cpg induced myeloid cd11b gr 1 cells efficiently suppress t cell mediated immunoreactivity and graft versus host disease in a murine model of allogeneic cell therapy

Biology of Blood and Marrow Transplantation 14:973-984 (2008) Q 2008 American Society for Blood and Marrow Transplantation 1083-8791/08/1409-0001$32.00/0 doi:10.1016/j.bbmt.2008.06.018 CpG-Induced Myeloid CD11b1Gr-11 Cells Efficiently Suppress T Cell–Mediated Immunoreactivity and Graft-Versus-Host Disease in a Murine Model of Allogeneic Cell Therapy Shoshana Morecki,1 Yael Gelfand,1 Elena Yacovlev,1 Osnat Eizik,1 Yehudit Shabat,1 Shimon Slavin2 Department of Bone Marrow Transplantation, Cancer Immunotherapy & Immunobiology Research Center, Hadassah University Hospital, Jerusalem, Israel, and The International Centre for Cell Therapy and Cancer, Tel Aviv (Souraski) Medical Center, Tel Aviv 64239, Israel Correspondence and reprint requests: Shoshana Morecki, PhD, Department of Bone Marrow Transplantation, Cancer Immunotherapy & Immunobiology Research Center, Hadassah University Hospital, Jerusalem 91120, Israel (e-mail: morecki@hadassah.org.il) Received April 28, 2008; accepted June 30, 2008 ABSTRACT Transplantation of mismatched allografts in irradiated recipients results in lethal graft- versus-host disease (GVHD) In our study, pretransplantation donor treatment with CpG, administered either alone or emulsified in incomplete Freund’s adjuvant, efficiently prevented GVHD in sublethally irradiated recipients of haploidentical (H-2b into H-2b/d) and fully mismatched (H-2b into H-2d) allografts CpG treatment of donor mice caused an accumulation of double-positive CD11bGr-1 cells in their blood and spleens, whereas treatment with CpG1IFA resulted in an even greater accumulation of these cells Isolated CD11b1 cells from the spleens of CpG1IFAtreated mice efficiently suppressed alloreactivity in vitro ( 92%), as determined by co-culturing these cells in mixed lymphocyte reactions After CpG1IFA treatment, a T cell–depleted fraction enriched with CD11b1Gr-11 cells, acting as myeloid suppressor cells, was able to efficiently prevent GVHD induced by naăve T cells in the sublethally irradiated recipients: 20/21 mice remained GVHD-free survivors for more than 200 days Splenocytes from CpG1IFA-treated mice displayed enhanced interleukin (IL)-6, IL-10, and interferong production, reduced T cell allogeneic and mitogenic responses, as well as failure of T cells to induce GVHD In summary, CpG treatment led to impaired T cell function, enriched myeloid suppressor cells and regulatory cytokine production, which together appear to suppress alloreactivity and protect against the development of GVHD We hypothesize that similar immunoregulatory effects could be applied experimentally in a clinical setting when inhibition of alloreactivity is required in recipients of stem cell allografts Ó 2008 American Society for Blood and Marrow Transplantation KEY WORDS Immunosuppression  Myeloid Suppressor Cells  Graft versus Host Disease  CpG INTRODUCTION Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs (CpG) mimic bacterial/ viral DNA sequences and are recognized as nonself by Toll-like receptors (TLRs) expressed on various immune cells of mammalian and other vertebrates [1,2] The TLRs are key components of the innate immune system, and ligation of CpG to these receptors leads to activation of B lymphocytes, natural killer (NK) cells, and antigen-presenting cells, such as macrophages and dendritic cells (DCs) [2-6] CpG is known to be a potent nonspecific immunomodulator that also plays an important role in adaptive immunity directed to achieve Th 1-mediated immune responses after vaccination with viral- or cancer-derived peptides [7-13], as well as in modification of Th-2– associated disorders toward a Th response in allergies and autoimmune diseases [14-16] CpG’s immunomodulatory effect and ability to activate various immune cell subsets led us to investigate its capability to modify the 973 974 complex network of cell subpopulations and the Th 1/ Th 2-type cytokine pattern of cell sources currently being applied in clinical protocols of allogeneic cell therapy and induction of hematopoietic chimerism Allogeneic cell therapy and donor lymphocyte infusion after allogeneic stem cell transplantation in the clinic, as well as in experimental models, are frequently associated with acute and chronic graft-versus-host disease (GVHD), which diminishes the benefit of the allogeneic reaction required to achieve an efficient response in patients with genetic disorders and malignant diseases [17-21] Although most of the strategies aimed at preventing or modifying the intensity of GVHD are based on posttransplantation immunosuppression, the only effective modality to prevent GVHD requires removal of donor T cells before transplantation or the use of only purified CD341 stem cells Unfortunately, elimination of T cells results in increased risk of graft rejection unless conditioning is myeloablative and highly immunosuppressive, leading to delayed immune reconstitution, with a concomitant increase in opportunistic infections and increased risk of relapse of the underlying malignant disease [22,23] Recently we have shown that pretransplantation donor treatment with various immunomodulators, such as complete Freund’s adjuvant (CFA), lipopolysaccharide (LPS), and CpG, can indeed prevent the development of GVHD in sublethally irradiated host mice inoculated with haploidentical donor cells [24] The present study aimed to clarify how CpG-induced immunomodulation occurs and to ascertain the role of cells and cytokines in the prevention of and/or protection against alloreactivity in vitro and in vivo This strategy was also tested on another donor genotype in an experimental model of GVHD across fully mismatched major histocompatibility complex (MHC) barriers MATERIALS AND METHODS Mice Female BALB/c H-2d (BALB), C57BL/6 H-2b (C57), and (BALB/c  C57BL/6)F1 H-2d/b (F1) mice age 10-12 weeks, weighing 22-24 g, were used in this study All mice were purchased from Harlan, Israel and maintained in the animal facility of the Hadassah University Hospital in full compliance with the regulations for the protection of animal rights Donor Pretreatment CpG (ODN #1826) or non-CpG control (ODN #2138) (Coley Pharmaceutical Group, Kanata, Canada) at a dose of 100 mg, either alone or as an emulsion in incomplete Freund’s adjuvant (IFA) (Difco Laboratories, Detroit, MI), was injected subcutaneously into sites in naive mice (0.1 mL/site) The day of splenocyte harvest is specified in each experiment S Morecki et al Experimental Design for GVHD Induction F1 recipient mice were conditioned with total body irradiation of 5.5 Gy, using a 6-MeV linear accelerator at a dose rate of 1.9 Gy/min The nonlethally irradiated recipients were inoculated intravenously 48 h later with 20-40 106 splenocytes derived from either naăve or pretreated mice, as specified in each experiment Flow Cytometry Analysis For fluorescence-activated cell sorting (FACS) analysis, anti-mouse CD80 (phycoerythrin [PE]), CD86 (PE), CD25 (PE), CD11c (antigen-presenting cell [APC] or fluorescein isothiocyanate [FITC]), CD45R/B220 (PE), CD3 (FITC), CD19 (APC), Gr1/Ly6-G (FITC), and CD11b/Mac-1 (peridinin-chlorophyll-protein complex) were purchased from BD Biosciences (San Diego, CA) Anti-mouse CD14 (PE), F4/80 (APC), CD8 (APC), and TLR9 (FITC) were purchased from eBioscience (San Diego, CA) Before fluorescence staining, red blood cells were lysed with mouse erythrocyte lysing buffer (R&D Systems, Minneapolis, MN) Splenocytes were washed with phosphate-buffered saline (PBS) (Biological Industries, Beit Haemek, Israel) and resuspended in staining buffer (1% bovine serum albumin [purchased from Sigma-Aldrich, Rehovot, Israel] and 0.03% sodium azide in PBS) Splenocytes (5  105) were incubated for minutes with mouse Fc blocker (CD32/16) antibody (eBioscience) to prevent nonspecific staining Staining with specified antibody was carried out for 20 minutes on ice, then washed with staining buffer, fixed for 30 minutes with 1% formaldehyde solution in PBS, and resuspended in PBS for reading on the next day For intracellular staining, splenocytes after lysis (5  105) were fixed with 4% formaldehyde solution in PBS for 20 minutes at room temperature, washed with permeabilization buffer (0.1% saponin, 0.09% sodium azide in distilled water) and then stained with TLR9 antibody for 20 All samples were analyzed with a FACSCalibur flow cytometer and CellQuest software (BD Biosciences) Chimerism Assay The mice were anesthetized (100 mg/kg ketamine and 1.2 mg/kg dihydrobenzperidol administered intraperitoneally), and blood samples were obtained from the retro-orbital sinus of the eye Peripheral blood mononuclear cells were isolated using lympholyte-M gradient (Cedar Lane Laboratories, Ontario, Canada), and donor cell percentages were detected by FACS analysis using PE anti-H-2d antibodies (BD Biosciences) The percentage of H-2b donor cells in the F1 (H-2d/b) hosts was determined by measuring the disappearance of host cells carrying H-2d antigen, according to the following formula: 100% - %H-2d1 cells %H-2b1 cells 975 CpG-Induced Myeloid Suppressor Cells The percentage of H-2d donor cells in the C57 (H-2b) hosts was determined directly by using PE anti-H-2d antibodies Magnetic Cell Sorting Magnetic cell separation was carried out from suspensions of splenocytes derived from either naăve or pretreated C57 mice, as specified in each experiment T cells were isolated by depletion of non-T cells (negative selection) with the mouse Pan T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) The T cell–depleted (TCD) fraction (negative selection) was obtained through T cell depletion with mouse CD90 (Thy 1.2) MicroBeads (Miltenyi Biotec) The CD11b1 fraction (positive selection) was isolated with BD IMag Anti-Mouse CD11b Particles-DM (BD Bioscience) All isolations were carried out according to the manufacturers’ protocols In brief, for isolated T cells and TCD fractions, splenocytes were labeled with the corresponding antibody and then loaded onto a MACS LS column placed in the magnetic field of a MACS cell separator (Miltenyi Biotec) For CD11b1 fraction isolation, splenocytes were labeled and then placed in a test tube within the magnetic field of the BD IMag cell separator Mixed Lymphocyte Reaction The mixed lymphocyte reaction (MLR) and suppressive activity were carried out as described previously [25], with minor changes In brief,  105 isolated T cells derived from naăve or pretreated mice were incubated for days with  106 irradiated (50 Gy) splenocytes in 0.2-0.25 mL of medium containing 5% human inactivated AB1 serum To evaluate suppressive activity,  105 irradiated (15 Gy) CD11b1 or CD11b2 cells from either naăve or CpG1IFA-pretreated mice were added as a co-culture to the MLR test, as specified in each experiment After 72 h, cultures were pulsed with 185 GBq/ mmol 1m Ci[methyl3H]thymidine (Amersham, GE Healthcare, UK) for 18 h, and response was monitored by measuring radioactivity uptake (in CPM) The percentage of suppression was calculated, after subtraction of syngeneic response, by the following formula: 100 - [CPM of allogeneic response in the presence of co-cultured isolated CD11b2 or CD11b1 cells/ CPM of allogeneic response in the presence of co-cultured naăve unseparated cells] 100 ionophore (Ca11 Iono) or 50 mg/mL of LPS (SigmaAldrich, Rehovot, Israel), or mg/mL of mouse antiCD3 monoclonal antibody (clone 2C11, kindly provided by G Gross, Migal, Kiryat Shmona, Israel) in 0.2mL of RPMI 1640 supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY), mmol/L of glutamine, 100 mg/mL of streptomycin, 100 U/mL of penicillin, 0.25 mg/mL of amphotericin B, and  1025 M of 2-mercaptoethanol (Sigma-Aldrich, Rehovot, Israel) After 48 h, cultures were pulsed with 1mCi [methyl3H]thymidine for 18 h and then harvested Response was monitored by measuring radioactivity uptake Cytokine Profile C57 splenocytes from either naăve mice or mice pretreated with CpG alone or CpG1IFA were depleted of red blood cells with mouse erythrocyte lysing buffer, washed with PBS, and resuspended in RPMI 1640 supplemented with 10% fetal bovine serum (Gibco), mmol of glutamine, 100 mg/mL of streptomycin, 100 U/mL of penicillin, 0.25 mg/mL of amphotericin B (Biological Industries), and  1025 M of 2-mercaptoethanol (Sigma-Aldrich) Cells (4  106 cell/mL/well) were incubated with or without 2.5 mg/mL of Con-A for 48 h in a 24-well plate in a humidified incubator at 37 C and 5% CO2 Supernatants were collected and kept frozen at 280 C until testing by enzyme-linked immunosorbent assay (ELISA) (Ready-SET-Go! Kit; eBioscience), according to the manufacturer’s directions The following cytokines were tested: interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-g, and tumor necrosis factor-a Absorption was measured using a microplate reader (Spectra Fluor Plus; Tecan, Durham, NC) at 450 nm after the value of the wavelength (570 nm) was subtracted Statistical Analysis The Kaplan-Meier method [26] was used to calculate the probability of survival as a function of time after cell inoculation The statistical significance between pairs of Kaplan-Meier curves was evaluated using the log-rank test [27] Statistical significance in the difference of mitogenic response and cytokine production observed in experimental groups compared with control groups was evaluated using the standard 2-tailed, unpaired Student t-test RESULTS Mitogenic Response Splenocytes (2-4 10 ) isolated from naăve or pretreated C57 mice were cultured in a flat 96-well plate (Nunc, Roskilde, Denmark) with 10 mg/mL of concanavalin-A (Con-A) or ng/mL of phorbol 12-myristate acetate (PMA), and 0.2 mmol of calcium Prevention of GVHD in C57 Mice by Pretransplantation Treatment of Fully Mismatched BALB Donor Mice Injection of CpG1IFA into BALB donor mice resulted in enlarged spleens and increased numbers of splenocytes, which reached a maximum on day after 976 S Morecki et al Table Effect of Pretransplantation Donor Treatment with CpG on GVHD Induction in C57 Host Mice Inoculated with BALB Splenocytes Donor Pretreatment (BALB) Survival, Days,Median (Range), n — 14 (8-33), 14 IFA 34 (8-131), 14 Non-CpG1IFA (7-55), CpG1IFA 200 (89-.200), 17 GVHD-Related Disease-Free Death Survivors 14 14 0 15 Sublethally irradiated (6.5 Gy) C57 mice were inoculated intravenously with 40  106 BALB splenocytes after irradiation BALB donors were treated with CpG 1IFA days before spleen harvest Results were pooled from independent experiments P 00 comparing the CpG1IFA experimental group with all other groups injection (333  106 68 cells vs 93  106 13 cells in naăve mice) (data not shown) Treatment of BALB mice serving as donors for C57 recipients was carried out days before transplantation Inoculation with 40  106 splenocytes from naăve BALB mice into sublethally irradiated C57 recipients induced severe GVHD, with 100% mortality (14/14) between days and 33 after cell inoculation In contrast, inoculation with 40  106 splenocytes from BALB donor mice pretreated with CpG1IFA did not cause GVHD in C57 recipients; 88% (15/17) of the recipients remained healthy and free of GVHD for more than 200 days In 5/5 mice tested for chimerism on day 100 after cell inoculation, 70% donor-type cells were documented, suggesting the induction of stable chimerism rather than rejection (data not shown) Control mice inoculated with donor cells pretreated with IFA or non-CpG1IFA died of severe GVHD (median survival, and 34 days, respectively) (Table 1) Phenotypic Analysis of Spleen Cells Responding to CpG In vivo stimulation of C57 mice with CpG alone led to a slight rise (7%) in the number of CD11b1Gr-11 cells in the spleen, compared with 15% after stimulation with CpG emulsified in IFA (Figure 1) The greatest increase in CD11b1Gr-11 cells was observed days after CpG inoculation and 10 days after CpG1IFA treatment (data not shown) To characterize the CD11b1Gr-11 cell subpopulation after CpG1IFA treatment, we isolated CD11b1 cells by positive selection using magnetic beads and carried out a detailed phenotypic analysis after gating for Gr-11 cells in the FACS The following cell surface markers were checked and their expression on the CD11b1Gr-11 cells was determined 10 days after CpG1IFA treatment: CD80 (8%) and CD86 (5%) for the detection of co-stimulatory molecules, B220 (10%) for all stages of B lymphocytes, CD11c (5%) for DCs, CD14 (4%) for detection of the LPS macrophage receptor, and F4/80 (3%) expressed on mature macrophages (Figure 2) Cell surface markers indicative of T lymphocytes were very low: 7% CD3 and 0% CD8 (data not shown) Taken altogether, the CD11b1Gr-11 cells isolated and gated after Figure Accumulation of CD11b1/GR-11 cells in spleens of CpG-treated mice CpG or CpG1IFA was injected subcutaneously into C57 mice or 10 days, respectively, before flow cytometry analysis Injections of non-CpG or non-CpG1IFA or IFA alone served as controls and were given in parallel to their relevant opposite (ie, CpG vs non-CpG and CpG1IFA vs non-CpG1IFA) The results shown represent experiment out of experiments conducted CpG-Induced Myeloid Suppressor Cells 977 Figure Characterization of CD11b1 cells isolated from spleens of GpG1IFA-treated mice Flow cytometry was carried out on a CD11b1 fraction isolated by magnetic beads from spleens of mice treated with CpG1IFA 10 days earlier (A) Positive fraction of isolated CD11b cells (B) Isolated CD11b cells gated for GR-1 cells (C)-(H) Various phenotypic markers of the CD11b-isolated Gr-1 gated cells The results shown represent experiment out of experiments conducted CpG1IFA treatment did not carry cell surface markers that could relate them to any subpopulation of DCs, T and B lymphocytes, or mature macrophages All of the aforementioned phenotypic analyses were carried out on spleen cells It was important to determine whether any major changes also occurred in cells derived from other tissues The most prominent change in CD31 cells was found in lymph nodes, whereas a significant increase (28%) in the percentage of CD11b1 cells, Gr-11 cells, and double-positive CD11b-Gr-1 cells was detected in the blood (Figure 3) No major changes were found in the distribution of these subpopulations in the bone marrow of treated mice compared with naăve bone marrow cells (data not shown) The Role of CD11b1 Cells in the Immunoregulation of Alloreactivity Isolated CD11b1 cells from C57 mice were added to the MLR of C57-derived cells responding to BALB splenocytes CD11b1 cells from naăve mice and from mice treated with non-CpG1IFA 10 days earlier caused MLR suppression of 80% and 70%, respectively The greatest MLR suppression (98%) was induced by CD11b1 cells isolated from mice treated with CpG1IFA 10 days earlier In parallel, the negative cell fraction (non-CD11b1) caused only weak or absolutely no suppression, as shown in Table Similar to the C57-derived cells, CD11b1 cells from BALB mice suppressed the MLR of BALB cells responding to C57 splenocytes Table shows that co-cultured CD11b1 cells derived from BALB mice treated with CpG1IFA days earlier strongly suppressed the MLR (92%), whereas CD11b1 cells derived from naăve or non–CpG1IFA-treated BALB mice showed significantly less suppressive activity (45% and 55%, respectively) The negative cell fraction (nonCD11b1) of BALB mice was totally nonsuppressive in the MLR Effect of CpG1IFA Pretreatment on Immunologic Reactivity In Vitro Mitogenic and allogeneic responses were tested in C57 splenocytes from mice treated with CpG1IFA 10 days earlier (Figure 4) Compared with naăve splenocytes, reduced proliferation was observed in response to allogeneic stimuli, Con-A, and PMA1Ca11Iono (response of 41%, 23%, and 16%, respectively), whereas the responses to anti-CD3 and LPS remained almost the same as those of the naăve control 978 S Morecki et al Figure Distribution of T cells and myeloid cells after CpG treatment Peripheral blood cells, splenocytes, and lymph node–derived C57 cells were analyzed by flow cytometry to detect T cells (CD3) and/or myeloid cells (CD11b and Gr-1) 10 days after inoculation of CpG1IFA (100 mg) Non-CpG1IFA inoculations served as controls The results shown represent experiment out of experiments conducted splenocytes (74% and 90%, respectively) The responses of splenocytes from mice treated with nonCpG1IFA to all of the mitogenic stimuli tested were similar to those of naăve cells, except for the response to PMA1Ca11Iono, which was very low (45%) A statistically significant difference between CpG1IFA and non-CpG1IFA treatment was observed only in the MLR and in the response to Con-A (P 049 and 016, respectively) These findings indicate that treatment with CpG1IFA affected allogeneic and mitogenic T cell responses to Con-A but had no effect on B cell mitogenic response as tested by LPS stimulation Cytokine Secretion after CpG Treatment The effect of CpG treatment on cytokine production was measured in supernatants of splenocytes derived from C57 mice pretreated 10 or days earlier with either CpG or non-CpG with or without IFA The results, presented in Figure 5, show that secretion of IL-10 and IL-6 was significantly higher after treatment with CpG than after treatment with non-CpG Secretion of IFN-g did not differ significantly after treatment with CpG and with non-CpG Inoculation of CpG or non-CpG emulsified in IFA resulted in significantly increased amounts of IL-6 and IL-10 and, to Table Suppression of C57 Anti-BALB Alloreactivity In Vitro by CD11b1 Cells CD11b2 Cells Pretreatment of Co-Cultured C57 Cells None Non-CpG1IFA CpG1IFA SE, standard error CD11b1 Cells CPM ± SE % Suppression CPM ± SE % Suppression 49,120 ± 1,956 71,748 ± 1,840 55,969 ± 11,415 22 10 13,591 ± 1,149 19,091 ± 374 1,569 ± 525 80 70 98 979 CpG-Induced Myeloid Suppressor Cells Table Suppression of BALB Anti-C57 Alloreactivity In Vitro by CD11b1 Cells CD11b2 Cells Pretreatment of CoCultured BALB Cells None Non-CpG1IFA CpG1IFA CD11b1 Cells CPM ± SE % Suppression CPM ± SE % Suppression 65,890 ± 1,458 64,617 ± 7,966 55,260 ± 10,745 0 31,610 ± 5,745 25,570 ± 3,464 4,778 ± 1,552 45 55 92 SE, standard error a lesser degree, of IFN-g in splenocytes derived from CpG1IFA-treated mice These findings demonstrate that treatment with CpG affected cytokines known to play a role in immunoregulation Effect of CpG1IFA Treatment on T Cell Reactivity In Vivo T cells isolated from C57 donors treated with CpG1IFA 10 days earlier did not induce GVHD when inoculated into sublethally irradiated F1 mice Almost all of the mice (13/14) remained GVHD-free for a follow-up period of more than 250 days (Table 4), and only 2/14 mice contained donor cell genotype when evaluated 80-100 days after cell inoculation Control groups of mice inoculated with T cells isolated from naăve C57 donors or donors treated 10 Figure In vitro mitogenic responses of splenocytes pretreated with CpG1IFA Splenocytes derived from C57 mice treated with either CpG1IFA or non-CpG1IFA 10 days earlier were tested for various mitogenic responses (LPS, Con-A, PMA1Ca11Iono, and anti-CD3) Results are presented as response percentage in relation to 100% response of naăve nontreated splenocytes Response percentages were calculated from 3HTdR uptake in proliferation assays of days of mitogenic stimulation and days for the MLR of C57derived T cells responding to BALB splenocytes P 049 and 016 for the comparison of non-CpG 1IFA and CpG1IFA treatments in the MLR and in response to Con-A, respectively; P 105, 12, and 235 for the comparison of non-CpG 1IFA and CpG1IFA treatments in response to PMA1Ca11Iono, anti-CD3, and LPS, respectively The results represent the mean standard error of separate experiments days earlier with IFA or non-CpG1IFA all died of severe GVHD, with a median survival of 18, 20, and 22 days, respectively (Table 4) Testing of long-term chimerism in these control groups was not feasible on day 80 after T cell inoculation These findings demonstrate that treatment with CpG1IFA led to impaired T cell function, which did not allow an alloreactive response in vivo or induction of long-term chimerism Inhibition of GVHD by a TCD Spleen Subpopulation Isolated from CpG1IFA-Treated Mice Naăve T cells isolated from the spleens of naăve C57 mice and injected into sublethally irradiated F1 mice led to severe GVHD and 100% (21/21) mortality, with a median survival of 19 days The TCD cell fraction isolated from splenocytes derived from C57 mice treated with CpG1IFA 10 days earlier almost totally prevented GVHD when co-injected with naăve T cells The isolated TCD fraction contained CD11b1Gr-11 cells and was negative for Thy-1 and CD3 cells as tested by FACS analysis (data not shown) The TCD cell fraction conferred the most efficient protection against GVHD induction, and 20/21 mice remained disease-free survivors over a follow-up period of more than 200 days (Table 5) It is interesting to note that 14 of the 15 F1 mice tested had 85%-100% donor-type cells in peripheral blood samples obtained more than 170 days after cell inoculation, indicating a stable state of chimerism In the absence of T cells, inoculation of TCD cell fraction alone, derived from either naăve or C57 mice pretreated with CpG1IFA, did not result in chimerism in the sublethally irradiated F1 hosts (data not shown) These findings demonstrate that after CpG1IFA treatment, the cell subpopulation contains cellular constituents that enable engraftment and at the same time suppress alloreactivity by the enriched CD11b1Gr-11 cells in the TCD fraction DISCUSSION We have documented that treatment of donor mice before allogeneic cell therapy with immunomodulator CpG and, more significantly, with a combination of CpG IFA led to engraftment with no GVHD Challenging mice with CpG resulted in 980 S Morecki et al Figure Cytokine secretion after CpG treatment Splenocytes from C57 mice treated or 10 days earlier with CpG (A) or CpG1IFA (B) were stimulated with Con-A for 48 h The cytokine levels in the supernatants of the various cultures were measured The results indicate P 001, 007, and 36 for the comparison of CpG versus non-CpG for secretion of IL-10, IL-6, and IFN-g, respectively, and P 048, 013, and 076 for the comparison of CpG1IFA versus non-CpG1IFA for secretion of IL-10, IL-6, and IFN-g, respectively a reduced number of CD31 cells and accumulation of CD11b1Gr-11 myeloid suppressor cells (MSCs) able to suppress allogeneic response in vitro and in vivo The overall results of our phenotypic analyses carried out after in vitro stimulation demonstrate that the CpG 1826 that we used served as a ligand for TLR9 (data not shown), which is known for its role in providing signals that trigger innate and adaptive immune responses In vivo stimulation led to an increase in the myeloid cell population expressing both the myeloid differentiation antigen Gr-1 and the Mac-1 (CD11b) cell surface markers The time of splenocyte harvest after donor treatment was determined by parameters: (1) maximum increase in the total cell numbers in the spleen, (2) decrease in number of CD31 cells, and (3) maximum increase in the number of CD11b1Gr-11 cells Taken altogether, these parameters were achieved on day after CpG treatment of C57 donors or CpG1IFA treatment of BALB/c donors and on day 10 after CpG1IFA treatment of C57 donors Kinetic experiments revealed not a constant rate of change, but rather the existence of a regulatory network triggered by the CpG stimulus The ability of this CD11b1Gr-11 cell population, functioning as MSCs, to inhibit alloreactivity led us to use it as a donor cell source for allogeneic cell therapy in murine models of GVHD across haploidentical and fully mismatched alloantigenic barriers Because CpG ODN mimics DNA sequences of infective microbial agents, ligation to its TLR led to a transient splenomegaly, likely due to increased splenic hematopoiesis, teleologically aimed at combating bacterial or viral infection [28] Similar to the effect of CpG on CD11b1Gr-11 enrichment observed in our study, accumulation of CD11b1Gr-11 cells with immune suppressive capability has been reported previously in various circumstances of immune stress, such as in the spleens of tumor-bearing mice [29,30] or in the peripheral blood of cancer patients [31], during polymicrobial sepsis [32] or chronic inflammation [33], and after myelosuppressive conditioning by either cyclophosphamide or irradiation [34,35] In accordance with our findings, and as reported by others, the CD11b1Gr-11 cell population is phenotypically heterogenous, consisting Table Impaired T Cell Alloreactivity In Vivo after Donor Pretreatment T Cell Pretreatment None IFA Non-CpG1IFA CpG1IFA Survival, Days, Median (Range), n GVHD-Related Death GVHD-Free Survivors Chimerism 18 (10-118), 17 20 (15-54), 11 22 (16-.250), 250 (107-.250), 14 17/ 17 11/11 7/9 1/14 0/17 0/11 2/9 13/14 NA NA ND 2*/14 NA, not applicable; ND, not done T cells were isolated from spleens of naăve or C57 mice pretreated with CpG1IFA 10 days earlier Pretreatment with IFA or non-CpG1IFA served as controls Isolated T cells (3  106) were inoculated intravenously into sublethally irradiated F1 mice Results were pooled from independent experiments P 00 for the comparison of the CpG1IFA experimental group versus all other groups *65% and 95% chimerism were determined in blood samples obtained 80-100 days after cell inoculation 981 CpG-Induced Myeloid Suppressor Cells Table Prevention of GVHD by the TCD Fraction of Splenocytes from Mice Pretreated with CpG1IFA Experimental Group Survival, Days, Median (Range), n GVHD-Related Deaths GVHD-Free Survivors* Naăve T cells Naăve T cells TCD fraction of CpG1IFA 19 (10-118), 21 200 (14-.200), 21 21/21 1/21 0/17 20/21 Naăve C57 T cells (3 106) were injected into sublethally irradiated F1 mice with or without TCD (20  106) cell fraction of spleens derived from C57 donor mice treated with CpG1IFA 10 days earlier Results were pooled from independent experiments All GVHD-free survivors tested displayed 85%-100% donor cell genotype (H-2b) 170 days after cell inoculation P 00 for comparison of the experimental groups *GVHD-free survivors were evaluated on day 200 after cell inoculation mainly of early immature myeloid progenitor cells [29,36] and lacking the very early CD34 hematopoietic marker, the B220 B cell marker, and the monocytic and DC differentiation markers CD14 and CD11c [36] The increased number of CD11b1Gr-11 MSCs is associated with impaired immune functions, such as a reduced number of CD31 cells [29,32,33,36,37] and diminished proliferative response of T lymphocytes to mitogenic or alloantigenic stimuli, as was also confirmed in the present study Splenocytes from CpG1IFA-treated mice exhibited a significantly lower response to alloantigens and Con-A stimuli compared with splenocytes from naăve or nonCpG1IFAtreated mice In contrast to naăve cells, spleen cells from treated donors did not cause GVHD after injection as either unseparated bulk cells or isolated T cells This indicates that in addition to the reduced number of T cells in treated mice, impaired T cell function also results from CpG treatment, possibly associated with down-regulation of the T cell receptor zeta chain, as has been found in an experimental model of chronic inflammation [33] Both ConA and anti-CD3 antibodies are known to serve as T cell mitogenic stimulators, but, as shown in the present study, CpG treatment caused a decreased ConA response, while anti-CD3 activation remained unaffected The different responses of these mitogens might be due to various aspects of their stimulatory reactions (eg, binding to the specific receptor), triggering various pathways of signal transduction, and the outcome of production/ secretion of various cytokines, chemokines, or other microenvironmental agents Indeed, ConA is a lectin that binds to both T cells and non–T cells through glucose and mannose receptors, whereas anti-CD3 antibody is specific and binds only to T cell receptors expressed exclusively on T cells Binding and activation of macrophages by ConA leads to nitric oxide production, which is believed to be involved in macrophage-mediated cytotoxicity [38,39] Because splenocytes derived from CpG-treated mice contained an increased number of CD11b1 cells, ConA stimulation may have activated these cells to produce the cytotoxic component nitric oxide, thereby affecting the proliferation of T cell cultures It is noteworthy that, as has been reported by others, because of a differential sensitivity to iron re- quirements, the response to anti-CD3 antibody is much less affected than the response to ConA in a situation of ferric deficiency [40] Changes induced in the culture might have different effects on the response to different stimuli The activation of various cell subsets by CpG also has a stimulatory effect on gene expression and production of a cytokine pattern, depending on the type and dose of CpG used for stimulation [41] The CpG 1826 used in our study is identified as class B, which is known for its stimulatory activity on B cells, NK cells, and monocytes, as well as for its potent ability to induce proinflammatory and regulatory cytokines derived from these activated cells [2,41-46] Indeed, as shown in the present study, the secretion of immunoregulatory IL-10, IL-6 and, to a lesser degree, IFN-g was greater after CpG treatment with and without IFA than that in the relevant non-CpG control treatment or naăve cells The source of these cytokines might be lymphocytes, NK cells (IFN-g, IL-10), or monocytes (IL-6), all of which, however, are known for their immunomodulatory activity that might affect various cell types, including down-regulation of Th and Th cells These cytokines are probably responsible for the reduced mitogenic and allogeneic responses in vitro and may play a role in the prevention of alloreactivity in vivo when CpGactivated donor cells are triggered on inoculation into the irradiated hosts CpG ODN has been applied by others [10] as an adjuvant capable of inducing preferential Th1 immune responses It has been suggested that CpG may provide greater immune protection and that it may replace the toxic mycobacteria included in CFA [12] Whereas injection of antigen emulsified in CFA induces a Th1-dominated response and injection of antigen emulsified in IFA induces a Th2-dominated response, the combination of antigen emulsified in IFA and CpG induces a stronger immune response than either of these [11] In the present study as well, an increased number of double-positive CD11bGr-1 myeloid cells and greater immunoregulatory cytokine secretion was observed after inoculation of an admix of CpG and IFA compared with inoculation by CpG alone; consequently, we focused more on donor treatment with CpG1IFA and its mode of action 982 The suppression of allogeneic response in vitro by CD11b1 cells provides evidence of the role of these cells in immunoregulation; however, there is no direct evidence of their role in vivo The fact that a TCD fraction derived from CpG1IFA-treated donors efficiently prevented GVHD allows us to exclude the role of T immunoregulatory cells in our experimental model and to regard the TCD fraction as an efficient, feasible source of immunoregulatory cells when aiming to prevent GVHD in clinical applications Phenotypic analysis of the TCD fraction revealed the presence of CD11b1Gr-11 cells and the absence of CD31 or Thy1.21 cells (data not shown) Prevention of GVHD by pretransplantation donor treatment with CpG1IFA was the outcome of the reduced number and functional impairment of T cells, the presence of enriched MSCs and/or the increased potential for immunoregulatory cytokine secretion, or a combination of these factors Protection from lethal GVHD clearly was not the result of allograft rejection, because inoculation of the unseparated splenocytes or TCD fraction co-injected with naăve T cells led to full and long-lasting engraftment, with documented chimerism in the sublethally irradiated hosts The finding that inoculation of TCD fraction alone did not result in engraftment can be explained by the fact that after nonmyeloablative conditioning, the number of donor T cells is critical for stable hematopoietic engraftment due to facilitation of engraftment by immunocompetent T cells [47] Although CpG’s adjuvant effect and its ability to stimulate a Th1-type immune response [7-13] have been demonstrated, several reports have shown CpG-induced immunosuppression mediated through CD191 DCs [48] or through an anti-inflammatory effect based on a T cell–independent increased level of IFN-g [49] CpG’s stimulatory and protective activities are both mediated through TLR9 activation, but insufficient data are available at present to evaluate the exact circumstances under which these pathways are triggered [49] Similarly, it has been shown that CpG treatment given to irradiated host mice in an experimental model of acute GVHD caused acceleration of the allogeneic reaction [44,50], whereas CpG given to host mice with chronic GVHD conferred a therapeutic benefit [45] CpG given to irradiated hosts in our model of acute GVHD also proved ineffective in preventing lethal GVHD [24] It is important to note that although these previous studies reported the effect of CpG treatment on host mice, our strategy was based on pretransplantation treatment of donors in a murine model of acute GVHD We have previously discussed the rationale and advantages of donor treatment over host treatment [24], and this approach has now proven to be most efficient in a murine GVHD model across fully mismatched MHC alloantigens S Morecki et al It has been shown that isolated bone marrow–derived CD11b1 DCs given with splenocytes to highdose irradiated recipient mice, as well as spleen-derived CD11b1 DCs administered after nonmyeloablative conditioning and co-stimulatory blockade, can induce transplant tolerance without GVHD induction [51,52] These findings are in accordance with our observation of the capability of CD11b1-derived cells to prevent alloreactivity Here we have presented evidence showing that after CpG treatment, regulation of GVHD is carried out by myeloid cells, acting as MSCs However, several studies have reported immunomodulation of GVHD achieved by T regulatory CD41CD251 cells [53] or NK T cells [25] A comparison of myeloid cells and regulatory CD41CD251 T cells carried out side by side in murine experimental models revealed the myeloid cells’ superior suppressive strength [36]; however, their efficacy can be determined only in well- controlled clinical trials The use of allogeneic cell therapy in tumor-bearing mice or cancer patients aims to induce a graft-versus-tumor response without inducing GVHD Although a graft-versus-leukemia response is often coexpressed with GVHD, various strategies are available to achieve effective separation of these reactivities [51,54] Thus, it was of utmost importance to test our strategy of pretransplantation donor treatment with CpG in tumor-bearing mice A report on this study is currently in preparation In accordance with our previous observations using parental C57 cells inoculated into F1 mice [24], the present study provides further insight into the effect of CpG pretransplantation donor treatment on the prevention of GVHD induction when another strain of donor mice (BALB/c) is used across fully mismatched MHC antigen barriers Detailed phenotypic and biological activity analysis of cell subset populations revealed cellular and humoral immunologic constituents that allow us to explain the mode of action and to design an improved defined protocol with the TCD fraction that also eventually may be useful in clinical applications Our data provide evidence of the feasibility of controlling GVHD by pretransplantation donor treatment with CpG Similar to the triggering of TLR9 by CpG, we have previously reported the triggering of TLR4 by its ligand LPS Appling the same strategy of pretransplantation donor treatment, a significant (albeit less effective) reduction of GVHD incidence was observed after LPS donor treatment [24] The finding of a substantial accumulation of MSCs in the peripheral blood after CpG treatment facilitates the collection of these cells and makes our strategy feasible for clinical application This finding may justify preliminary clinical application of a similar strategy in patients with an absolute indication for stem cell transplantation with genetic disorders or malignant diseases at risk for GVHD or CpG-Induced Myeloid Suppressor Cells otherwise at risk due to long-lasting posttransplantation immunosuppression with consenting donors, because treatment with CpG with or without IFA before harvesting of stem cells in a normal donor is certainly less hazardous than uncontrolled GVHD in the recipient ACKNOWLEDGMENTS We wish to thank the Danny Cunniff Leukemia Research Laboratory for its continuous support of our ongoing basic and clinical research REFERENCES Krieg AM CpG motif in bacterial DNA and their immune effect Annu Rev Immunol 2002;20:709-760 Vollmer J, Weeratna R, Payette P, et al Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities Eur J Immunol 2004;34:251-262 Krieg AM, Yi AK, Matson S, et al CpG motif in bacterial DNA trigger direct B-cell activation Nature 1995;374:546-549 Roda JM, Parihar R, Carson WE CpG-containing oligodeoxynucleotides 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Front Biosci 2007;12:4568-4594  ... in CD11b1 Gr- 11 cells was observed days after CpG inoculation and 10 days after CpG1 IFA treatment (data not shown) To characterize the CD11b1 Gr- 11 cell subpopulation after CpG1 IFA treatment, we... number of CD11b1 Gr- 11 cells Taken altogether, these parameters were achieved on day after CpG treatment of C57 donors or CpG1 IFA treatment of BALB/c donors and on day 10 after CpG1 IFA treatment of. .. contained CD11b1 Gr- 11 cells and was negative for Thy -1 and CD3 cells as tested by FACS analysis (data not shown) The TCD cell fraction conferred the most efficient protection against GVHD induction,