100 l of luminescence-generating reagent (ATP detection reagent) was added into each well to detect ATP. After 3 to 10 minutes, the plates were read by a luminometer. RESULTS C1H Treatment of nT-BMC Does Not Alter the Ability to Induce T-Cell Anergy We have previously demonstrated that a primary effect of coculturing PBL with BMC is the induction of nonresponsiveness (anergy) in the T-cell population [17]. One key event that occurs in anergized T cells is impaired signaling through the T-cell receptor (TCR)-CD3 complex [21]. In the present experiment, the proliferative response to immobilized anti-CD3 of CD3⫹ cells derived from cultures of PBL ⫹ C1H-treated nT-BMC was suppressed to the same degree as that of CD3⫹ cells from cultures of PBL and C1H-untreated nT-BMC (Figure 1). The addition of IL-2 to CD3⫹ cells from either C1Htreated or untreated nT-BMC cultures caused only a slight increase in the response. This reduced responsiveness indicated reduced signaling through the CD3 complex, which was also accompanied by a ⬃50% reduction in cellular ATP levels (Figure 2), i.e., a lower capacity for energy
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C1H-treated nT-BMC was suppressed ⬃70% when compared with CD3⫹ cells derived from PBL cultures. C1H Treatment of BMC Does Not Alter Their Ability to Induce T reg Cells In a previous study we reported that BMC induced regulatory T cells in vitro that suppressed both autologous and allogeneic T-cell responses to viral antigens and alloantigens, respectively [17]. To examine if C1H affects this process, putative T reg cells were induced by three rounds of stimulation of CD3⫹ cells with either nT-BMC or C1H-treated nT-BMC (see Materials and Methods). The T cells from both nT-BMC– containing cultures were predominantly of the CD3/CD4/CD25high phenotype when compared with the T cells from the PBL controls (p ⫽ 0.002) (Figure 4). The average percentages of CD3/CD4/CD25high cells from both groups (n ⫽ 4) were 33.95 ⫾ 3.6% and 35.74 ⫾ 3.86% for C1Htreated and untreated nT-BMC, respectively. To test for immune regulatory function, these T cells from either C1H-treated or untreated nT-BMC cultures (Figure 5) were added into primed cultured cell lines containing their cognate alloantigen specific effector T cells. Cytotoxicity was equivalently downregulated by T regs inFIGURE 3 C1H-treated nT-BMC has equivalent capacity to untreated nT-BMC to inhibit an autologous T-cell cytotoxic response to alloantigen. Three groups of cultures were prepared with autologous PBL: (1) PBL ⫹ nT-PBL (controls); (2) PBL ⫹ untreated nT-BMC; and (3) PBL⫹C1H treated nT-BMC—all at a ratio of 2:1. CD3⫹ cells retrieved from PBL/nT-BMC and PBL/C1H-treated nT-BMC were equally inhibited in their CTL responses compared with CD3⫹ cells from the control cultures at an E/T ratio of 40:1 (p ⫽ 0.04, PBL/nT-PBL versus PBL/nTBMC, n ⫽ 5; and p ⫽ 0.03, PBL/nT-PBL vs. PBL/C1H-treated nT-BMC, n ⫽ 5). Data are expressed as means ⫾ SE.
transformation, which was similar in T cells derived from either C1H-treated or untreated nT-BMC. C1H Treatment of BMC Does Not Inhibit Suppression of CD3ⴙ Cytotoxic Function We reported in previous studies that CD3⫹ cells cocultured with autologous nT-BMC exhibited a profound reduction in the ability to respond in mixed lymphocyte and CTL reactions [16, 18]. In order to determine whether C1H had any effect on this BMC cytotoxic inhibitory function, we induced cytotoxic T cells after first culturing PBL with C1H-treated nT-BMC versus untreated nT-BMC. CD3⫹ cells derived from cultures of PBL ⫹ autologous nT-PBL were tested as controls. The CD3⫹ cells from the experimental and control cultures were stimulated with allogeneic EBV-transformed B cell blasts (5:1 ratio) to engender cytotoxic T cells. As illustrated in Figure 3, the cytotoxicity of both untreated and
FIGURE 2 ATP levels. After stimulation with immobilized anti-CD3, the (anergized) T cells of PBL/nT-BMC and PBL/ C1H-treated nT-BMC, demonstrated ATP levels that were approximately half that of control PBL/nT-PBL (p ⫽ 0.005, PBL/nT-PBL vs. PBL/nT-BMC, n ⫽ 4; p ⫽ 0.006, PBL/nTPBL vs. PBL/C1H-treated nT-BMC, n ⫽ 4; p ⫽ 0.16, PBL/ nT-BMC vs. PBL/C1H-treated nT-BMC). Data are expressed as means ⫾ SE.
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Materials and Methods). This experiment (Figure 6) indicated that T cells after encountering either treated or untreated allogeneic nT-BMC had at least twofold reduced alloreactivity compared with the controls at E/T ratios of 40:1 and 20:1 (n ⫽ 6 per group). As depicted, the C1H-treated (allogeneic) nT-BMC unexpectedly caused statistically even stronger inhibition than the untreated (allogeneic) nT-BMC.
FIGURE 5 C1H treatment does not remove nT-BMC potential to induce T reg. T cells cocultured with autologous C1H-treated (T reg-C1H) or untreated (T reg) nT-BMC were added into the cognate (primed) T-cell alloantigen-specific CTL assays. Alloreactivity was downregulated with either T reg C1H-treated nT-BMC (n ⫽ 4) or T reg nT-BMC (n ⫽ 4). p Values are 0.006 control versus T reg–C1H–treated nTBMC, n ⫽ 4, and 0.002 control versus T reg nT-BMC, n ⫽ 4. T reg C1H-treated nT-BMC and T reg nT-BMC exhibited equivalent inhibitory capacity (p ⫽ 0.44). Data are expressed as means ⫾ SE.
DISCUSSION Several reports from these laboratories have demonstrated that human BMC markedly downregulate in vitro and ex vivo (see previous definitions) responses to both autologous and allogeneic T cells [16 –18], and have confirmed that C1H, the humanized anti-CD52 monoclonal antibody, has a profound and prolonged effect on T-cell levels in vivo, which has led to its use in organ transplantation [9, 10]. We proposed that combining DBMC infusions and C1H in renal transplant recipients would lead to a more potent tolerogenic effect, but the question remained if the elimination of CD52-expressing hematopoietic cells from BMC would dampen their ability to downregulate T-cell responses. In previous studies, BMC in a contact-dependent mechanism, effectively reduced the ability of T cells to be stimulated through the TCR-CD3 complex [17]. We therefore tested the proliferative response of the CD3⫹ cells evoked by anti-CD3 monoclonal antibody, a CD3
duced by both C1H-treated and untreated autologous nT-BMC (Figure 5). The controls consisted of T cells derived from cultures of PBL, similarly treated. C1H Treatment of (Allogeneic) BMC May Enhance Their Ability to Regulate T-Cell Function The in vitro experimental model that uses CD3⫹ cells and autologous nT-BMC to induce regulatory T cells does not completely reflect the potential effect of (allogeneic rather than autologous) donor BMC infusions in kidney allograft recipients in clinical studies being performed at this and other centers [11, 12, 14, 22]. Visà-vis, infused donor BMC directly encounter recipient allogeneic rather than autologous immunoreactive cells. In order to attempt to duplicate this in vitro, we examined the T-cell response (recipient) to allogeneic BMC (donor) by coculturing PBL with allogeneic nT-BMC treated and untreated with C1H, and (recipient) T cells retrieved from these cultures were stimulated with a mature DC-enriched population derived from the same BMC donor in order to generate cytotoxic effectors (see
FIGURE 4 T reg phenotypic markers. A high percentage of CD25highCD4⫹ T cells could be induced by multiple rounds of stimulation with autologous nT-BMC in the presence or absence of C1H treatment. The average percentage of CD3/ CD4/CD25high was 33.95% ⫾ 3.6% for T reg C1H-treated nT-BMC versus 35.74% ⫾ 3.86% for T reg nT-BMC (n ⫽ 4 in each group, p ⫽ 0.30). One representative of four sets of histograms is shown. In contrast, there were 4.97% ⫾ 0.87% seen after incubation with control PBL cultures (p ⫽ 0.002).
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complex signal transduction-dependent pathway, after coculturing with nT-BMC versus C1H-treated nT-BMC. The results (Figure 1) demonstrated that CD3⫹ cells derived from cocultures with either C1H-treated or untreated nT-BMC were equally suppressed, and that the addition of IL-2 did not restore the proliferative activity to control levels. Intriguingly, the CD3⫹ cells from cultures with C1H-treated nT-BMC were slightly less responsive in the IL-2 environment, suggesting a possible differential amplification effect of IL-2 on PBL/ntBMC versus the PBL/C1H-treated nT-BMC, i.e., perhaps even on a non-TCR signal transduction pathway. This difference, though not significant, may warrant further investigation and characterization. Nonetheless, in an experiment involving T-cell proliferation signal transduction, in which second messengers (cAMP) are generated from phosphorylated precursor ATP, the ATP levels of CD3⫹ cells from PBL/nTBMC versus PBL/C1H-treated nT-BMC after stimulation with anti-CD3⫹ monoclonal antibody, exhibited an equivalent ⬃40% decrease, i.e., a downstream effect of this treatment that was apparently equivalent. Because earlier studies have demonstrated that BMC markedly reduced T-cell cytotoxic function, we examined the C1H nT-BMC effect on this immune function. When compared with the controls (PBL/nT-PBL), both BMC cultures (with or without C1H), equally suppressed the cytotoxic response of autologous T cells (Figure 3). Over the past several years, there has been a spate of reports dealing with T reg cells in transplantation tolerance [23]. There have even been reports of veto cells generated by (contact with) CD34⫹ bone marrow-derived stem cells [24] and other inhibitory effects of bone marrow mesenchymal cells [25]. The putative T reg cells in the present report were generated by repeated stimulation of CD3⫹ cells with autologous nT-BMC in the presence of IL-2, with C1H treatment of the nT-BMC not altering T reg generation. This repeated stimulation of CD3⫹ cells with C1H-treated or untreated nT-BMC produced a high percentage of CD4/CD25high T cells (Figure 4), which were potent inhibitors of a “primed” cytotoxic immune response (Figure 5). To determine whether C1H treatment had any impact on the ability of BMC to downregulate an allogeneic response, we cocultured PBL with 1-DR matched C1Htreated or untreated allogeneic nT-BMC (analogous to the donor/recipient match inclusion criteria in our clinical studies [11]). The CD3⫹ T cells obtained from these cocultures were then stimulated with a mature DCenriched population derived from the BMC donor, i.e., a culture condition mimicking what might occur when an organ recipient receives an allogeneic DBMC infusion and a kidney from the same donor. At E/T ratios of 40:1
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FIGURE 6 C1H treatment of BMC enhances their ability to induce alloimmune regulatory function. T cells retrieved from PBL/autologous nT-PBL (as control), PBL/allogeneic nTBMC, or PBL/C1H-treated allogeneic nT-BMC were all stimulated with a mature DC-enriched population derived from the BMC donor of the coculture. The T cells that were cocultured with either C1H-untreated or treated allogeneic nTBMC exhibited at least twofold reduced cytotoxic alloreactivity against the bone marrow donor (n ⫽ 6) in each group (p values are 0.007 and 0.045 for PBL/nT-PBL versus PBL/nTBMC at E/T ratio of 40:1 and 20:1, respectively; p values are 0.004 and 0.032 for PBL/nT-PBL versus PBL/C1H-treated nT-BMC at E/T ratio of 40:1 and 20:1, respectively). p Values between PBL/nT-BMC and PBL/C1H-treated nT-BMC also exhibited statistical differences— 0.046 and 0.043 at the different E/T ratios. Data are expressed as means ⫾ SE.
and 20:1, inhibition of C1H-treated nT-BMC derived CD3⫹ cell cytotoxicity was even greater than that of nT-BMC-derived CD3⫹ cells (Figure 6), so that it appeared that in this allogeneic system, C1H treatment of nT-BMC enhanced the suppressive effect. Data from this study therefore suggest that C1Hresistant cells in the BMC compartment are able to provide a tolerogenic environment, consistent with the notion of a potential clinical application of donor BMC infusion accompanying C1H administration. Further work on (differential) CD52 expression on specific subsets of BMCs appears warranted. ACKNOWLEDGMENTS
This work was supported by research grants from the NIH (5 R01-DK25243-24), the VA Medical Research Services of the Miami VA Medical Center, Fujisawa, and Roche Pharmaceuticals.
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14. Ciancio G, Miller J, Garcia-Morales RO, Carreno M, Burke GW 3rd, Roth D, Kupin W, Tzakis AG, Ricordi C, Rosen A, Fuller L, Esquenazi V: Six-year clinical effect of donor bone marrow infusions in renal transplant patients. Transplantation 71:827, 2001. 15. Ricordi C, Karatzas T, Nery J, Webb M, Selvaggi G, Fernandez L, Khan FA, Ruiz P, Schiff E, Olson L, Fernandez H, Bean J, Esquenazi V, Miller J, Tzakis AG: High-dose donor bone marrow infusions to enhance allograft survival: the effect of timing. Transplantation 63:7, 1997. 16. 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. 17. Jin Y, Fuller L, Carreno M, Esquenazi V, Blomberg BB, Wei YT, Ciancio G, Burke GW 3rd, Tzakis A, Ricordi C, Miller J: Functional and phenotypic properties of peripheral T cells anergized by autologous CD3⫹ depleted bone marrow cells. Hum Immunol 63:567, 2002. 18. Mathew JM, Garcia-Morales R, Fuller L, Rosen A, Ciancio G, Burke GW, Carreno M, Temple D, Tzakis AG, Ricordi C, Miller J, Esquenazi V: Donor bone marrow– derived chimeric cells present in renal transplant recipients infused with donor marrow. I. Potent regulators of recipient antidonor immune responses. Transplantation 70:1675, 2000. 19. Jin Y, Fuller L, Esquenazi V, Blomberg BB, Rosen A, Tzakis AG, Ricordi C, Miller J: Bone marrow cells inhibit the generation of autologous EBV-specific CTL. Hum Immunol 61:538, 2000. 20. Jin Y, Fuller L, Esquenazi V, Ciancio G, Burke GW 3rd, Tzakis AG, Ricordi C, Miller J: Antigen presentation and immune regulatory capacity of immature and matureenriched antigen presenting (dendritic) cells derived from human bone marrow. Hum Immunol 65:93, 2004. 21. Migita K, Eguchi K, Kawabe Y, Tsudada T, Ichinose Y, Nafarake S, Ochi A: Defective TCR-mediated signaling in anergic T cells. J Immunol 155:5083, 1995. 22. Millan MT, Shizuru JA, Hoffmann P, Dejbakhsh-Jones S, Scandling JD, Grumet FC, Tan JC, Salvatierra O, Hoppe RT, Strober S. Mixed chimerism and immunosuppressive drug withdrawal after HLA-mismatched kidney and hematopoietic progenitor transplantation. Transplantation 73:1386, 2002. 23. Wood KJ, Sakaguchi S: Regulatory T cells in transplantation tolerance. Nat Rev Immunol 3:199, 2003. 24. Reisner Y, Gur H, Reich-Zeliger S, Martelli MF, BacharLustig E. Hematopoietic stem cell transplantation across major genetic barriers: tolerance induction by megadose CD34 cells and other veto cells. Ann N Y Acad Sci 996:72, 2003. 25. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105:1815, 2005.
From the Lillian Jean Kaplan Renal Transplant Center of the Division of Transplantation of the Department of Surgery (Y.J., L.F., A.R., G.C., G.W.B., C.R., A.G.T., J.M., V.E.), the Department of Microbiology/ Immunology (L.F., C.R., J.M., V.E.), and the Diabetes Research Institute (C.R.) at the University of Miami, Leonard M. Miller School of Medicine; and the Miami Veterans Affairs Medical Center (J.M., V.E.), Miami, FL, USA. Address reprint requests to: Dr. Y. Jin, Department of Surgery, Division of Transplantation, University of Miami School of Medicine, P.O. Box 012440 (R-440), Miami, FL 33101; Tel: (305) 355-5100; Fax: (305) 3555134; E-mail: [email protected]. Received February 2, 2005. Human Immunology 66, 637– 643 (2005) © American Society for Histocompatibility and Immunogenetics, 2005 Published by Elsevier Inc.
treated and untreated nT-BMC cocultures. The ability of C1H-treated or untreated nT-BMC to suppress autologous T-cell cytotoxic function was also equivalent, with a marked, but equivalent, capacity to induce CD4/CD25high T regs from CD3⫹ cells, which effectively downregulated cytotoxic T cells. To mimic the clinical infusion of DBMC into (allogeneic) recipients, peripheral blood mononuclear cells were also cultured with allogeneic C1H-treated and untreated nT-BMC. T cells derived from these cultures secondarily stimulated with the same-donor mature antigen-presenting cells exhibited suppressed cytotoxicity by 85% and 54%, respectively. These in vitro studies suggest that C1H does not abrogate BMC immunoregulation and thus may allow its lympho-depleting effect to be synergistic. Human Immunology 66, 637– 643 (2005). © American Society for Histocompatibility and Immunogenetics, 2005. Published by Elsevier Inc. KEYWORDS: Campath-1H; bone marrow antigenpresenting cell; regulatory function
EBV nT-BMC PBL T regs TCR
Epstein-Barr virus human BMC depleted of T cells peripheral blood mononuclear cells T regulatory cells T-cell receptors
INTRODUCTION Alemtuzumab (Campath-1H, or C1H; Ilex Oncology, San Antonio, TX) is a humanized monoclonal antibody that recognizes the CD52 epitope, a small glycosylphosphatidylinositol glycoprotein expressed at a high level on T and B lymphocytes and to a lesser extent on other mononuclear cells [1– 4]. It appears that the CD52 epitope is close to the cell membrane, so that antigenantibody binding will easily lead to cell lysis through the complement or antibody-dependent cell-mediated cytotoxicity pathways [5, 6]. On this basis, C1H has been used in the past to treat lymphoid malignancies and rheumatoid arthritis [3, 4]. CD52 has also been reported 0198-8859/05/$–see front matter doi:10.1016/j.humimm.2005.03.005
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to be expressed on human dendritic cells (DC), vis-à-vis administering C1H to help eliminate host antigen-presenting DC so as to reduce graft-versus-host disease after bone marrow transplantation has also been considered as an ameliorating mechanism [5], in addition to host immunodepletion to facilitate engraftment [6]. Recently, C1H was also found to be a very effective immunosuppressive agent in organ transplantation [7–10]. We and others have previously reported on the effects of donor bone marrow cells (DBMC) infusions in kidney and liver transplant recipients [11–15]. Several in vitro and ex vivo studies1 demonstrated that bone marrow cells (BMC) downregulate immune responses [16 –18]. We questioned whether C1H would have any impact on this BMC immune regulatory function. As such, would DBMC infusion, combined with C1H administration, have additive benefit if used clinically, or would the C1H-depleting effect somehow cancel tolerogenic mechanisms engendered by DBMC? In the present report, C1H-treated BMC were therefore compared with untreated BMC to assess immune downregulatory effects on either autologous or allogeneic T-cell activation and on inducing T regulatory cells (T regs) (infectious tolerance). MATERIALS AND METHODS Induction of Modulatory Effects on the CD3ⴙ Cells of Peripheral Blood Mononuclear Cells by Autologous C1H-Treated or Untreated BMC in Short-Term Culture Human BMC or peripheral blood mononuclear cells (PBL) were depleted of CD3⫹ cells (nT-PBL or human BMC depleted of T cells [nT-BMC]) and placed in tissue culture as previously described [17] in the following experimental protocols: One group of nT-BMC had been cultured in the presence of 20 g/ml of C1H and 15% rabbit complement for 4 days. On day 4, an additional 20 g/ml of C1H with 15% rabbit complement was added with fresh medium, and the cells were further cultured for 2 days. On day 6, the cells were washed three times with RPMI-1640. The (untreated) nT-PBL and (untreated) nT-BMC were cultured under similar conditions without C1H and complement. Three groups of autologous mixed cultures were then prepared: PBL ⫹ nT-PBL (control), PBL ⫹ nT-BMC, and PBL ⫹ nTBMC treated with C1H (the latter as described above). The ratio of PBL/nT-PBL (control) or PBL/nT-BMC was 2:1 for all cultures. The cultures were then incubated in RPMI-1640 containing 10% FCS in 5% CO2 at 37°C for an additional 7 days. Cells that were CD3⫹ were then 1
In vitro indicates cells tested from volunteers (nondonor or recipient specific); ex vivo indicates cells derived from specific clinical organ transplant donors and recipients.
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isolated from each of the three groups of mixed cultures by negative selection with the Pan-T kit (Miltenyi Biotec, Auburn, CA) as per the manufacturer’s instructions. These three different CD3⫹ cell populations (⬎90% purity) were tested for their immune function. Determination of Proliferative Cytotoxic Response of CD3ⴙ Cells Derived From Autologous Cocultures of PBL Stimulated With and Without C1H-Treated nT-BMC Proliferative response to immobilized anti-CD3 monoclonal antibody. CD3⫹ T cells were retrieved from the above three (autologous) cocultures and their proliferative response via signaling through the CD3 complex was tested. Concentrations of 105 cells/well from each group were prepared in triplicate in flat-bottom 96-well plates coated or uncoated with OKT3 (Ortho Biotech Inc, Raritan, NJ); the wells without OKT3 coating were considered background after 3H-thymidine treatment and processing, as previously described [17, 19]. In addition, 100 U/well of exogenous interleukin 2 (IL-2; Cetus, Emeryville, CA) was added to determine whether this was able to restore the response. Cytotoxic T-cell response to alloantigens. CD3⫹ T cells retrieved from the above (three) autologous cocultures were tested for their cytotoxic response to alloantigenic stimulation. Mitomycin C–treated allogeneic Epstein-Barr virus (EBV) blasts were used as the stimulator cells to generate alloantigen-specific cytotoxic lymphocytes (CTL) at a ratio of 5:1 (responder vs. stimulator). After 7 days in the culture, 4-hour 51Cr release assays were performed against the specific targets (the same EBVtransformed cells used as the stimulators) as previously described [19] at an effector-to-target (E/T) cell ratio of 40:1. Generation of T reg by Multiple Stimulation With C1H-Treated or Untreated nT-BMC in LongTerm Culture CD3⫹ cells were isolated from PBL by negative selection with the Pan-T kit (Miltenyi Biotec, Auburn, CA). The CD3⫹ cells were cultured with mitomycin C–treated autologous C1H-treated or untreated nT-BMC at a ratio of 5:1 in the presence of 20 U/ml of exogenous IL-2. Autologous Ficoll-Hypaque gradient isolated peripheral blood mononuclear cells cultured simultaneously without BMC were similarly treated with 20 U/ml of IL-2 and served as controls (PBL controls). The experimental (CD3⫹ cells mixed with C1H-treated or untreated nTBMC) and control (PBL) cells were cultured for 5 days, then maintained in newly replenished medium for an additional 5 days. The CD3⫹ cell cultures were then restimulated with the two (experimental) respective au-
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tologous mitomycin C C1H-treated or untreated nTBMC for a total of three such cycles. The PBL controls were subjected to the same cycles without additional cell supplementation, and finally after the total culture period, CD3⫹ (control) cells were isolated by negative selection. The CD3⫹ cells, cultured with C1H-treated or untreated BMC, and the CD3⫹ control cells were analyzed for phenotypic markers by flow cytometry. The CD3/CD4/CD25high phenotype of the putative T reg was determined with three-color immunocytometry analysis with CD3-FITC, CD25-PE, and CD4-PerCp (BD Biosciences, San Jose, CA). In functional studies, the effect of T reg generated with C1H-treated or untreated nT-BMC on the primed (effector) phase of the CTL response was determined as follows. Primed CTL against allogeneic EBV-transformed B cell blasts were established as previously described [19]. Briefly, PBL autologous to the T reg were stimulated with allogeneic EBV B cell blasts at a 5:1 ratio for 7 days. The resulting PBL primed (cytotoxic) to the allogeneic EBV cells were added to 96-well round-bottom plates. The T reg cells derived from culture with C1H-treated or untreated BMC were added at a 1:1 effector to T reg ratio 1 day before the addition of the 51Cr-labeled EBV target cells. Control cultures contained the added PBL cultured for the same three cycles as the putative CD3⫹ T reg cells. Cytotoxic Response of CD3ⴙ Cells Derived From Cocultures of PBL With Allogeneic C1H-Treated or Untreated nT-BMC Cocultures of PBL stimulated with (1-DR mismatched) allogeneic C1H-treated or untreated nT-BMC were performed, with PBL versus nT autologous PBL again serving as the responder control. After 7 days of coculture, CD3⫹ cells were isolated by negative selection (with Pan-T Kit, Miltenyi Biotec, Auburn, CA). These were then restimulated with mature dendritic cell (mDC)enriched populations (generated as previously described [20]) from the same (allogeneic) BMC donor at a 5:1 responder to stimulator ratio for 7 days. Cytotoxic activity against the EBV target cells again from the same donor was tested in 4-hour 51Cr release assays. Measurement of Adenosine Triphosphate Levels of CD3ⴙ Cells Derived From Cocultures With Autologous PBL Versus C1H-Treated or Untreated nT-BMC Adenosine triphosphate (ATP) levels of the T cells retrieved from these cocultures were measured as described by the ImmuKnow kit manufacturer (Cylex, Columbia, MD). Briefly, 3 days after incubation with immobilized anti-CD3 monoclonal antibody (vide supra), 105 CD3⫹ cells were collected, pelleted, and treated with 100 l of lysis reagent to release ATP. A total of 50 l of cell
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FIGURE 1 C1H treatment does not alter BMC capacity to anergize T cells. T cells retrieved from cocultures of PBL/nTBMC or PBL/C1H-treated nT-BMC exhibited an equivalent reduction in proliferation to immobilized anti-CD3, compared with T cells retrieved from controls (PBL/nT-PBL). Exogenous IL-2 exhibited a slight rescue effect, perhaps to a lesser degree on T cells from PBL/C1H-treated nT-BMC (n ⫽ 4). In the absence of IL-2, p ⫽ 0.003 (PBL/nT-PBL vs. PBL/nT-BMC) and p ⫽ 0.002 (PBL/nT-PBL vs. PBL/C1H-treated nT-BMC). In the presence of IL-2, p ⫽ 0.039 (PBL/nT-PBL vs. PBL/nTBMC) and p ⫽ 0.019 (PBL/nT-PBL vs. PBL/C1H-treated nT-BMC). Data are expressed as means ⫾ SE.
lysates were transferred into a measurement plate, and 100 l of luminescence-generating reagent (ATP detection reagent) was added into each well to detect ATP. After 3 to 10 minutes, the plates were read by a luminometer. RESULTS C1H Treatment of nT-BMC Does Not Alter the Ability to Induce T-Cell Anergy We have previously demonstrated that a primary effect of coculturing PBL with BMC is the induction of nonresponsiveness (anergy) in the T-cell population [17]. One key event that occurs in anergized T cells is impaired signaling through the T-cell receptor (TCR)-CD3 complex [21]. In the present experiment, the proliferative response to immobilized anti-CD3 of CD3⫹ cells derived from cultures of PBL ⫹ C1H-treated nT-BMC was suppressed to the same degree as that of CD3⫹ cells from cultures of PBL and C1H-untreated nT-BMC (Figure 1). The addition of IL-2 to CD3⫹ cells from either C1Htreated or untreated nT-BMC cultures caused only a slight increase in the response. This reduced responsiveness indicated reduced signaling through the CD3 complex, which was also accompanied by a ⬃50% reduction in cellular ATP levels (Figure 2), i.e., a lower capacity for energy
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C1H-treated nT-BMC was suppressed ⬃70% when compared with CD3⫹ cells derived from PBL cultures. C1H Treatment of BMC Does Not Alter Their Ability to Induce T reg Cells In a previous study we reported that BMC induced regulatory T cells in vitro that suppressed both autologous and allogeneic T-cell responses to viral antigens and alloantigens, respectively [17]. To examine if C1H affects this process, putative T reg cells were induced by three rounds of stimulation of CD3⫹ cells with either nT-BMC or C1H-treated nT-BMC (see Materials and Methods). The T cells from both nT-BMC– containing cultures were predominantly of the CD3/CD4/CD25high phenotype when compared with the T cells from the PBL controls (p ⫽ 0.002) (Figure 4). The average percentages of CD3/CD4/CD25high cells from both groups (n ⫽ 4) were 33.95 ⫾ 3.6% and 35.74 ⫾ 3.86% for C1Htreated and untreated nT-BMC, respectively. To test for immune regulatory function, these T cells from either C1H-treated or untreated nT-BMC cultures (Figure 5) were added into primed cultured cell lines containing their cognate alloantigen specific effector T cells. Cytotoxicity was equivalently downregulated by T regs inFIGURE 3 C1H-treated nT-BMC has equivalent capacity to untreated nT-BMC to inhibit an autologous T-cell cytotoxic response to alloantigen. Three groups of cultures were prepared with autologous PBL: (1) PBL ⫹ nT-PBL (controls); (2) PBL ⫹ untreated nT-BMC; and (3) PBL⫹C1H treated nT-BMC—all at a ratio of 2:1. CD3⫹ cells retrieved from PBL/nT-BMC and PBL/C1H-treated nT-BMC were equally inhibited in their CTL responses compared with CD3⫹ cells from the control cultures at an E/T ratio of 40:1 (p ⫽ 0.04, PBL/nT-PBL versus PBL/nTBMC, n ⫽ 5; and p ⫽ 0.03, PBL/nT-PBL vs. PBL/C1H-treated nT-BMC, n ⫽ 5). Data are expressed as means ⫾ SE.
transformation, which was similar in T cells derived from either C1H-treated or untreated nT-BMC. C1H Treatment of BMC Does Not Inhibit Suppression of CD3ⴙ Cytotoxic Function We reported in previous studies that CD3⫹ cells cocultured with autologous nT-BMC exhibited a profound reduction in the ability to respond in mixed lymphocyte and CTL reactions [16, 18]. In order to determine whether C1H had any effect on this BMC cytotoxic inhibitory function, we induced cytotoxic T cells after first culturing PBL with C1H-treated nT-BMC versus untreated nT-BMC. CD3⫹ cells derived from cultures of PBL ⫹ autologous nT-PBL were tested as controls. The CD3⫹ cells from the experimental and control cultures were stimulated with allogeneic EBV-transformed B cell blasts (5:1 ratio) to engender cytotoxic T cells. As illustrated in Figure 3, the cytotoxicity of both untreated and
FIGURE 2 ATP levels. After stimulation with immobilized anti-CD3, the (anergized) T cells of PBL/nT-BMC and PBL/ C1H-treated nT-BMC, demonstrated ATP levels that were approximately half that of control PBL/nT-PBL (p ⫽ 0.005, PBL/nT-PBL vs. PBL/nT-BMC, n ⫽ 4; p ⫽ 0.006, PBL/nTPBL vs. PBL/C1H-treated nT-BMC, n ⫽ 4; p ⫽ 0.16, PBL/ nT-BMC vs. PBL/C1H-treated nT-BMC). Data are expressed as means ⫾ SE.
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Materials and Methods). This experiment (Figure 6) indicated that T cells after encountering either treated or untreated allogeneic nT-BMC had at least twofold reduced alloreactivity compared with the controls at E/T ratios of 40:1 and 20:1 (n ⫽ 6 per group). As depicted, the C1H-treated (allogeneic) nT-BMC unexpectedly caused statistically even stronger inhibition than the untreated (allogeneic) nT-BMC.
FIGURE 5 C1H treatment does not remove nT-BMC potential to induce T reg. T cells cocultured with autologous C1H-treated (T reg-C1H) or untreated (T reg) nT-BMC were added into the cognate (primed) T-cell alloantigen-specific CTL assays. Alloreactivity was downregulated with either T reg C1H-treated nT-BMC (n ⫽ 4) or T reg nT-BMC (n ⫽ 4). p Values are 0.006 control versus T reg–C1H–treated nTBMC, n ⫽ 4, and 0.002 control versus T reg nT-BMC, n ⫽ 4. T reg C1H-treated nT-BMC and T reg nT-BMC exhibited equivalent inhibitory capacity (p ⫽ 0.44). Data are expressed as means ⫾ SE.
DISCUSSION Several reports from these laboratories have demonstrated that human BMC markedly downregulate in vitro and ex vivo (see previous definitions) responses to both autologous and allogeneic T cells [16 –18], and have confirmed that C1H, the humanized anti-CD52 monoclonal antibody, has a profound and prolonged effect on T-cell levels in vivo, which has led to its use in organ transplantation [9, 10]. We proposed that combining DBMC infusions and C1H in renal transplant recipients would lead to a more potent tolerogenic effect, but the question remained if the elimination of CD52-expressing hematopoietic cells from BMC would dampen their ability to downregulate T-cell responses. In previous studies, BMC in a contact-dependent mechanism, effectively reduced the ability of T cells to be stimulated through the TCR-CD3 complex [17]. We therefore tested the proliferative response of the CD3⫹ cells evoked by anti-CD3 monoclonal antibody, a CD3
duced by both C1H-treated and untreated autologous nT-BMC (Figure 5). The controls consisted of T cells derived from cultures of PBL, similarly treated. C1H Treatment of (Allogeneic) BMC May Enhance Their Ability to Regulate T-Cell Function The in vitro experimental model that uses CD3⫹ cells and autologous nT-BMC to induce regulatory T cells does not completely reflect the potential effect of (allogeneic rather than autologous) donor BMC infusions in kidney allograft recipients in clinical studies being performed at this and other centers [11, 12, 14, 22]. Visà-vis, infused donor BMC directly encounter recipient allogeneic rather than autologous immunoreactive cells. In order to attempt to duplicate this in vitro, we examined the T-cell response (recipient) to allogeneic BMC (donor) by coculturing PBL with allogeneic nT-BMC treated and untreated with C1H, and (recipient) T cells retrieved from these cultures were stimulated with a mature DC-enriched population derived from the same BMC donor in order to generate cytotoxic effectors (see
FIGURE 4 T reg phenotypic markers. A high percentage of CD25highCD4⫹ T cells could be induced by multiple rounds of stimulation with autologous nT-BMC in the presence or absence of C1H treatment. The average percentage of CD3/ CD4/CD25high was 33.95% ⫾ 3.6% for T reg C1H-treated nT-BMC versus 35.74% ⫾ 3.86% for T reg nT-BMC (n ⫽ 4 in each group, p ⫽ 0.30). One representative of four sets of histograms is shown. In contrast, there were 4.97% ⫾ 0.87% seen after incubation with control PBL cultures (p ⫽ 0.002).
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complex signal transduction-dependent pathway, after coculturing with nT-BMC versus C1H-treated nT-BMC. The results (Figure 1) demonstrated that CD3⫹ cells derived from cocultures with either C1H-treated or untreated nT-BMC were equally suppressed, and that the addition of IL-2 did not restore the proliferative activity to control levels. Intriguingly, the CD3⫹ cells from cultures with C1H-treated nT-BMC were slightly less responsive in the IL-2 environment, suggesting a possible differential amplification effect of IL-2 on PBL/ntBMC versus the PBL/C1H-treated nT-BMC, i.e., perhaps even on a non-TCR signal transduction pathway. This difference, though not significant, may warrant further investigation and characterization. Nonetheless, in an experiment involving T-cell proliferation signal transduction, in which second messengers (cAMP) are generated from phosphorylated precursor ATP, the ATP levels of CD3⫹ cells from PBL/nTBMC versus PBL/C1H-treated nT-BMC after stimulation with anti-CD3⫹ monoclonal antibody, exhibited an equivalent ⬃40% decrease, i.e., a downstream effect of this treatment that was apparently equivalent. Because earlier studies have demonstrated that BMC markedly reduced T-cell cytotoxic function, we examined the C1H nT-BMC effect on this immune function. When compared with the controls (PBL/nT-PBL), both BMC cultures (with or without C1H), equally suppressed the cytotoxic response of autologous T cells (Figure 3). Over the past several years, there has been a spate of reports dealing with T reg cells in transplantation tolerance [23]. There have even been reports of veto cells generated by (contact with) CD34⫹ bone marrow-derived stem cells [24] and other inhibitory effects of bone marrow mesenchymal cells [25]. The putative T reg cells in the present report were generated by repeated stimulation of CD3⫹ cells with autologous nT-BMC in the presence of IL-2, with C1H treatment of the nT-BMC not altering T reg generation. This repeated stimulation of CD3⫹ cells with C1H-treated or untreated nT-BMC produced a high percentage of CD4/CD25high T cells (Figure 4), which were potent inhibitors of a “primed” cytotoxic immune response (Figure 5). To determine whether C1H treatment had any impact on the ability of BMC to downregulate an allogeneic response, we cocultured PBL with 1-DR matched C1Htreated or untreated allogeneic nT-BMC (analogous to the donor/recipient match inclusion criteria in our clinical studies [11]). The CD3⫹ T cells obtained from these cocultures were then stimulated with a mature DCenriched population derived from the BMC donor, i.e., a culture condition mimicking what might occur when an organ recipient receives an allogeneic DBMC infusion and a kidney from the same donor. At E/T ratios of 40:1
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FIGURE 6 C1H treatment of BMC enhances their ability to induce alloimmune regulatory function. T cells retrieved from PBL/autologous nT-PBL (as control), PBL/allogeneic nTBMC, or PBL/C1H-treated allogeneic nT-BMC were all stimulated with a mature DC-enriched population derived from the BMC donor of the coculture. The T cells that were cocultured with either C1H-untreated or treated allogeneic nTBMC exhibited at least twofold reduced cytotoxic alloreactivity against the bone marrow donor (n ⫽ 6) in each group (p values are 0.007 and 0.045 for PBL/nT-PBL versus PBL/nTBMC at E/T ratio of 40:1 and 20:1, respectively; p values are 0.004 and 0.032 for PBL/nT-PBL versus PBL/C1H-treated nT-BMC at E/T ratio of 40:1 and 20:1, respectively). p Values between PBL/nT-BMC and PBL/C1H-treated nT-BMC also exhibited statistical differences— 0.046 and 0.043 at the different E/T ratios. Data are expressed as means ⫾ SE.
and 20:1, inhibition of C1H-treated nT-BMC derived CD3⫹ cell cytotoxicity was even greater than that of nT-BMC-derived CD3⫹ cells (Figure 6), so that it appeared that in this allogeneic system, C1H treatment of nT-BMC enhanced the suppressive effect. Data from this study therefore suggest that C1Hresistant cells in the BMC compartment are able to provide a tolerogenic environment, consistent with the notion of a potential clinical application of donor BMC infusion accompanying C1H administration. Further work on (differential) CD52 expression on specific subsets of BMCs appears warranted. ACKNOWLEDGMENTS
This work was supported by research grants from the NIH (5 R01-DK25243-24), the VA Medical Research Services of the Miami VA Medical Center, Fujisawa, and Roche Pharmaceuticals.
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