Biology of Blood and Marrow Transplantation
Volume 14, Issue 9 , Pages 973-984, September 2008

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

  • Shoshana Morecki

      Affiliations

    • Department of Bone Marrow Transplantation, Cancer Immunotherapy & Immunobiology Research Center, Hadassah University Hospital, Jerusalem, Israel
    • Corresponding Author InformationCorrespondence and reprint requests: Shoshana Morecki, PhD, Department of Bone Marrow Transplantation, Cancer Immunotherapy & Immunobiology Research Center, Hadassah University Hospital, Jerusalem 91120, Israel.
    • ,
  • Yael Gelfand

      Affiliations

    • Department of Bone Marrow Transplantation, Cancer Immunotherapy & Immunobiology Research Center, Hadassah University Hospital, Jerusalem, Israel
    • ,
  • Elena Yacovlev

      Affiliations

    • Department of Bone Marrow Transplantation, Cancer Immunotherapy & Immunobiology Research Center, Hadassah University Hospital, Jerusalem, Israel
    • ,
  • Osnat Eizik

      Affiliations

    • Department of Bone Marrow Transplantation, Cancer Immunotherapy & Immunobiology Research Center, Hadassah University Hospital, Jerusalem, Israel
    • ,
  • Yehudit Shabat

      Affiliations

    • Department of Bone Marrow Transplantation, Cancer Immunotherapy & Immunobiology Research Center, Hadassah University Hospital, Jerusalem, Israel
    • ,
  • Shimon Slavin

      Affiliations

    • The International Centre for Cell Therapy and Cancer, Tel Aviv (Souraski) Medical Center, Tel Aviv 64239, Israel

Received 28 April 2008; accepted 30 June 2008.

Article Outline

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 CpG+IFA resulted in an even greater accumulation of these cells. Isolated CD11b+ cells from the spleens of CpG+IFA-treated mice efficiently suppressed alloreactivity in vitro (> 92%), as determined by co-culturing these cells in mixed lymphocyte reactions. After CpG+IFA treatment, a T cell–depleted fraction enriched with CD11b+Gr-1+ cells, acting as myeloid suppressor cells, was able to efficiently prevent GVHD induced by naïve T cells in the sublethally irradiated recipients: 20/21 mice remained GVHD-free survivors for more than 200 days. Splenocytes from CpG+IFA-treated mice displayed enhanced interleukin (IL)-6, IL-10, and interferon-γ 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.

Key Words: Immunosuppression, Myeloid Suppressor Cells, Graft versus Host Disease, CpG

 

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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, 3, 4, 5, 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, 8, 9, 10, 11, 12, 13, as well as in modification of Th-2– associated disorders toward a Th 1 response in allergies and autoimmune diseases 14, 15, 16. CpG's immunomodulatory effect and ability to activate various immune cell subsets led us to investigate its capability to modify the 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, 18, 19, 20, 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 CD34+ 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.

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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 μg, either alone or as an emulsion in incomplete Freund's adjuvant (IFA) (Difco Laboratories, Detroit, MI), was injected subcutaneously into 2 sites in naive mice (0.1 mL/site). The day of splenocyte harvest is specified in each experiment.

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 naïve 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), Gr-1/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 5 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 min. 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-2d+ cells = %H-2b+ 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 naïve 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 CD11b+ 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 CD11b+ 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, 5 × 105 isolated T cells derived from naïve or pretreated mice were incubated for 4 days with 1 × 106 irradiated (50 Gy) splenocytes in 0.2-0.25 mL of medium containing 5% human inactivated AB+ serum. To evaluate suppressive activity, 5 × 105 irradiated (15 Gy) CD11b+ or CD11b− cells from either naïve or CpG+IFA-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 1μ 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 CD11b− or CD11b+ cells/CPM of allogeneic response in the presence of co-cultured naïve unseparated cells] × 100.

Mitogenic Response 

Splenocytes (2-4 × 105) isolated from naïve or pretreated C57 mice were cultured in a flat 96-well plate (Nunc, Roskilde, Denmark) with 10 μg/mL of concanavalin-A (Con-A) or 2 ng/mL of phorbol 12-myristate acetate (PMA), and 0.2 μmol of calcium ionophore (Ca++ Iono) or 50 μg/mL of LPS (Sigma-Aldrich, Rehovot, Israel), or 1 μg/mL of mouse anti-CD3 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), 2 mmol/L of glutamine, 100 μg/mL of streptomycin, 100 U/mL of penicillin, 0.25 μg/mL of amphotericin B, and 5 × 10−5 M of 2-mercaptoethanol (Sigma-Aldrich, Rehovot, Israel). After 48 h, cultures were pulsed with 1μCi [methyl3H]thymidine for 18 h and then harvested. Response was monitored by measuring radioactivity uptake.

Cytokine Profile 

C57 splenocytes from either naïve mice or mice pretreated with CpG alone or CpG+IFA 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), 2 mmol of glutamine, 100 μg/mL of streptomycin, 100 U/mL of penicillin, 0.25 μg/mL of amphotericin B (Biological Industries), and 5 × 10−5 M of 2-mercaptoethanol (Sigma-Aldrich). Cells (4 × 106 cell/mL/well) were incubated with or without 2.5 μg/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 −80°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)-γ, and tumor necrosis factor-α. 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.

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Results 

Prevention of GVHD in C57 Mice by Pretransplantation Treatment of Fully Mismatched BALB Donor Mice 

Injection of CpG+IFA into BALB donor mice resulted in enlarged spleens and increased numbers of splenocytes, which reached a maximum on day 6 after injection (333 × 106 ± 68 cells vs 93 × 106 ± 13 cells in naïve mice) (data not shown). Treatment of BALB mice serving as donors for C57 recipients was carried out 6 days before transplantation. Inoculation with 40 × 106 splenocytes from naïve BALB mice into sublethally irradiated C57 recipients induced severe GVHD, with 100% mortality (14/14) between days 8 and 33 after cell inoculation. In contrast, inoculation with 40 × 106 splenocytes from BALB donor mice pretreated with CpG+IFA 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-CpG+IFA died of severe GVHD (median survival, 9 and 34 days, respectively) (Table 1).

Table 1. 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), nGVHD-Related DeathDisease-Free Survivors
---14 (8-33), 14140
IFA34 (8-131), 14140
Non-CpG+IFA9 (7-55), 770
CpG+IFA>200 (89->200), 17215

Sublethally irradiated (6.5 Gy) C57 mice were inoculated intravenously with 40 × 106 BALB splenocytes after irradiation. BALB donors were treated with CpG +IFA 6 days before spleen harvest. Results were pooled from 2 independent experiments. P = .00 comparing the CpG+IFA experimental group with all other groups.

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 CD11b+Gr-1+ cells in the spleen, compared with 15% after stimulation with CpG emulsified in IFA (Figure 1). The greatest increase in CD11b+Gr-1+ cells was observed 6 days after CpG inoculation and 10 days after CpG+IFA treatment (data not shown). To characterize the CD11b+Gr-1+ cell subpopulation after CpG+IFA treatment, we isolated CD11b+ cells by positive selection using magnetic beads and carried out a detailed phenotypic analysis after gating for Gr-1+ cells in the FACS. The following cell surface markers were checked and their expression on the CD11b+Gr-1+ cells was determined 10 days after CpG+IFA 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 CD11b+Gr-1+ cells isolated and gated after CpG+IFA treatment did not carry cell surface markers that could relate them to any subpopulation of DCs, T and B lymphocytes, or mature macrophages.

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  • Figure 1. 

    Accumulation of CD11b+/GR-1+ cells in spleens of CpG-treated mice. CpG or CpG+IFA was injected subcutaneously into C57 mice 6 or 10 days, respectively, before flow cytometry analysis. Injections of non-CpG or non-CpG+IFA or IFA alone served as controls and were given in parallel to their relevant opposite (ie, CpG vs non-CpG and CpG+IFA vs non-CpG+IFA). The results shown represent 1 experiment out of 5 experiments conducted.

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  • Figure 2. 

    Characterization of CD11b+ cells isolated from spleens of GpG+IFA-treated mice. Flow cytometry was carried out on a CD11b+ fraction isolated by magnetic beads from spleens of mice treated with CpG+IFA 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 1 experiment out of 3 experiments conducted.

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 CD3+ cells was found in lymph nodes, whereas a significant increase (28%) in the percentage of CD11b+ cells, Gr-1+ 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 naïve bone marrow cells (data not shown).

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  • Figure 3. 

    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 CpG+IFA (100 μg). Non-CpG+IFA inoculations served as controls. The results shown represent 1 experiment out of 2 experiments conducted.

The Role of CD11b+ Cells in the Immunoregulation of Alloreactivity 

Isolated CD11b+ cells from C57 mice were added to the MLR of C57-derived cells responding to BALB splenocytes. CD11b+ cells from naïve mice and from mice treated with non-CpG+IFA 10 days earlier caused MLR suppression of 80% and 70%, respectively. The greatest MLR suppression (98%) was induced by CD11b+ cells isolated from mice treated with CpG+IFA 10 days earlier. In parallel, the negative cell fraction (non-CD11b+) caused only weak or absolutely no suppression, as shown in Table 2.

Table 2. Suppression of C57 Anti-BALB Alloreactivity In Vitro by CD11b+ Cells
CD11b− CellsCD11b+ Cells
Pretreatment of Co-Cultured C57 CellsCPM ± SE% SuppressionCPM ± SE% Suppression
None49,120 ± 1,9562213,591 ± 1,14980
Non-CpG+IFA71,748 ± 1,840019,091 ± 37470
CpG+IFA55,969 ± 11,415101,569 ± 52598

SE, standard error.

Similar to the C57-derived cells, CD11b+ cells from BALB mice suppressed the MLR of BALB cells responding to C57 splenocytes. Table 3 shows that co-cultured CD11b+ cells derived from BALB mice treated with CpG+IFA 6 days earlier strongly suppressed the MLR (92%), whereas CD11b+ cells derived from naïve or non–CpG+IFA-treated BALB mice showed significantly less suppressive activity (45% and 55%, respectively). The negative cell fraction (non-CD11b+) of BALB mice was totally nonsuppressive in the MLR.

Table 3. Suppression of BALB Anti-C57 Alloreactivity In Vitro by CD11b+ Cells
CD11b− CellsCD11b+ Cells
Pretreatment of Co-Cultured BALB CellsCPM ± SE% SuppressionCPM ± SE% Suppression
None65,890 ± 1,458031,610 ± 5,74545
Non-CpG+IFA64,617 ± 7,966025,570 ± 3,46455
CpG+IFA55,260 ± 10,74544,778 ± 1,55292

SE, standard error.

Effect of CpG+IFA Pretreatment on Immunologic Reactivity In Vitro 

Mitogenic and allogeneic responses were tested in C57 splenocytes from mice treated with CpG+IFA 10 days earlier (Figure 4). Compared with naïve splenocytes, reduced proliferation was observed in response to allogeneic stimuli, Con-A, and PMA+Ca++Iono (response of 41%, 23%, and 16%, respectively), whereas the responses to anti-CD3 and LPS remained almost the same as those of the naïve control splenocytes (74% and 90%, respectively). The responses of splenocytes from mice treated with non-CpG+IFA to all of the mitogenic stimuli tested were similar to those of naïve cells, except for the response to PMA+Ca++Iono, which was very low (45%). A statistically significant difference between CpG+IFA and non-CpG+IFA 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 CpG+IFA affected allogeneic and mitogenic T cell responses to Con-A but had no effect on B cell mitogenic response as tested by LPS stimulation.

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  • Figure 4. 

    In vitro mitogenic responses of splenocytes pretreated with CpG+IFA. Splenocytes derived from C57 mice treated with either CpG+IFA or non-CpG+IFA 10 days earlier were tested for various mitogenic responses (LPS, Con-A, PMA+Ca++Iono, and anti-CD3). Results are presented as response percentage in relation to 100% response of naïve nontreated splenocytes. Response percentages were calculated from 3HTdR uptake in proliferation assays of 3 days of mitogenic stimulation and 4 days for the MLR of C57-derived T cells responding to BALB splenocytes. P = .049 and .016 for the comparison of non-CpG +IFA and CpG+IFA treatments in the MLR and in response to Con-A, respectively; P = .105, .12, and .235 for the comparison of non-CpG +IFA and CpG+IFA treatments in response to PMA+Ca++Iono, anti-CD3, and LPS, respectively. The results represent the mean ± standard error of 3 separate experiments.

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 6 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-γ 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 a lesser degree, of IFN-γ in splenocytes derived from CpG+IFA-treated mice. These findings demonstrate that treatment with CpG affected cytokines known to play a role in immunoregulation.

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  • Figure 5. 

    Cytokine secretion after CpG treatment. Splenocytes from C57 mice treated 6 or 10 days earlier with CpG (A) or CpG+IFA (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-γ, respectively, and P = .048, .013, and .076 for the comparison of CpG+IFA versus non-CpG+IFA for secretion of IL-10, IL-6, and IFN-γ, respectively.

Effect of CpG+IFA Treatment on T Cell Reactivity In Vivo 

T cells isolated from C57 donors treated with CpG+IFA 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 naïve C57 donors or donors treated 10 days earlier with IFA or non-CpG+IFA 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 CpG+IFA led to impaired T cell function, which did not allow an alloreactive response in vivo or induction of long-term chimerism.

Table 4. Impaired T Cell Alloreactivity In Vivo after Donor Pretreatment
T Cell PretreatmentSurvival, Days, Median (Range), nGVHD-Related DeathGVHD-Free SurvivorsChimerism
None18 (10-118), 1717/ 170/17NA
IFA20 (15-54), 1111/110/11NA
Non-CpG+IFA22 (16->250), 97/92/9ND
CpG+IFA>250 (107->250), 141/1413/142∗/14

NA, not applicable; ND, not done.

T cells were isolated from spleens of naïve or C57 mice pretreated with CpG+IFA 10 days earlier. Pretreatment with IFA or non-CpG+IFA served as controls. Isolated T cells (3 × 106) were inoculated intravenously into sublethally irradiated F1 mice. Results were pooled from 2 independent experiments. P = .00 for the comparison of the CpG+IFA experimental group versus all other groups.

∗65% and 95% chimerism were determined in blood samples obtained 80-100 days after cell inoculation.

Inhibition of GVHD by a TCD Spleen Subpopulation Isolated from CpG+IFA-Treated Mice 

Naïve T cells isolated from the spleens of naïve 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 CpG+IFA 10 days earlier almost totally prevented GVHD when co-injected with naïve T cells. The isolated TCD fraction contained CD11b+Gr-1+ 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 naïve or C57 mice pretreated with CpG+IFA, did not result in chimerism in the sublethally irradiated F1 hosts (data not shown). These findings demonstrate that after CpG+IFA treatment, the cell subpopulation contains cellular constituents that enable engraftment and at the same time suppress alloreactivity by the enriched CD11b+Gr-1+ cells in the TCD fraction.

Table 5. Prevention of GVHD by the TCD Fraction of Splenocytes from Mice Pretreated with CpG+IFA
Experimental GroupSurvival, Days, Median (Range), nGVHD-Related DeathsGVHD-Free Survivors∗
Naïve T cells19 (10-118), 2121/210/17
Naïve T cells + TCD fraction of CpG+IFA>200 (14->200), 211/2120/21

Naïve 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 CpG+IFA 10 days earlier. Results were pooled from 3 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 2 experimental groups.

∗GVHD-free survivors were evaluated on day 200 after cell inoculation.

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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 a reduced number of CD3+ cells and accumulation of CD11b+Gr-1+ 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 3 parameters: (1) maximum increase in the total cell numbers in the spleen, (2) decrease in number of CD3+ cells, and (3) maximum increase in the number of CD11b+Gr-1+ cells. Taken altogether, these parameters were achieved on day 6 after CpG treatment of C57 donors or CpG+IFA treatment of BALB/c donors and on day 10 after CpG+IFA 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 CD11b+Gr-1+ 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 CD11b+Gr-1+ enrichment observed in our study, accumulation of CD11b+Gr-1+ 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 CD11b+Gr-1+ cell population is phenotypically heterogenous, consisting 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 CD11b+Gr-1+ MSCs is associated with impaired immune functions, such as a reduced number of CD3+ 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 CpG+IFA-treated mice exhibited a significantly lower response to alloantigens and Con-A stimuli compared with splenocytes from naïve or non–CpG+IFA-treated mice. In contrast to naïve 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 CD11b+ 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 requirements, 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, 42, 43, 44, 45, 46. Indeed, as shown in the present study, the secretion of immunoregulatory IL-10, IL-6 and, to a lesser degree, IFN-γ was greater after CpG treatment with and without IFA than that in the relevant non-CpG control treatment or naïve cells. The source of these cytokines might be lymphocytes, NK cells (IFN-γ, 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 1 and Th 2 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 CpG-activated 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 CpG+IFA and its mode of action.

The suppression of allogeneic response in vitro by CD11b+ 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 CpG+IFA-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 CD11b+Gr-1+ cells and the absence of CD3+ or Thy1.2+ cells (data not shown). Prevention of GVHD by pretransplantation donor treatment with CpG+IFA 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 naïve 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, 8, 9, 10, 11, 12, 13 have been demonstrated, several reports have shown CpG-induced immunosuppression mediated through CD19+ DCs [48] or through an anti-inflammatory effect based on a T cell–independent increased level of IFN-γ [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.

It has been shown that isolated bone marrow–derived CD11b+ DCs given with splenocytes to high-dose irradiated recipient mice, as well as spleen-derived CD11b+ 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 CD11b+-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 CD4+CD25+ cells [53] or NK T cells [25]. A comparison of myeloid cells and regulatory CD4+CD25+ 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 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.

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Acknowledgments 

We wish to thank the Danny Cunniff Leukemia Research Laboratory for its continuous support of our ongoing basic and clinical research.

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PII: S1083-8791(08)00278-4

doi:10.1016/j.bbmt.2008.06.018

Biology of Blood and Marrow Transplantation
Volume 14, Issue 9 , Pages 973-984, September 2008

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