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Keywords:

  • dendritic cells;
  • multiple myeloma;
  • interleukin-12;
  • transforming growth factor β;
  • immunotherapy

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Blood DC assay
  5. Up-regulation of CD80 and CD86 by huCD40LT
  6. Cytokine inhibition and neutralization studies
  7. Blood DC subsets
  8. Results
  9. Number of DC in blood of patients with myeloma
  10. Inhibition of CD80 up-regulation on DC is due to TGFβ1 and IL-10
  11. Correction of CD80 expression on DC by IL-12 and IFN-γ
  12. Effect of cytokines on DC subsets
  13. Discussion
  14. Acknowledgments
  15. References

The poor response to immunotherapy in patients with multiple myeloma (MM) indicates that a better understanding of any defects in the immune response in these patients is required before effective therapeutic strategies can be developed. Recently we reported that high potency (CMRF44+) dendritic cells (DC) in the peripheral blood of patients with MM failed to significantly up-regulate the expression of the B7 co-stimulatory molecules, CD80 and CD86, in response to an appropriate signal from soluble trimeric human CD40 ligand. This defect was caused by transforming growth factor β1 (TGFβ1) and interleukin (IL)-10, produced by malignant plasma cells, and the defect was neutralized in vitro with anti-TGFβ1. As this defect could impact on immunotherapeutic strategies and may be a major cause of the failure of recent trials, it was important to identify a more clinically useful agent that could correct the defect in vivo. In this study of 59 MM patients, the relative and absolute numbers of blood DC were only significantly decreased in patients with stage III disease and CD80 up-regulation was reduced in both stage I and stage III. It was demonstrated that both IL-12 and interferon-γ neutralized the failure to stimulate CD80 up-regulation by huCD40LT in vitro. IL-12 did not cause a change in the distribution of DC subsets that were predominantly myeloid (CD11c+ and CDw123−) suggesting that there would be a predominantly T-helper cell type response. The addition of IL-12 or interferon-γ to future immunotherapy trials involving these patients should be considered.

The numerous reports of idiotype vaccination in patients with lymphoma and multiple myeloma (MM) have been recently reviewed (Ruffini et al, 2002). Idiotype vaccination in patients with follicular lymphoma has resulted in many clinically significant responses with up to 73% of patients reported to achieve a molecular remission in one series (Bendandi et al, 1999). In contrast, the clinical response in patients with MM to immunotherapeutic strategies using idiotype has been modest and only minimal anti-tumour activity has been noted (Ruffini et al, 2002). This sub-optimal response in patients with MM suggests that both the cells and antigens involved with antigen presentation need to be investigated before new strategies are implemented.

Dendritic cells (DC) are highly specialized antigen-presenting cells, which play a critical role in the activation and potentiation of antigen-specific responses. Resting and immature DC capture and process soluble antigens in late endosomal and lysosomal compartments that are rich in major histocompatibility complex (MHC) molecules (Nijman et al, 1995; Kleijmeer et al, 1997). During migration and activation, maturing DC up-regulate the expression of peptide/MHC class I and II complexes that can be recognized by antigen-specific T cells. Many other cell types including B cells, monocytes and even tumour cells can present antigen but the potency and effectiveness of these cells as antigen-presenting cells is much less than DC (Egner et al, 1993; Fearnley et al, 1997). As most idiotype vaccination strategies have used relatively crude mononuclear cell preparations to present antigen, it is quite likely that the lack of a significant response to immunotherapy is related to, at least in part, the type of antigen-presenting cells used.

The ongoing studies of DC differentiation pathways and DC subsets have been accompanied by the development of a range of in vitro-generated DC populations and some problems with standardization of DC biology (Hart, 1997). In most previous in vitro studies, workers derived DC either from monocytes after 5-d culture with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4 followed by 2–3 d with tumour necrosis factor-α (TNFα) or else DC have been derived in culture from CD34+ cells. We studied peripheral blood DC on the basis that these may have more relevance to clinical applications. Blood DC quickly express CMRF44, an antigen found on high potency DC (Fearnley et al, 1997; Hart, 1997). In a previous study, we demonstrated that in patients with myeloma, high potency blood DC failed to up-regulate the expression of the co-stimulatory molecule CD80 in response to stimulation by soluble trimeric human CD40 ligand (huCD40LT) (Brown et al, 2001). It was shown by neutralization studies that the inhibition of CD80 up-regulation was due to transforming growth factor-β (TGF-β) and IL-10, produced by malignant plasma cells. Furthermore, when recombinant (r) TGF-β was added to normal DC, these also failed to up-regulate CD80 expression. As anti-TGF-β is not suitable for clinical use, we have sought another agent for use during immunotherapy to correct the functional defect in the DC of these patients without changing the distribution of DC subsets.

Blood DC assay

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Blood DC assay
  5. Up-regulation of CD80 and CD86 by huCD40LT
  6. Cytokine inhibition and neutralization studies
  7. Blood DC subsets
  8. Results
  9. Number of DC in blood of patients with myeloma
  10. Inhibition of CD80 up-regulation on DC is due to TGFβ1 and IL-10
  11. Correction of CD80 expression on DC by IL-12 and IFN-γ
  12. Effect of cytokines on DC subsets
  13. Discussion
  14. Acknowledgments
  15. References

Blood DC were enumerated in samples collected after informed consent using a previously described standardized assay (Fearnley et al, 1997; Hart, 1997; Brown et al, 2001). Briefly, the blood DC assay involved separating mononuclear cells from 5 ml of EDTA blood on ficoll-paque (Amersham Biosciences, Little Chalfont, UK), which were then cultured in Roswell Park Memorial Institute (RPMI) medium (ICN) with 10% fetal calf serum (FCS; CSL) for 24 h at 37°C in 7·5% CO2 prior to assay (Fearnley et al, 1997). Cells were harvested, mixed with anti-CMRF44 followed by anti-mouse Ig fluorescein isothiocyanate (FITC) (Chemicon) and then anti-CD19 phycoerythrin cyanin  5 (PECy5; Becton Dickinson) and anti-CD14 PECy5 (Becton Dickinson). The control tube contained anti-IgG1-PE (Becton Dickinson) and anti-CMRF-75, an isotype-matched control, stained with anti-mouse Ig-FITC (Chemicon). At least 100 000 propidium iodide (PI)-negative events were analysed on a Coulter EPICS XL flow cytometer (Beckman Coulter). Absolute numbers of DC were calculated using relative DC numbers in conjunction with absolute and differential leucocyte counts.

Up-regulation of CD80 and CD86 by huCD40LT

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Blood DC assay
  5. Up-regulation of CD80 and CD86 by huCD40LT
  6. Cytokine inhibition and neutralization studies
  7. Blood DC subsets
  8. Results
  9. Number of DC in blood of patients with myeloma
  10. Inhibition of CD80 up-regulation on DC is due to TGFβ1 and IL-10
  11. Correction of CD80 expression on DC by IL-12 and IFN-γ
  12. Effect of cytokines on DC subsets
  13. Discussion
  14. Acknowledgments
  15. References

Ficoll-paque separated blood mononuclear cells were cultured with or without human soluble trimeric CD40 ligand (huCD40LT, 5 μg/ml) and rIL-2 (0·1 μg/ml) (R&D Systems) in 1 ml cultures of RPMI containing 10% FCS (CSL) at 37°C in 7·5% CO2. After 24 h, cells were harvested and assayed for CMRF44 antigen expression on CD19, CD14, PI cells using a Coulter EPICS XL flow cytometer. Four colour staining with anti-CD80-PE (Becton Dickinson) or anti-CD86-PE (Becton Dickinson), anti-CD19-PE-Cy5 plus anti-CD14-PE-Cy5, CMRF44 (indirect FITC) on viable cells (PI) was performed to demonstrate the expression of CD80 and CD86 after culture with and without huCD40LT (R&D Systems) and rIL-2.

Cytokine inhibition and neutralization studies

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Blood DC assay
  5. Up-regulation of CD80 and CD86 by huCD40LT
  6. Cytokine inhibition and neutralization studies
  7. Blood DC subsets
  8. Results
  9. Number of DC in blood of patients with myeloma
  10. Inhibition of CD80 up-regulation on DC is due to TGFβ1 and IL-10
  11. Correction of CD80 expression on DC by IL-12 and IFN-γ
  12. Effect of cytokines on DC subsets
  13. Discussion
  14. Acknowledgments
  15. References

Cultures of peripheral blood cells were established (as per DC assay) with and without monoclonal anti-TGF-β1 (clone 9016), anti-IL-10 or anti-interferon-γ (IFN-γ; R&D Systems) at concentrations of 0·12–12 μg/ml. Other cultures were established with rIL-12 or IFN-γ (R&D Systems) at concentrations between 1 ng/ml and 1 μg/ml. CD80 expression on DC was assayed after 24 h as described above.

Blood DC subsets

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Blood DC assay
  5. Up-regulation of CD80 and CD86 by huCD40LT
  6. Cytokine inhibition and neutralization studies
  7. Blood DC subsets
  8. Results
  9. Number of DC in blood of patients with myeloma
  10. Inhibition of CD80 up-regulation on DC is due to TGFβ1 and IL-10
  11. Correction of CD80 expression on DC by IL-12 and IFN-γ
  12. Effect of cytokines on DC subsets
  13. Discussion
  14. Acknowledgments
  15. References

Mononuclear cells were incubated with 10 ng/ml rIL-12 (R&D Systems). Cells were stained with anti-CMRF-44 followed by sheep anti-mouse Ig FITC (Chemicon), anti-CD14 PECy5 and anti-CD19 PeCy5 (Immunotech) as well as either anti-CD11c PE (Pharmingen) to detect DC1 or anti-CDw123 PE (Pharmingen) to detect DC2. At least 50 000 PI-negative events were analysed.

Number of DC in blood of patients with myeloma

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Blood DC assay
  5. Up-regulation of CD80 and CD86 by huCD40LT
  6. Cytokine inhibition and neutralization studies
  7. Blood DC subsets
  8. Results
  9. Number of DC in blood of patients with myeloma
  10. Inhibition of CD80 up-regulation on DC is due to TGFβ1 and IL-10
  11. Correction of CD80 expression on DC by IL-12 and IFN-γ
  12. Effect of cytokines on DC subsets
  13. Discussion
  14. Acknowledgments
  15. References

The assay for blood DC (Fig 1A) using anti-CMRF44 expression on CD19 CD14 cells has been standardized and several laboratories have established similar normal ranges (D. Hart, personal communication). In the current study, there was no significant difference between either the mean number of blood DC in all patients with MM (n = 59) or patients in stage I disease (Durie & Salmon, 1975) and a normal control group (n = 13). However, there was a significant decrease in the mean of the relative (t = 2·1; P < 0·05) (Fig 2A) and absolute (t = 2·6; P < 0·02) (Fig 2B) number of blood DC in patients in stage III disease (n = 16) compared with the normal control group. The mean absolute blood DC numbers were 5·0 × 106/l for normal, 5·1 × 106/l for stage I MM, 4·9 × 106/l for stage II MM and 2·3 × 106/l for stage III.

image

Figure 1. Blood dendritic cell (DC) assay and the up-regulation of CD80 expression on DC from a representative patient with multiple myeloma stage I. (A) Blood DCs are assayed as CMRF44+, CD19, CD14 and PI cells. (B) Isotype control for CD80 expression on DC. (C) CD80 expression on DC after 24 h incubation. (D) Up-regulation of CD80 expression on DC after 24 h incubation with huCD40LT and IL-2.

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image

Figure 2. The relative (A) and absolute (B) blood dendritic cell (DC) counts in patients with myeloma (n = 59) at different stages of their disease compared with a normal control group. NS, not significant.

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image

Figure 3. Up-regulation of CD80 on dendritic cell (DC). CD80 expression on DC from patients with myeloma (n = 50) and a normal control group is shown both with and without stimulation with huCD40LT. NS, not significant.

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Inhibition of CD80 up-regulation on DC is due to TGFβ1 and IL-10

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Blood DC assay
  5. Up-regulation of CD80 and CD86 by huCD40LT
  6. Cytokine inhibition and neutralization studies
  7. Blood DC subsets
  8. Results
  9. Number of DC in blood of patients with myeloma
  10. Inhibition of CD80 up-regulation on DC is due to TGFβ1 and IL-10
  11. Correction of CD80 expression on DC by IL-12 and IFN-γ
  12. Effect of cytokines on DC subsets
  13. Discussion
  14. Acknowledgments
  15. References

We previously reported (Brown et al, 2001) that the up-regulation of CD80 on the DC of patients with MM after hCD40LT stimulation (Fig 1) was impaired and that this is could be neutralized in vitro by antibodies to TGFβ1 and IL-10. Ongoing studies, now involving 50 patients have confirmed the inability of huCD40LT to adequately stimulate the up-regulation of CD80 expression on the blood DC of patients with MM (Fig 3). Stimulation with huCD40LT significantly increased CD80 expression on DC in all normal control blood samples (t = 6·3; P < 0·003). Up-regulation of the surface expression of CD80 on DC of patients with MM was variable but was often either decreased or absent on the DC of patients in stage I MM and was even more compromised in patients with stage III MM. Indeed there was no significant increase in mean CD80 expression on DC after huCD40LT stimulation in the patients in stage III (t = 1·7; P = n.s.). Mean CD80 expression on normal DC after huCD40LT stimulation (67%) was significantly greater than on DC of both stage I (32%) and stage III patients (36%) (P < 0·001).

Correction of CD80 expression on DC by IL-12 and IFN-γ

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Blood DC assay
  5. Up-regulation of CD80 and CD86 by huCD40LT
  6. Cytokine inhibition and neutralization studies
  7. Blood DC subsets
  8. Results
  9. Number of DC in blood of patients with myeloma
  10. Inhibition of CD80 up-regulation on DC is due to TGFβ1 and IL-10
  11. Correction of CD80 expression on DC by IL-12 and IFN-γ
  12. Effect of cytokines on DC subsets
  13. Discussion
  14. Acknowledgments
  15. References

Previously we established that either anti-TGFβ1 or anti-IL-10 could neutralize the inhibition of CD80 up-regulation (Brown et al, 2001). Following a search for other agents that could restore CD80 up-regulation on blood DC, it was found that the addition of either IL-12 or IFN-γ at a concentration of 10 ng/ml or greater could restore the up-regulation of CD80 as effectively as anti-TGFβ (Fig 4). Preliminary studies using different concentrations of IL-12 and IFN-γ were performed on DC from seven patients. Although there was heterogeneity in the responses between patients, a representative patient is shown in Fig 4(A). It was decided that 10 ng/ml was a suitable concentration. The ability of either 10 ng/ml IL-12 or IFN-γ to up-regulate CD80 expression on the DC of a total of 15 patients was studied. Although there was some heterogeneity in the response between patients and also to different cytokine concentrations, there was no significant difference in the mean CD80 expression on DC after incubation with either IL-12 or IFN-γ. Fig 4(B) illustrates the results from three patients in stage III who failed to up-regulate CD80 in response to huCD40LT.

image

Figure 4. Neutralization of the inhibition of CD80 up-regulation on patient with multiple myeloma in stage III. (A) Dose response curves for CD80 expression after 24 h stimulation with 10, 100 and 1000 ng/l of interleukin-12 (IL-12) and interferon-γ (IFN-γ) added to huCD40LT compared with huCD40LT alone, αTGF-β and control. Data are from a single patient in stage III. (B) Data from three representative patients, whose DC failed to up-regulate CD80 expression, is shown. Anti-TGFβ, IL-12 (10 ng/ml) and IFN-γ (10 ng/ml) at least partly corrected the defect.

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Effect of cytokines on DC subsets

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Blood DC assay
  5. Up-regulation of CD80 and CD86 by huCD40LT
  6. Cytokine inhibition and neutralization studies
  7. Blood DC subsets
  8. Results
  9. Number of DC in blood of patients with myeloma
  10. Inhibition of CD80 up-regulation on DC is due to TGFβ1 and IL-10
  11. Correction of CD80 expression on DC by IL-12 and IFN-γ
  12. Effect of cytokines on DC subsets
  13. Discussion
  14. Acknowledgments
  15. References

CD11c and CDw123hi expression was determined on CMRF44+ blood DC of eight patients both with and without IL-12 stimulation to determine if IL-12 had an effect on DC subset distribution. There was no significant difference in the mean percentage of CD11c+ DC with (62%) or without IL-12 (62%). The percentage of CDw123hi DC was also not significantly different with (16%) or without (10%) IL-12. Thus, IL-12 had no significant effect on the distribution of DC subsets.

Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Blood DC assay
  5. Up-regulation of CD80 and CD86 by huCD40LT
  6. Cytokine inhibition and neutralization studies
  7. Blood DC subsets
  8. Results
  9. Number of DC in blood of patients with myeloma
  10. Inhibition of CD80 up-regulation on DC is due to TGFβ1 and IL-10
  11. Correction of CD80 expression on DC by IL-12 and IFN-γ
  12. Effect of cytokines on DC subsets
  13. Discussion
  14. Acknowledgments
  15. References

The poor response of patients with MM to regular vaccination procedures was first demonstrated 50 years ago (Zinneman & Hall, 1954). More recently, it has been shown that viral antigen-specific CD8+ T-cell responses are impaired in patients with MM (Maecker et al, 2003) and that protective titres against influenza, Streptococcus pneumoniae and Haemophilus influenzae type B are achieved in only 19% of patients with MM (Robertson et al, 2000). Other studies have demonstrated a reversible defect in the natural killer cells of patients with MM (Dhodapkar et al, 2003). Early murine studies suggested that the poor immune response was due to a defect in antigen-presenting cells (Joshua et al, 1979) and more recently a defect in the DC of patients with MM has been defined by our group and others (Brown et al, 2001; Ratta et al, 2002). Thus, the poor response to idiotype vaccination and other active immunotherapy strategies in these patients (Ruffini et al, 2002) should have been expected. It is surprising that there have not been strategies developed to adequately address the defect in antigen presentation in these patients. The current study presented a clinically relevant solution to overcome a defect in the DC of patients with MM.

Expression of the B7 molecules, CD80 and CD86, on antigen-presenting cells provides an important ‘second signal’ that determines the fate of each T cell – apoptosis, anergy or productive immunity. During DC maturation prior to antigen presentation, DC up-regulate the expression of the B7 molecules in response to an appropriate signal. Two early studies failed to detect any defects in the DC of patients with MM (Pfeiffer et al, 1997; Raje et al, 1999). Subsequent studies (Brown et al, 2001; Ratta et al, 2002) demonstrated that there is a defect in the DC of these patients, which is related to the expression of the B7 co-stimulatory factors. It has been reported that 4-1BBL/4-1BB interaction enhances IL-12 mRNA expression (Laderach et al, 2003) and provides co-stimulatory signals to T cells independent of CD80 signalling through the CD28 receptor and that 4-1BBL is inducible by CD40 (DeBenedette et al, 1997). It is not known whether 4-1BB/4-1BBL signalling is impaired in MM. Another report (Xie et al, 2003) suggested that β-2-microglobulin levels of greater than 10 μg/ml retard the generation of DC in patients with MM; however, more than 90% of the patients in this study had β-2-microglobulin levels of less than 8 mg/l. There was no relationship between β-2-microglobulin levels and the failure to up-regulate CD80 on DC and there is no reason why the cytokines used would have an effect on β-2-microglobulin. We demonstrated that the DC of patients with MM failed to up-regulate the expression of CD80 and CD86 due to TGFβ1 and IL-10 secreted by malignant plasma cells (Brown et al, 2001). The failure of previous studies (Pfeiffer et al, 1997; Raje et al, 1999) to detect functional defects in DC of patients with MM is probably related to the fact that these studies usually cultured the cells in vitro for at least 7 d in the presence of high concentrations of various cytokines prior to their evaluation, while in our studies (Brown et al, 2001) DC were studied within 24 h of collection and more closely represents the in vivo situation.

We sought to identify a clinically available biological modifier that, like anti-TGFβ, could restore the up-regulation of CD80 expression on DC. Both IL-12 and IFN-γ meet these criteria, however, IL-12 appears to be more consistent (Fig 4). Unlike granulocyte colony stimulating factor (Barrow et al, 2003; Vuckovic et al, 2003), IL-12 did not appear to alter the DC1:DC2 ratio, which could change the response from a Th1 to a Th2 response. Although this agent had considerable toxicity in early studies, the immunomodulatory potential of IL-12 has been harnessed without the in vivo-associated toxicity by ex vivo exposure of cells (Emtage et al, 2003). This latter strategy could easily be applied to DC prepared for immunotherapy.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Blood DC assay
  5. Up-regulation of CD80 and CD86 by huCD40LT
  6. Cytokine inhibition and neutralization studies
  7. Blood DC subsets
  8. Results
  9. Number of DC in blood of patients with myeloma
  10. Inhibition of CD80 up-regulation on DC is due to TGFβ1 and IL-10
  11. Correction of CD80 expression on DC by IL-12 and IFN-γ
  12. Effect of cytokines on DC subsets
  13. Discussion
  14. Acknowledgments
  15. References
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