DC2 Cells Research Articles

Regulation of alloimmune responses by dendritic cell subsets

Dendritic cells (DCs) are powerful mediators of immune responses. We have demonstrated that the content of plasmacytoid (type 2) dendritic cells (DC2) within allogeneic bone marrow grafts impacts survival and graft-vs-host disease following transplantation. In order to better understand the effect of DC subsets on regulation of immunity, we tested the effect of DC subsets on T cells in a model of indirect antigen presentation to mimic presentation of host-type alloantigen by donor-type DC. Volunteers underwent apheresis without cytokine priming, and DC1, DC2, naïve, and memory T cells were purified by immunomagnetic bead and fluorescein-activated cell sorting. Purified DC1 and DC2 cells were cultured with third-party irradiated blood mononuclear cells and either naïve or memory homologous T cells in mixed lymphocyte reactions. Myeloid (type 1) dendritic cells (DC1) induced significant proliferation of homologous T cells and were more effective in priming naïve T-cell responses than memory T cells responding to alloantigen. DC2 cells induced minimal T-cell proliferation regardless of the T-cell subset used as the responding fraction. Secondary mixed lymphocyte reaction studies demonstrated that DC2 primed T cells remained hyporesponsive even when challenged with a third-party alloantigen. The immunostimulatory effect of DC1 required DC-to-T-cell contact, and induced interleukin-12 secretion, while DC2 cells induced interferon-gamma secretion. Polymerase chain reaction analysis of DC2-primed T cells demonstrated a significant increase in Foxp3 expression, supporting induction of a regulatory T-cell population. DC1 and DC2 cells induced divergent T-cell responses using homologous cells. Better understanding of DC2-mediated T-cell suppression may yield strategies that overcome tumor-specific immune tolerance and regulate graft-vs-host disease.

Enhancement of DNA Vaccine Potency through Coadministration of CIITA DNA with DNA Vaccines via Gene Gun

Administration of DNA vaccines via gene gun has emerged as an important form of Ag-specific immunotherapy. The MHC CIITA is a master regulator of MHC class II expression and also induces expression of class I molecules. We reasoned that the gene gun administration of CIITA DNA with DNA vaccines employing different strategies to improve MHC I and II processing could enhance DNA vaccine potency. We observed that DC-1 cells transfected with CIITA DNA lead to higher expression of MHC I and II molecules, leading to enhanced Ag presentation through the MHC I/II pathways. Furthermore, our data suggested that coadministration of DNA-encoding calreticulin (CRT) linked to human papillomavirus (HPV) 16 E6 Ag (CRT/E6) with CIITA DNA leads to enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. In addition, coadministration of the combination of CRT/E6 DNA with CIITA DNA and DNA encoding the invariant chain (Ii) linked to the pan HLA-DR-reactive epitope (Ii-PADRE) further enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. Treatment with the combination vaccine was also shown to enhance the antitumor effects and to prolong survival in TC-1 tumor-bearing mice. Vaccination with the combination vaccine also led to enhanced E6-specific CD8(+) memory T cells and to long-term protection against TC-1 tumors and prolonged survival in vaccinated mice. Thus, our findings suggest that the combination of CIITA DNA with CRT/E6 and Ii-PADRE DNA vaccines represents a potentially effective means to combat tumors in the clinical setting.

Maturation pathways of dendritic cells determine TAP1 and TAP2 levels and cross‐presenting function

Cross‐presentation of exogenous antigens in the HLA class I pathway is key to the antigen presenting function of mature tumor‐loaded dendritic cells (DC). Conditions of DC maturation have been shown to influence the ability of DCs to induce T cell effector functions. However, it remains unknown if different pathways of DC maturation are associated with modulation in the ability of DCs to cross‐present tumor‐associated antigens. We compared DC matured with three clinically relevant cytokine protocols: IL‐1β, TNF‐α, IL‐6 (DC‐0), DC‐0+PGE‐2, or IFN‐γ, IFN‐α, TNF‐α, and IL1‐β (DC‐1). We found that these DC vary in the ability to cross‐present tumor antigens to CTL, with DC‐1 protocol being significantly more effective than the other protocols. This level of TA cross presentation was correlated with the level of expression of the antigen processing machinery (APM) components, TAP1 and TAP2, and to a lesser extent, tapasin, indicating that these components could be used to standardize DC preparations for optimal function. However, upregulation of TAP1/2 was not sufficient to explain the strongest cross‐presentation ability induced by DC‐1 cells, since IFN‐γ did not generate DC as effective at cross‐presentation as the full DC‐1 cytokine cocktail. These data indicate that DC maturation pathways modulate APM component expression and that DC vary in the ability to cross‐present TA to HLA class I‐restricted CTL.

Polarized Type-1 Dendritic Cells (DC1) Producing High Levels of IL-12 Family Members Rescue Patient TH1-type Antimelanoma CD4+ T cell Responses In Vitro

Deficiencies in TH1-type immunity in patients with cancer may facilitate tumor progression and limit the effectiveness of current immunotherapy approaches. We hypothesized that Type-1 polarized dendritic cells (DC1) might be able to recondition patient antitumor CD4+ T cell responses toward the TH1-type in vitro. Although DC1 have been previously demonstrated to prime TH1 responses from naive CD4+ T cells, their impact on antigen-experienced TH responses remains unknown. We confirmed our own earlier observations that patient CD4+ T cell reactivity against melanoma-associated antigens (MAA) was weaker and less Type-1-polarized than their corresponding antiviral responses. Stimulation of patient CD4 T cells with peptide-pulsed DC1 (producing multiple IL-12 family member cytokines, including IL-12p70, IL-23, and IL-27) promoted robust TH1-type, epitope-specific T cell responses. Addition of exogenous IL-12 family member cytokines alone, or in combination, to nonpolarized DC was insufficient to equate to the benefits associated with DC1-based stimulation; however, IL-27 and IL-12p70 blockade neutralized the ability of DC1 cells to enhance TH1-type antitumor immunity in vitro. Notably, DC1-based stimulation seemed capable of "revitalizing" defective TH1-type responses within the CD45RO+ subset of antigen-experienced CD4+ T cells in melanoma patients. In addition to promoting elevated levels of IFN-gamma from responder CD4+ T cells, DC1-based stimulation also led to increased levels of IL-12Rbeta2 and t-bet expression by TH cells. These results suggest that preexisting CD4+ T cell immunity to cancer is not relegated to Type-1 insufficiency and may be corrected via the application of DC1-based vaccination protocols.

A Randomized Trial To Evaluate the Impact of Cytokines (G-CSF or GM + G-CSF) on Dendritic Cell and T-Cell Content and Function When Mobilizing Normal Donors for Allogeneic Progenitor Cell Transplant.

Introduction: Optimal cellular immunity following allogeneic HPC transplant is a balance between effective anti-tumor immunity and the avoidance of life threatening GvHD. Our group previously demonstrated that DC content following allogeneic BMT can impact EFS (Waller et al, Blood 2001), and that mobilization cytokines can impact DC content of the autologous PBSC graft (Lonial et al, BBMT 2004). The current trial was designed with the hypothesis that the use of G + GM-CSF will result in fewer tolerogenic DC2 cells within the graft, and increased Th1 phenotype among the mobilized T-cell when compared with normal donors mobilized with G-CSF alone.Methods: 43 normal donors were randomized to receive G-CSF alone (7.5 mcg/kg BID) or G-CSF (7.5mcg/kg qd) and GM-CSF (7.5 mcg/kg qd) which was administered for 5 days or longer if additional days of apheresis were necessary. Side effects between the 2 cytokine regimens were documented using a questionnaire that was administered within 2 weeks of successful collection. Graft content was evaluated using multi-color flow cytometry, and pre-mobilization blood was collected to test for T-cell and DC content as well as T-cell function. DC1 (myeloid DC) were defined as Lin−/HLA-DR+/CD11C+/CD123−, while DC2 (lymphoid DC) were defined as Lin−/HLA-DR+/CD11C−/CD123+. T-cell proliferation was assessed using thymidine incorporation assays following mitogen exposure, and T-cell activation profile was assessed using ELISA assays of secreted cytokines and intracellular cytokine measurements following mitogen stimulation.Results: 19 patients received G+GM-CSF and 24 patients received G-CSF alone. All 43 donors were successfully mobilized, though more patients in the G-CSF arm required multiple days of collection (mean number of collections 1.4 G-CSF vs 1.1 G+GM-CSF, p=0.06). There was no difference in %CD34+ or CD34+ content of the grafts between groups. There was a marked reduction in DC2 content as measured by percent (0.33% vs 0.45%, p=.03) absolute DC2 content (1.98e6 vs 3.66e6, p=0.0003), and delivered DC2 dose e6/kg (2.8 vs 5.2, p=.0002) for the donors randomized to received the combination of GM/G-CSF vs G-CSF alone. There was no statistical difference in DC1 content measured in an analogous fashion. Additionally, grafts collected with G-CSF alone had significantly greater numbers of total T-cells than compared with G+GM-CSF (379e6/kg vs 230e6/kg, p=0.0007). This percent reduction in total T-cell content was identical to the reduction in the CD4 and CD8 subsets between the 2 randomized groups. Interestingly, cells collected from the recipients of G+GM-CSF on the day of mobilization secreted higher amounts of IL-2 at rest, though when stimulated with PMA, G-CSF mobilized cells secreted significantly higher amounts of IL-2. Other secreted cytokines were measured, and to date do not show a clear trend towards a difference. Side effects between the 2 randomized arms were similar with 1 patient in the G-GM-CSF arm having to stop growth factors secondary to bone pain and fever.Conclusion: The combination of G+GM-CSF mobilizes significantly fewer DC2 cells as well as 40–50% fewer T-cells, and is more likely to result in a successful single PBSC collection than the use of G-CSF alone. Further laboratory evaluation of the graft, survival, and GvHD data following transplant will also be presented.

A Pilot Phase II Trial of a Synthetic Tumor-Specific Breakpoint Peptide Vaccine in Patients with Chronic Myeloid Leukemia (CML) and Minimal Residual Disease.

Peptides derived from BCR-ABL have been used to trigger immune responses in CML pts. The native peptides may have low immunogenicity because they bind with low affinity to the major histocompatibility complex (MHC) molecules. Synthetic peptides containing mutations in the sequences of these peptides can bind with higher affinity than parental peptides and may generate a cross-reactive T cell response to the native sequence (known as a heteroclitic response). We used a mixture of heteroclitic and native peptides derived from both b3a2 and b2a2 sequences in a pilot study to vaccinate pts with CML in complete cytogenetic remission on imatinib (IM) therapy with stable bcr-abl transcript levels. Montanide ISA51 and GM-CSF were used as immunological adjuvants. Pts were required to have received IM for ≥12 mo with no change in dose for at least 6 mo and were not allowed any dose increases after registration. Pts were vaccinated with the peptides corresponding to their BCR-ABL breakpoint as follows: every 2 wks x4, then once 3 wks later, followed by 10 monthly vaccinations for a total of 15 vaccinations in 12 mo. After registration, pts had 3 additional measures of bcr-abl transcripts to better define baseline values. 8 pts have been vaccinated. At enrollment, pts had a median age of 45 yrs (range 29–63) and had been receiving IM for a median of 63 mo (range 35–68 mo) on a median dose of 600mg (range 300–800mg). Pts have been followed for a median of 15 wks (range 2–20) and have received a median of 7 vaccinations (range 1 to 8). To determine the effect of vaccination on immune leukocytes, we measured peripheral blood T-cell subsets, B, NK, and dendritic cells by FACS at baseline and at 3-month intervals thereafter. The percentage of myeloid DC (DC1) and plasmacytoid DC (DC2) cells were identified by the mutually exclusive expression of CD11c and CD123; Treg cells by the surface co-expression of CD3, CD4 and CD25 together with cytoplasmic expression of CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and FoxP3 (forkhead/winged helix transcription factor). Vaccinations have been well tolerated. The only adverse event attributable to the vaccine is local site reactions grade 1 or 2 in 5 pts. Among the 5 pts who have had an evaluation at 3 mo from the 1st vaccination, 1 has achieved a major molecular response (bcr-abl/abl <0.05%). The Wilcoxon signed ranks test was used to assess changes in the immunophenotype of peripheral leukocytes of 5 pts evaluated for leukocyte FACS analyses at baseline and after 3 mo on study. Compared with baseline, there were significant decreases in the percentages of B (p = 0.042) and NKT (p = 0.043) cells, and significant increases in the percentages of DC2 (p = 0.043) and Treg (p = 0.043) cells. All pts continue receiving vaccination as planned. We conclude that vaccination with b2a2 and b3a2 synthetic peptides is well tolerated. Further follow-up is required to determine the immunogenecity and efficacy of the vaccine in this setting.

Immunomodulating effect of vitamin D3 derivatives on type-1 cellular immunity

1 alpha,25-dihydroxyvitamin D3 [Calcitriol or 1,25(OH)(2)D(3)] is an important active metabolite involved in multiple functions but its calcemic effect in vivo limits its therapeutic applications. On the other hand, 22-oxa-1 alpha,25-dihydroxyvitamin D(3) (22-oxacalcitriol or 22-Oxa-1 alpha,25-D(3)), a low calcemic analog of vitamin D3 (VitD3), has been widely used as a drug for the secondary hyperparathyroidism. Here, we investigated immunomodulating effect of these two VitD3 derivatives on the differentiation of type-1 immunoregulatory cells such as dendritic cells (DC1), cytotoxic T cells (Tc1) and helper T cells (Th1). BALB/c mouse bone marrow-derived DC (BMDC1) induced by culture with Th1 condition (GM-CSF, IL-3, IL-12 and IFN-gamma) expressed higher levels of MHC Class I and Class II molecules and co-stimulatory molecules compared with BMDC0 induced by neutral condition (GM-CSF+IL-3). In addition, BMDC1 showed stronger immunostimulating activity to induce alloantigen (H-2(d))-specific cytotoxic T lymphocytes (CTL) compared with BMDC0. However, if VitD3 derivatives were added into the culture for BMDC1 induction, the expression of functional molecules and type-1 IFNs were greatly inhibited. Moreover, VitD3 derivative-treated BMDC1 lost their immunostimulating activity to induce alloantigen-specific IFN-gamma-producing Tc1. In addition, it was demonstrated that the addition of VitD3 derivatives inhibited the differentiation of IFN-gamma-producing Th1 cells from ovalbumin (OVA)-specific naive Th cells, while it rather augmented the differentiation of IL-4- or IL-10-producing Th2 cells. There was no significant difference in immunomodulating activity between 1,25(OH)(2)D(3) and 22-Oxa-1 alpha,25-D(3). Thus, VitD3 derivatives are demonstrated to inhibit the functional differentiation of DC1, Tc1 and Th1 cells, which play a critical role in type-1 cellular immune responses.

A Randomized Trial To Evaluate the Impact of Cytokines (G-CSF or GM + G-CSF) on Dendritic Cell and T-Cell Content and Function When Mobilizing Normal Donors for Allogeneic Progenitor Cell Transplant.

Introduction: Optimal cellular immunity following allogeneic HPC transplant is a tenuous balance between effective anti-tumor immunity and the avoidance of life threatening GvHD. The events necessary for potentially curative GvL are related to tumor burden, T-cell content, NK cell content, and dendritic cell content. Our group previously demonstrated that DC content following allogeneic BMT can impact EFS (Waller et al, Blood 2001), and that cytokines used during mobilization impact DC content of the autologous graft (Lonial et al, BBMT 2004). The current trial was designed with the hypothesis that the use of G + GM-CSF will result in fewer tolerogenic DC2 cells within the graft, and increased Th1 phenotype among the mobilized T-cells.Methods: 40 normal donors were randomized to receive G-CSF alone (7.5 mcg/kg BID) or G-CSF (7.5mcg/kg qd) and GM-CSF (7.5 mcg/kg qd) for 5 days or longer if additional apheresis were necessary. Side effects between the 2 cytokine regimens were documented using a questionnaire that was administered within 2 weeks of successful collection. Graft content was evaluated using multi-color flow cytometry, and pre-mobilization blood was collected to test for T-cell and DC content as well as T-cell function. T-cell proliferation was assessed using thymidine incorporation assays following mitogen exposure, and T-cell activation profile was assessed using ELISA assays of secreted cytokines following mitogen stimulation. DC1 (myeloid DC) were defined as Lin-/HLA-DR+/CD11C+/CD123−, while DC2 (lymphoid DC) were defined as Lin-/HLA-DR+/CD11C−/CD123+.Results: 18 patients received G+GM-CSF and 22 patients received G-CSF alone. All 40 donors were successfully mobilized, though more patients in the G-CSF arm required multiple days of collection (mean number of collections 1.5 G-CSF vs 1.0 G+GM-CSF, p=0.01). Donors mobilized with G-CSF alone had a higher overall cell count (765e8/ml compared with 615e8/ml for G+GM-CSF, p=0.04), though the CD34 content of the grafts were comparable. Again, there was a significant reduction in delivered DC2 content of graft mobilized with G+GM-CSF when compared with G-CSF alone (5.1e6/kg compared with 2.85e6/kg, p=0.001), with a trend towards fewer DC1 cells as well (2.6e6/kg vs 1.7e6/kg, p=0.05). Overall, the DC1:DC2 ratio favors DC1 among donors mobilized with G+GM-CSF. Additionally, grafts collected with G-CSF alone had nearly twice the total T-cell content of grafts mobilized with G+GM-CSF (420e6/kg vs 220e6/kg, p=0.0001). The reduction in total T-cells was evenly distributed between CD4 and CD8 cells with similar magnitude of reduction in both subsets amongst those receiving G+GM-CSF. Interestingly, grafts collected from the recipients of G+GM-CSF produced more IL-2 at rest, and secreted significantly more IL-12 in response to mitogen. Toxicity between the 2 arms were similar with 1 patient in the G+GM-CSF arm having to stop growth factors due to bone pain and fever.Conclusion: The combination of G+GM-CSF mobilizes fewer DC2 cells as well as 40–50% fewer T-cells, and is more likely to result in a successful single PBSC collection than the use of G-CSF alone. Preliminary data suggests that the combination of G+GM-CSF shifts T-cells towards a Th1 phenotype, despite fewer overall T-cells. Further laboratory evaluation of the graft, survival, and GvHD data following transplant will also be presented.

Dendritic cells in autologous hematopoietic stem cell transplantation for diffuse large B-cell lymphoma: graft content and post transplant recovery predict survival

Allograft dendritic cell (DC) content has been identified as a predictor of relapse and event-free survival after allogeneic bone marrow transplantation. However, the prognostic importance of DCs has not been evaluated in the setting of autologous hematopoietic stem cell transplantation (HSCT). We prospectively determined pre-transplant and post transplant DC levels, including DC1 and DC2 subset levels, in 53 patients with diffuse large B-cell non-Hodgkin's lymphoma (DLBC NHL) undergoing autologous HSCT. Pre-transplant DCs were measured in the collected stem cell products and were therefore indicative of cell numbers infused directly into patients; post transplant analysis of DCs was performed on the peripheral blood of patients 6 weeks after the infusion of autologous stem cells. Higher pre-transplant levels of DC1 cells and total DCs were significantly associated with improved survival. Similarly, greater post transplant levels of total DCs and both subsets were significantly associated with survival. These findings suggest a relationship between DC reconstitution and survival following autologous HSCT for DLBC NHL. Strategies to increase autograft DC content or accelerate DC recovery after autologous HSCT might improve outcomes in this setting.

Allogeneic peripheral blood stem cell graft composition affects early T‐cell chimaerism and later clinical outcomes after non‐myeloablative conditioning

We have studied the influence of cell subsets [CD34, CD3, CD4, CD8, CD14, CD20, natural killer (NK; CD3(-)/CD56(+)), NKT (CD3(+)/CD56(+)), DC1, and DC2 cells] of granulocyte colony-stimulating factor mobilized peripheral blood stem cells (PBSC) on early T-cell chimaerism and later clinical outcomes in 125 patients with haematological malignancies who received human leucocyte antigen (HLA)-matched related grafts after non-myeloablative conditioning. Conditioning consisted of 2 Gy total body irradiation (TBI) alone (n = 28), or 2 Gy TBI preceded by either 90 mg/m(2) fludarabine (n = 62) or planned autologous haematopoietic cell transplantation (HCT) (n = 35). Post-transplant immunosuppression included mycophenolate mofetil and ciclosporin. Multivariate analysis showed that higher numbers of grafted NK cells predicted higher early T-cell chimaerism (P = 0.03), while higher numbers of B cells were associated with better clinical outcomes and a higher risk for chronic graft-versus-host disease (P = 0.05). Higher numbers of CD14(+) cells were associated with worse overall survival (P = 0.03), while higher numbers of CD34(+) cells showed better survival (P = 0.03). The addition of fludarabine or autologous HCT predicted higher early T-cell chimaerism (P = 0.001), while advanced donor age predicted lower chimaerism (P < or = 0.02). Patients with aggressive diseases were at higher risk for relapse/disease progression, and shorter progression-free and overall survival (P < 0.01). These results suggest that the dosing of certain cellular subsets of PBSC products can influence important outcomes post-HCT after non-myeloablative conditioning.

A randomized trial comparing the combination of granulocyte-macrophage colony-stimulating factor plus granulocyte colony-stimulating factor versus granulocyte colony-stimulating factor for mobilization of dendritic cell subsets in hematopoietic progenitor cell products

The ability of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) administration to increase the content of blood leucocytes and hematopoietic progenitor cells (HPCs) is well established, yet the effect of these cytokines on immune function is less well described. Recent data indicate that plasmacytoid dendritic cells (DC2) may inhibit cellular immune response. We hypothesized that administration of the combination of G-CSF and GM-CSF after chemotherapy would reduce the type 2, or plasmacytoid, DC2 content of the autologous blood HPC grafts compared with treatment with G-CSF alone. To test this hypothesis, 35 patients with lymphoma and myeloma were randomized to receive either G-CSF or the combination of G-CSF plus GM-CSF after chemotherapy, and blood HPC grafts were collected by apheresis. Cytokine-related adverse events between the 2 groups were similar. More than 2 x 10(6)CD34 + cells per kilogram were collected by apheresis in 14 of 18 subjects treated with G-CSF and in 16 of 17 subjects treated with GM-CSF plus G-CSF ( p = not significant). There were minor differences between the 2 groups with respect to the content of T cells and CD34 + cells in the apheresis products. However, grafts collected from recipients of the combination of GM-CSF plus G-CSF had significantly fewer DC2 cells and similar numbers of DC1 cells compared with recipients treated with G-CSF alone. A third cohort of patients received chemotherapy followed by the sequential administration of G-CSF and the addition of GM-CSF 6 days later. Grafts from these patients had a markedly reduced DC2 content compared with those from patients treated either with G-CSF alone or with the concomitant administration of both cytokines. These data, and recent data that cross-presentation of antigen by DC2 cells may induce antigen-specific tolerance among T cells, suggest that GM-CSF during mobilization of blood HPC grafts may be a clinically applicable strategy to enhance innate and acquired immunity after autologous and allogeneic HPC transplantation.

Comparison between G-CSF and GM-CSF for Mobilization of Dendritic Cells, CD34+ Cells, and CD3+ Cells in Normal Donors.

Donor lymphocyte infusions (DLI) can induce an allo-immune mediated graft-versus-malignancy (GVM) effect in patients (pts) that relapse after allogeneic stem cell transplantation (ASCT). Dendritic cells (DC) are effective antigen presenting cells and an important regulatory element of T-cell mediated immune responses. Previously, we have shown that NHL pts given GM-CSF mobilized up to 5 fold more immature DC (CD14+CD80+ cells) compared to G-CSF mobilization (Gazitt et al. J. Hematother. Stem Cell Res. 2001. 10:177). We hypothesized that increasing the DC populations in DLI may augment the GVM effect, particularly if GM-CSF mobilization resulted in increased DC1 cells. We designed a pilot study to treat pts with relapsed disease after ASCT with GM-CSF mobilized DLI. Total DC, DC1, DC2, and CD34+cells from the same donor were compared from G-CSF mobilized peripheral blood stem cell (PBSC) collections and GM-CSF mobilized DLI collections. Donors received G-CSF 10 ug/kg/day for 5 days prior to PBSCH, and GM-CSF 500 ug/day for 5 days prior to DLI collection. All donors were HLA matched related siblings and DLI was collected when pts demonstrated relapse or persistent disease after ASCT. Total DCs were defined as CD80+CD14- cells, DC1 cells as CD11c+HLADR+lin-, and DC2 cells as CD 123+HLADR+lin-. Analysis was done by flow cytometry using a 3 color assay. No unexpected adverse events occured to the donors who received GM-CSF prior to DLI collection or to the pts during DLI administration. Diseases of patients prior to ASCT were CML accelerated phase (n=1), high risk acute leukemia (n=4), chemotherapy refractory NHL (n=2) and refractory multiple myeloma (n=1). Four of 8 pts developed aGVHD (grade II: n=3, and grade IV: n=1). Three patient had remissions post DLI for 1 to 6 months. Subsequently, seven pts died of progressive malignacy and 1 pt died of sepsis with no active GVHD or malignancy. The mean number of cell types collected for the G-CSF and GM-CSF apheresis collections are described in the table below. Mobilization with G-CSF resulted in up to a 10 fold larger number of CD34+ cells/kg and 3–5 fold larger number of total DC, DC1 and DC2 cells within the same donor compared to GM-CSF. The ratio of DC1 to DC2 in each donor remained constant in both the G-CSF and GM-CSF mobilized apheresis products. We conclude that GM-CSF mobilization results in adequate collection of CD3+ cells for DLI, and infusion of these cells is tolerated well. In this small sample of normal donors it appears that G-CSF mobilizes more CD34+, total DC, DC1 and DC2 cells within the same donor compared to GM-CSF, with no polarization by G-CSF or GM-CSF for either DC1 or DC2 cells.Mean cell counts/kg x 106 (range)CD3CD34Total DCDC1DC2ratio DC1/DC2G-CSFN/A4.6 (1.2–7.5)8.9 (2.6–13.1)5.7 (1.2–10.6)5.8 (0.6–10.1)1.1GM-CSF81.2 (50.6–133.7)0.4 (0.09–0.6)3.5 (1.4–7.2)1.8 (0.4–3.8)2.1 (0.5–3.9)0.9p value *N/A0.010.050.010.05N/A* student t-test

Improved Mobilization of CD34+ Peripheral Blood Stem Cells (PBSC) and Dendritic Cells (DCs) by AMD-3100 (AMD) in Hard to Mobilize Non-Hodgkin's Lymphoma (NHL) Patients (pts) Mobilized with Standard Dose of G-CSF.

PBSC mobilization for auto transplantation of NHL pts with multiple regimens of prior chemotherapy is hard to achieve and 25%–30% of patients experience mobilization failure. AMD is a small bicyclam compound which specifically binds to CXCR4 receptor and blocks signaling through SDF-1. Previous studies in normal donors suggested clear dose dependent CD34+ cell mobilization as a single agent as well as additively with G-CSF with little toxicity (ASH meeting, 2002; 2003). Similarly, previous studies with NHL and Myeloma patients resulted with a dose-dependent augmentation of CD34+ cell mobilization in pts receiving G-CSF with little toxicity. The exact mechanism of AMD-induced mobilization of CD34+ cells was not studied in patients and AMD was not used before for PBSC mobilization in hard to mobilize NHL patients. Furthermore, its effect on mobilization of DCs and lymphoma cells was not studied before. On November 2003, we initiated a phase II study of 10, hard to mobilize NHL patients, receiving 16ug/kg of G-CSF for 4 days and G-CSF followed by 240ug/kg of AMD on day 5, 9 hours before apheresis collection. G-CSF and AMD were continued for additional day or 2, as needed, in order to collect the target dose of ≥ 2x106 PBSC/kg. Ten liters of blood were exchanged in ~4hours of apheresis. Median age was 54 years (44 to 63 years). Of the 10 patients enrolled, 6 pts had diffuse large cell lymphoma and 3 had follicular histology with 8/10 received 2 regimens of chemotherapy, 2 of which received also radiation prior to mobilization. At mobilization, 5/9 pts were primary refractory, 3 pts were in 1st relapse, 1 pt in 2nd relapse and 1 pt in 2nd CR. We determined percent CD34+ cells and percent DC1 and DC2 cells as well as percent lymphoma cell mobilization (by Real time DNA-PCR) at baseline (before administration of G-CSF) and before and after AMD, in the blood and in the apheresis product. To date 7 pts were transplanted. Five pts collected in 1 day and 5 pts collected in 2 days. No adverse events were observed during mobilization. All patients collected ≥ 2x106 PBSC/kg and 7 pts have been transplanted with a dose of 2-7x106 PBSC/kg. All transplanted pts engrafted with a mean of 10 days (9 to 12 days) for ≥ 500ANC and with a mean of 13 days (12–14 days for 6/7 pts) to reach 20K of plts. One pt had a delayed plt engraftment and was engrafted on day 27. Addition of AMD to G-CSF, prior to the first or 2nd PBSC collection resulted in a mean increase of percent CD34+ cells from 0.11% to 0.17% ( p=0.017), with a similar mean increase in CD34+ cells/ul (35/ul to 81/ul; p=0.0001) followed by normalization of CD34+ cells/ul within 24 hours. Similarly, addition of AMD to G-CSF resulted in an increase in the mean of DC1 cells from 79/ul to 156/ul (p=0.009) and from a mean of 62/ul to164/ul (p=0.006) for DC2 cells. One pt had 0.08% lymphoma cells at baseline by DNA-PCR for the major breakage point of the Bcl-2 translocation sequence, with no detectable lymphoma cells in the blood or apheresis collection post AMD. All other pts were negative for lymphoma pre and post AMD by this test. Adverse events and sever adverse events related to study were minimal. We conclude that AMD is a safe drug with clinical benefit in increasing PBSC and DC mobilization with no detectable mobilization of lymphoma cells.

The Molecular Pathway of Lysosomal Antigen Processing in Peripheral Blood Dendritic Cells (DC) Significantly Differs from That in Monocyte-Derived DC Generated Ex Vivo.

Dendritic cells (DC) initiatiate immunity and maintain tolerance. They internalize exogenous antigen and convert it into immunogenic peptides by lysosomal proteolytic degradation, ultimately followed by presentation to CD4 T cells. Monocyte-derived DC (MO-DC) generated in vitro with GM-CSF and IL-4 serve as prototype DC to analyse the cellular biology and biochemistry of DC. However, different types of primary DC, whose functional role in vivo and relationship to MO-DC generated in vitro is unclear, reside in human tissue as well as peripheral blood. The composition of lysosomal proteases in these primary human DC1 and DC2-cells and the way they handle a clinically relevant antigen are unknown, and there is no comparison of the lysosomal processing of antigen by these primary DC to that in primary human B cells or MO-DC generated ex vivo. We have isolated human peripheral blood (PB) DC1 and DC2 cells as well as primary B lymphocytes by magnetic separation and isolated lysosomal compartments from these cells, as well as from MO-DC. Expression and activity of endocytic proteases were assessed by western blot and active site-restricted affinity labelling using a synthetic probe that selectively binds to the active centre of cysteine proteases and allows a simultaneous semiquantitaive assessment and identification of multiple active protease species. In this analysis, PB-DC1 and DC2-cells lacked significant active Cathepsins (Cat) S, L and B as well as asparagine-specific endoprotease AEP, the major enzymes involved in antigen processing in the MHC II-compartment. Surprisingly, lysosomal extracts from PB-DC1 were by far more effective than MO-DC in processing the muliple sclerosis-associated autoantigen myelin basic protein (MBP) in vitro. When analyzed on a molecular scale using mass spectrometry, MBP processing was dominated by CatS, CatD and AEP in MO-DC, as expected, similar to B-lymphoblastoid cells (BLC). PB-DC, however, did not generate proteolytic processing intermediates indicative of CatS or AEP activity but showed the same pattern as primary B-lymphocyte-derived lysosomes, i.e. processing was performed by two cleavage sites that can be reproduced by purified CatG in vitro, suggesting a CatG like dominant lysosomal protease. While active CatG was present in primary human B cells, PB-DC1 cells lacked CatG protein by western blot, suggesting the presence of an as yet unknown dominant endoprotease with CatG-like activity in PB-DC1. By cleaving MBP after pos F90 and F114, this protease directly eliminates the integrity of the major immunodominant MBP epitope MBP85-99. This might lead to poor presentation of this epitope to regulatory T cells resulting in inefficient silencing of MBP-autoreactive T cells during the development of autoimmunity. Our results emphasize the need to apply state-of-the-art biochemical tools to primary human types of APC for the understanding of antigen processing and the rational design of tolerogenic or immunotherapy approaches towards human malignant and autoimmune disorders.

A Randomized Trial to Evaluate the Impact of Cytokines on Dendritic Cell, T-Cell and T-Cell Function When Mobilizing Normal Donors for Allogeneic Progenitor Cell Transplant: An Interim Analysis.

Introduction:Optimal cellular immunity following allogeneic HPC transplant represents a balance between the induction of sufficient anti-tumor immunity to eradicate residual cancer cells without the induction of life-threatening GvHD. Dendritic cells are potent APCs with the ability to regulate immune responses. Our group has previously reported that increased numbers of donor DC2 result in inferior EFS following allo BMT (Waller et al, Blood 2001), and that myeloid cytokines used for mobilization modulate the DC content of the auto graft (Lonial et al, BBMT in press). The current trial was designed to evaluate the impact of different cytokine combinations on DC content and T-cell function in normal donors mobilized with either G-CSF or the combination of G-CSF + GM-CSF.Methods: 32 normal donors were randomized to mobilization with G-CSF (7.5 mcg/kg BID) or the combination of GM-CSF (7.5 mcg/kg qAM) + G-CSF (7.5 mcg/kg qPM) until completion of the stem cell collection. Side effects between the 2 regimens were documented using a questionnaire filled out by the donors within 2 weeks of stem cell collection. DC, T-cell, and other cell subsets were measured from the graft using flow cytometry. T-cell function was evaluated by measuring T-cell proliferation in response to PMA, Con A, PHA, and PWM. Cytokines (IL2, IL4, IL10,IL12, TNF, and INF) secreted in response to antigens were measured by ELISA. DC1 (myeloid DC) were defined as Lin-/HLA-DR+/CD11c+/CD123- while DC2 (lymphoid DC) were defined as Lin-/HLA-DR+/CD11c-/CD123+.Results: 28 patients have been successfully collected to date (G-CSF n=15, GM+G-CSF n=13). No donor has failed to mobilize in either group. Among the 15 donors mobilized with G-CSF alone, 5 required multiple days of apheresis as compared with 1 of 13 donors who received GM+G-CSF who required multiple days of apheresis (p=0.06). There was no difference in baseline values of T-cells or DC subsets in the peripheral blood prior to cytokine administration. Grafts collected with GM-CSF+ G-CSF contained significantly fewer DC2 cells and T-cells (median DC2 dose of 2.1 x 10E6/kg and CD3 dose of 197x 10E6/kg) compared with grafts from donors who received G-CSF alone (median DC2 dose of 3.8 x 10E6/kg (p=.01) and CD3 dose of 320 x 10E6/kg (p=0.001)). There was no difference in the content of CD34+ or DC1 in the grafts, nor in the ratio of CD4:CD8 T-cells between grafts collected with the 2 cytokine combinations. T-cell proliferation and cytokine secretion in response to mitogens was not different between grafts collected from the two groups. To date, there is no difference in the frequency of GvHD or relapse between the patients transplanted with the grafts collected from the 2 cytokine cohorts.Conclusions: The addition of GM-CSF to the mobilization regimen results in significantly fewer DC2 cells and T-cells in the blood HPC graft which could impact immune function and GvL following allogeneic HPC transplant. Clinical outcomes and further analysis of TH1/TH2 polarization of T-cells in grafts collected with either G-CSF or G-CSF+GM-CSF are in progress..

The Prognostic Importance of Pre and Post Dendritic Cells in Autologous Transplantation for Diffuse Large B Cell Lymphoma.

Pre and post transplant dendritic cell (DC) levels may correlate with survival, GVHD, and relapse in allogeneic BMT. Whether they have prognostic importance in autologous peripheral blood progenitor cell (PBPC) is unknown. We prospectively collected pre and post dendritic cell levels, including DC1 and DC2 levels in 53 patients with diffuse large B cell lymphoma (DLBCL) to investigate their potential prognostic importance. Pre-transplant analysis of DC1 and DC2 by flow cytometry was obtained 24 hours prior to the initiation of VP-16 (2 gm/m2) or G-CSF (10 mcg/kg/d) (n=4) for PBPC mobilization; post transplant samples were obtained 6 weeks after transplant. 51 of 53 (96%) were sensitive to chemotherapy at the time of transplant; 34% received prior radiation therapy; 15% had an elevated LDH at transplant. 49 of 53 received VP-16 + G-CSF for stem cell mobilization and 4 patients received G-CSF alone. 98% received a preparative regimen of Bu/Cy/VP and one patient received Busulfan alone. In a univariate analysis, higher numbers of pre-transplant DC1 cells and pre-transplant total DC (DC1 + DC2) cells correlated with improved survival, as shown graphically below: [Display omitted] Patients received a median CD34+ cell dose of 8.7 x 106/kg. DC values did not correlate with CD34+ cell yield. Post-transplant DC1 (p=0.03), DC2 (p=0.035), and post-transplant total DCs (p=0.04) also correlated with improved survival. In all cases, more DCs was associated with a better outcome. In a multivariable model, pre-transplant total DC number (p=0.028), and a normal LDH at the time of transplant (p=0.017) correlated with improved outcome. Median follow up of surviving patients is 15 months. Of 26 patients with pre-transplant total DC numbers greater than 9.10 x 106/mL, 77% of patients are alive, as compared to 56% of patients with pre-transplant total DC numbers less than 9.10 x 106/mL (n= 27)(p=0.022). The high DC group had fewer relapses and fewer late infections. We conclude that higher pre-transplant total DC numbers are associated with improved outcome in ABMT for DLBCL.

Dendritic cell expression of FcepsilonRI is regulated by serum IgE

Rationale Dendritic cells (DCs) are important in the initiation of Th1 and Th2 responses. In humans, the high affinity IgE receptor, FceRI, is expressed by type 1 DCs (DC1), whereas expression by DC2 cells has not been previously reported. We sought 1.) to characterize FceRI expression on DC subpopulations and 2.) to determine if DC expression of FceRI is regulated by serum IgE. Methods FceRI expression by DCs was determined by flow cytometry. Study A examined 21 non-allergic healthy controls and 23 allergic asthmatic subjects. Study B was a clinical trial of 24 subjects with ragweed allergic rhinitis who received omalizumab (anti-IgE) or placebo. Results Study A: FceRI was expressed by both DC1 and DC2 cells, and was found in both peripheral blood precursor DC (pDC) and mature tissue DC populations. FceRI expression by pDC1 and pDC2 cells was highly correlated to serum IgE and was significantly greater in subjects with allergic asthma compared to the non-atopic controls. Study B: Omalizumab treatment significantly decreased FceRI expression in both the pDC1 and pDC2 subsets (maximum decrease: 52% and 83%, respectively). The decrease in DC FceRI expression correlated with the drop in serum IgE. Conclusions FceRI is expressed by both the DC1 and DC2 subpopulations. The correlation of FceRI expression and serum IgE in Study A, and the drop in FceRI expression in Study B, support the conclusion that serum IgE is a major factor regulating DC expression of FceRI in vivo. These results suggest that FceRI expression by DC may be a useful therapeutic target.

Isolation and functional inhibitory affects of specific peptides bound to type 2 dendritic cells (DC2)

Introduction: The impact of immunoregulatory type 1 (DC1) and type 2 (DC2) dendritic cells in allogeneic hematopoetic progenitor cell (HPC) transplantation has increased in interest. We have shown that greater numbers of DC2s in allogeneic HPC transplanted grafts has resulted in an increase in post-transplant relapse and a reduction of graft vs host disease (GVHD). Isolation of dendritic cell specific peptides thru phage display technology provides a method of studying the immunoregulatory affects of these cells and perhaps engineer grafts with anti-tumor affects. Method: Random phage peptide libraries were incubated overnight at 4° C with column enriched peripheral blood DC2 cells. Unbound phage was removed by washing. FACS sorting of the Lin-, CD123+, HLA-Dr+ cells, further purified the DC2 population. DC2 bound phage were removed from the cell surface, eluted and purified. Three rounds of this phage selection process (panning) were performed. A portion of the round 2 phage purification was incubated with enriched monocytes prior to incubation with DC2 cells to assist in eliminating phage that recognized common epitopes on both cell types. Flow cytometry identified individual phage clones isolated from plaque assays that were highly DC2 selective. Selective clones were DNA sequenced revealing the random peptide insert. A 1-way MLR was performed with varying dilution of each amplified DC2 specific phage to demonstrate the cellular affects on allogeneic T-cell proliferation. Results: Plaque assays from the monocyte adsorbed or non-adsorbed linear random peptide library after the three rounds of panning revealed two consensus sequences in 76 of the 78 (97%) isolated clones that were DC2 selective and one sequence found twice (3%) that was non-DC2 selective. The employed circular random peptide library revealed no DC2 selective sequences from 15 isolated in clones from the monocyte adsorbed and non-adsorbed plaque assays. Preliminary MLR data shows a 35 % reduction in allogeneic T-cell proliferation with the DC2 specific phage compared to control phage. Conclusions: Data shows that phage display technology can result in isolating highly specific DC2 peptides from a library of 10,000 different phage clones. Preliminary data suggests that binding DC2 specific peptides may inhibit the function of these immunoregulatory cells, leading to enhanced anti-tumor affect of the transplanted donor graft product by shifting it towards an activated Th1 immune response.

Reversal of allergic airway inflammation and airway hyperresponsiveness by second-generation immunomodulatory oligonucleotide agents containing novel RpG motifs

The immunomodulatory oligonucleotide (IMO) agents containing novel RpG motif have been shown to potently modulate immune responses. Importantly, this novel motif has been shown to override species-specific receptor recognition observed with natural CpG motif. We have recently reported that second-generation IMOs containing a different stimulatory motif, CpR (R=2'-deoxy-7-deazaguanosine), effectively prevent key parameters of allergic asthma. Since most asthmatics are treated after the development of asthma, in the present study we examined whether the IMOs containing novel RpG motif can reverse established allergic airway inflammation. Mice sensitized and challenged with ovalbumin (OVA) were evaluated for airway hyperresponsiveness (AHR) to methacholine. Following this, sensitized mice were randomized and treated with placebo or two doses (30 μg and 60 μg/dose, s.c.) of RpG IMO during the following 10 days. Two days after the final treatment, mice were rechallenged with OVA and pulmonary functions were recorded. Mice treated with RpG IMO were significantly protected from both early (EAR) and late (LAR) allergic response, AHR to methacholine, BAL and peribronchial eosinophilia, BAL and serum IL-5, and total serum IgE as compared to vehicle-treated OVA-sensitized and challenged animals. There was a significant increase in immature lung dendritic cells (CD11c+CD45R+) together with increase in serum IL-10 levels and significant decrease in lung DC2 type cells (CD11c+ CD11b+CD8α-) following treatment with RpG IMO. These data suggest that the second-generation IMOs containing novel RpG motif are effective and potent in attenuating established allergic airway inflammation in bronchial asthma, and this effect could be mediated via decreasing lung DC2 cells and increasing immature DCs.

HYB 2055 – a second-generation immunomodulatory oligonucleotide containing CpR motifs potently reverses allergic airway inflammation and airway hyperresponsiveness in mouse models of established asthma

Rationale We have recently reported that second-generation immunomodulatory oligonucleotides (IMOs) containing CpR motifs prevent allergic asthma in mouse models. Since most asthmatics are treated after the development of asthma, we determined whether HYB 2055 can reverse established allergic airway inflammation in mouse models. Methods BALB/c mice sensitized and challenged with ovalbumin were evaluated for airway hyperresponsiveness to methacholine. Following ovalbumin sensitization, mice were randomized and treated with PBS or sc administration of HYB 2055 at two different dose levels during the following 10 days. Two days after the final treatment, mice were rechallenged with ovalbumin and pulmonary functions were recorded. Results HYB 2055 treatment significantly protected ovalbumin-sensitized mice from both early and late allergic response, airway hypersensitivity and hyperreactivity to methacholine compared with a control group of mice treated with PBS. A significant reduction in BAL eosinophilia, BAL and serum IL-5, and total serum IgE was observed in mice treated with HYB 2055 at both doses. Additionally, there was an increase in immature DCs (CD11c+CD45R+) with decrease in DC2 type cells (CD11c+ CD11b+CD8α-) in the lungs of the mice treated with HYB 2055 compared with PBS-treated control animals. Conclusions HYB 2055 is effective and potent in attenuating established allergic airway inflammation in bronchial asthma. The reversal of established asthma following HYB 2055 treatment could be as result of its ability to induce Th1 cytokine secretion, including IL-10, and its effect on decreasing lung DC2 cells and increasing immature DCs. Currently, HYB 2055 is under evaluation for its safety and immunopharmacology in healthy human volunteers.