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IMMUNOBIOLOGY
From the Deparment of Oncology and Immunology,
Newcastle Mater Hospital, Australia; and Section of Chemistry,
Faculty of Medicine, University of Brescia, Italy.
Heavy chain ferritin (H-ferritin) is a component of the
iron-binding protein, ferritin. We have previously shown that
H-ferritin inhibits anti-CD3-stimulated lymphocyte proliferation and
that this was due to increased production of interleukin-10 (IL-10). In
the present study we have shown that induction of IL-10 production was
due to effects of H-ferritin on adherent antigen-presenting cells
(APCs) in blood and monocyte-derived dendritic cells (MoDCs). IL-10 was
produced by a subpopulation of CD4 T cells, which expressed the CD25
component of the IL-2 receptor and the CTLA-4 receptor characteristic
of regulatory T cells. The changes induced in MoDCs were compared with
those induced by CD40L and their significance tested by inhibition with
monoclonal antibodies. These studies indicated that H-ferritin induced
relatively greater expression of CD86 and B7-H1 on MoDCs and that
monoclonal antibodies against their receptors, CTLA-4 and programmed
death receptor-1 (PD-1), inhibited IL-10 production from the regulatory
T cells. H-ferritin did not appear to induce direct production
of the cytokines IL-2, IL-4, IL-6, IL-10, IL-12, or interferon- The growth of melanoma, especially in its more
advanced stages, can be associated with suppression of immune responses
to the tumor and to other antigens.1,2 Induction of anergy
to melanoma antigens due to lack of costimulatory molecules or
production of reactive oxygen metabolites is one possible
mechanism for such changes.3 In addition, a number of
soluble factors produced by melanoma cells have been described that
down-regulate immune responses via direct effects on lymphocytes or on
antigen-presenting cells (APCs) in the vicinity of the tumor (reviewed
by Hersey4). Such factors include vascular endothelial
growth factor,5,6 interleukin-10
(IL-10),7 and transforming growth factor- We have recently reported that heavy chain ferritin (H-ferritin)
appears to be another factor released by melanoma cells that may
suppress immune responses by its ability to induce IL-10 production in
lymphocytes.10 Ferritin is a major tissue iron-binding
protein11 and in its native form has a molecular weight of
approximately 500 kd. It is composed of 24 subunits consisting of
acid/heavy (H) and basic/light (L) chains.12,13 The genes
encoding H-ferritin and light chain ferritin (L-ferritin) are found in
different chromosomes and are transcriptionally
independent.14 The 24-subunit polymer may form
isoferritins, which are either more acidic (H-rich) or more basic
(L-rich) depending on the relative proportions of H and L chains.
Liver and spleen ferritins are basic, because they are made up mainly
of light chains and very few heavy chains. In contrast, heart, kidney,
and placental ferritins are highly acidic, because they are composed
mostly of heavy chains.15 The ferritin found in cancer
cells was found to consist mainly of heavy chains.16,17
Marked variability was found in the ratios of H- to L-ferritin in
supernatants from melanoma cultures. Some melanoma cells released
predominantly H-ferritin, whereas others contained more equal
proportions of H- and L-ferritin.10 The immunosuppressive effects of the supernatants correlated with their content of
H-ferritin. These findings were consistent with previous reports that
H-ferritin could suppress proliferation of T cells18-20 and
E rosette formation.21 In our previous studies it was
found that monoclonal antibodies (mAbs) against H-ferritin or against
IL-10 could reverse the immunosuppressive effects. The latter was
produced from lymphocytes exposed to H-ferritin and was associated with
small decreases in IL-2 and IL-4 production. Interferon- In the present study we have examined the mechanism by which H-ferritin
induces IL-10 production and the cell types in blood responsible for
the production of IL-10. We report that H-ferritin appears to mediate
its effects on lymphocytes by action on adherent cells and dendritic
cells (DCs) generated from monocytes. The lymphocytes producing IL-10
appear to have the characteristics of regulatory T cells.
Antibodies and recombinant proteins
Preparation of T-cell populations
Generation of DCs DCs were generated as described by Romani et al,28 with minor modifications. Briefly, lymphocyte-depleted peripheral blood mononuclear cells (PBMCs) were cultured in DMEM containing 10% FCS (CSL Biosciences). GM-CSF and IL-4 were added at a final concentration of 800 and 500 U/mL, respectively. Cultures were fed every other day (days 2, 4, and 6). On day 7, nonadherent cells were either harvested or transferred to a 24-well flat-bottom plate (Falcon, Becton Dickinson, Franklin Lakes, NJ) and cultured further with GM-CSF and IL-4 in the presence or absence of H-ferritin, L-ferritin, or CD40L for a further 24 to 48 hours.Proliferation assay Proliferation assays were set up as described by Gray et al.10 Briefly, 1 × 105 nonadherent PBLs were cultured with adherent PBMCs or DCs in 200 µL DMEM containing 10% FCS (CSL Biosciences) in a 96-well U-bottom plate (Falcon, Becton Dickinson). H-ferritin, L-ferritin, or CD40L was added to the culture at 1 µg/mL along with 50 ng/mL anti-CD3. Cultures were then incubated in a 37°C incubator supplemented with 5% CO2 for 72 hours for proliferation studies or 24 hours for cytokine expression studies. For studies on the effect of purified CD25+ cells on lymphocyte proliferation, the purified subpopulations were cultured for 24 hours with H-ferritin, L-ferritin, or CD40L-pretreated DCs at a CD25+ to DC ratio of 10. The CD25+ subpopulation was then harvested and subsequently treated with 25 µg/mL mitomycin-C (Kyowa Hakko Kogyo, Tokyo, Japan) at 37°C for 1 hour, before addition to the proliferation assay. For blocking studies, mAbs against B7-H1, PD-1, CTLA-4, or CD28 were prebound to the plate with 10 µg/mL anti-CD3 in carbonate buffer (0.1 M NaHCO3, pH 8.2) overnight at 4°C. For proliferation studies, 2 µCi (74 KBq) of [125I]UdR (5-[125I]Iodo-2'-deoxy uridine) (Amersham, Aylesbury, United Kingdom) was added for the final 4 hours before cell harvesting. Incorporation of [125I]UdR was then measured on a CompuGamma CS gamma counter (Wallac, Oy, Finland). The degree of inhibition of anti-CD3 stimulation was calculated as previously described elsewhere.29Mixed lymphocyte response The mixed lymphocyte response (MLR) was set up as described by Romani et al,28 with minor modifications. Briefly, serial dilutions (1 × 104 to 1 cells per well) of DCs were cultured in triplicate with 1 × 105 allogeneic nonadherent PBLs in 200 µL DMEM containing 10% FCS (CSL Biosciences) in a 96-well U-bottom plate (Falcon, Becton Dickinson). Cultures were then incubated in a 37°C incubator supplemented with 5% CO2 for 5 days. Then, 2 µCi (74 KBq) of [125I]UdR (Amersham) was added for the final 8 hours before cell harvesting. Incorporation of [125I]UdR was then measured on a CompuGamma CS gamma counter (Wallac). The results are expressed as the mean of triplicate cultures.Flow cytometry Analysis was carried out using a Becton Dickinson (Mountain View, CA) FACScan flow cytometer. Appropriate concentrations of mAbs were added to the cells in 100 µL PBS containing 1% human serum and incubated for 30 minutes at 4°C. Cells were washed twice with PBS and analyzed. For indirect labeling, cells were incubated with F(ab')2 fragment affinity-isolated phycoerythrin- or fluorescein isothiocyanate-conjugated sheep antimouse immunoglobulin (Silenus, Amrad Biotech, Boronia, Australia) plus 1% human serum for 30 minutes at 4°C. Cells were then washed once in PBS before analysis. A minimum of 5000 cells was analyzed by flow cytometry. The percentage of positive cells was calculated as the difference in positive area between the positive and negative control histograms. The positive area was that to the right of the intersection of the 2 curves.30IL-6 bioassay The IL-6 bioassay was set up as described by Lu and Kerbel,31 with minor modifications. In brief, IL-6-dependent B9.9 murine cells were cultured in 100 µL DMEM containing 10% FCS (CSL Biosciences) in a 96-well U-bottom plate (Falcon, Becton Dickinson) at 5 × 103 cells per well, with or without recombinant IL-6 or supernatant collected at 24 hours from anti-CD3 proliferation assay. Cultures were then incubated in a 37°C incubator supplemented with 5% CO2 for 3 days. Then, 2 µCi (74 KBq) of [125I]UdR (Amersham) was added for the final 8 hours before cell harvesting. Incorporation of [125I]UdR was then measured on a CompuGamma CS gamma counter (Wallac). The IL-6 concentration in the supernatants was determined by comparing its proliferative activity to a known IL-6 standard. The results are expressed as the mean of triplicate cultures.Assay for IL-2, IL-4, IL-10, TGF-  1, IL-12p70, and IFN- were then assayed using a
biotinylated enzyme-linked immunosorbent assay (ELISA) system (PharMingen) according to the manufacturer's instructions for each cytokine.
Kinetics of IL-10 production by H-ferritin We have previously reported that H-ferritin induced a significant increase in IL-10 and IFN- production in anti-CD3-stimulated lymphocytes, which is consistent with a regulatory T-cell
response.10 Kinetic studies have demonstrated that
regulatory T cells produce IL-10 more rapidly than TH0, TH1, or TH2
cells.32 The kinetics of IL-10 production induced by
H-ferritin in anti-CD3-stimulated PBLs is shown in Figure
1. IL-10 was detectable by ELISA within 6 to 8 hours and peaked at 48 hours. IFN- was detectable by 14 hours
and peaked at 48 hours.
H-ferritin induces IL-10 production and inhibition of lymphocyte proliferation by effects on adherent cells/DCs from blood To determine which cell type was responsible for the effect of H-ferritin on lymphocyte proliferation and IL-10 production, we depleted PBLs from a normal subject of adherent cells and pretreated the populations with H-ferritin or L-ferritin at 1 µg/mL for 24 hours. As shown in Figure 2, pretreatment of the adherent and nonadherent cell populations separately had no significant effect on anti-CD3-stimulated proliferation at 3 days or IL-10 production at 24 hours, but when the pretreated adherent population was added to the nonadherent PBLs there was increased production of IL-10 and inhibition of lymphocyte proliferation induced by anti-CD3. Addition of pretreated nonadherent cells to the adherent cells did not affect their production of IL-10 or lymphocyte proliferation.
These experiments were repeated with DCs produced by culture of
monocytes in IL-4 and GM-CSF for 7 days. The results shown in Figure
3 indicated that addition of DCs
pretreated with H-ferritin to nonadherent PBLs was associated with
increased production of IL-10 and IFN-
IL-10 is produced predominantly by CD4 T cells To determine which population of lymphocytes produced the IL-10 and IFN-, DCs were added to unseparated PBLs and purified populations of CD4 and CD8 T cells. The CD4 populations were 95% pure
and the CD8 populations 94% pure. The results shown in Figure 4 again showed that pretreatment with
H-ferritin of nonadherent PBLs and CD4 and CD8 populations of T cells
had no direct effects on IL-10 production but did increase IFN-
production from nonadherent and CD8 T-cell populations. However, when
H-ferritin-pretreated DCs were added to the lymphoid populations it
was apparent that CD4 T cells were mainly responsible for the
production of IL-10, whereas IFN- was produced mainly by the CD8
T cells.
IL-10 is produced by CD25+ T cells, whereas
IFN-  populations. The CD25+ population was
83% pure. These were then added to H-ferritin-pretreated DCs. The
CD25+ population was 84.7% CD4+, 5.5%
CD8+, and 9.7% CD4CD8 T cells
(Table 1). The CD25+ cells
also expressed CTLA-4 and were CD45RO+ and
PD-1+. The results in Figure
5 show that the CD25+
population was mainly responsible for production of IL-10, whereas the
CD25 population produced IFN-. IFN- production from
the CD25 population exceeded that from the nonadherent
population, suggesting that the CD25+ cells may have been
inhibitory for IFN- production.
H-ferritin induces maturation of DCs that are phenotypically and functionally different than mature DCs induced by CD40L The results above suggested that H-ferritin induced changes in APCs, which preferentially induced induction of regulatory T cells. We examined whether these changes may have been reflected in expression of regulatory molecules on DCs and compared the results with phenotypic changes in DCs induced by CD40L. Pretreatment of DCs at 6 days for 24 hours with H-ferritin or CD40L was associated with similar clumping of the DCs. The phenotypes of these DCs are shown in Figure 6 (donor 1) and Table 2. Both H-ferritin and CD40L induced an activated mature DC phenotype, as shown by increased expression of CD83, but relative to the changes induced by CD40L, H-ferritin increased expression of CD86 and B7-H1. These results were repeated in subsequent experiments (donors 2 and 3) shown in Table 2. Statistical analysis on the median fluorescence intensity for all 3 donors found that H-ferritin significantly increased CD86 (paired F test, P = .021) and B7-H1 (P = .019). In contrast, the increase in expression induced by CD40L was not statistically significant for CD86 (P = .172) or B7-H1 (P = .091).
IL-10 production induced by H-ferritin-treated DCs is dependent on interaction with CTLA-4 and PD-1 on T cells, and B7-H1 ligand on adherent PBMCs To determine whether these changes in expression of CD86 or B7-H1 may be important in induction of regulatory T cells, we carried out blocking studies with mAbs against these ligands and their receptors (CD28/CTLA-4 for CD86) and the PD-1 for B7-H1. As shown in Figure 7, mAb against the ligand B7-H1 and its receptor, PD-1 (but not the isotype control), inhibited the production of IL-10 induced by DCs pretreated with H-ferritin. Similarly, mAb against CTLA-4 inhibited IL-10 production, whereas mAb against CD28 increased IL-10 production. The latter was consistent with the known costimulatory function of CD28.
H-ferritin pretreatment of DCs induces a different cytokine profile in nonadherent lymphocytes than CD40L-pretreated DCs We next examined whether H-ferritin may induce or suppress the production of cytokines that may be involved in induction of regulatory T cells and again compared the results to those induced by CD40L. Studies on nonadherent PBLs (Figure 8A) showed (as expected) that H-ferritin did not induce IL-10 production but did induce IFN- production. There was also the expected small
reduction in IL-2 and IL-4 production. In addition, production of IL-6
was markedly reduced, whereas IL-12 production was increased. These effects reflected direct effects of H-ferritin on the PBLs. In contrast, CD40L had no direct effects on the nonadherent PBLs except
for a small increase in IFN- production.
When DCs pretreated with H-ferritin were added to the nonadherent PBLs (Figure 8B) at a ratio of 10:1, the main change in cytokine production was the increase in IL-10 production and, to a lesser extent, IL-2 production. The direct effects on cytokine production from nonadherent PBLs noted in Figure 8A, such as decreased production of IL-4 and IL-6 and increased IL-12 production, was again seen. (Note: H-ferritin added to unseparated PBLs slightly inhibited IL-2 production, as noted previously.) As shown in Figure 8C, H-ferritin had no detectable direct effects on cytokine production from the DCs alone. This contrasted with the effect of CD40L, which increased IL-12, IL-10, and IL-6 production from the DCs. These results argue against the possibility that induction of regulatory T cells was due to H-ferritin-induced changes in cytokine production from DCs, but we cannot exclude that cytokines other than those measured may have been important. H-ferritin pretreatment of DCs stimulates a different response in T cells to CD40L-pretreated DCs To further assess the function of H-ferritin-treated DCs, they were used as stimulators in an MLR. As shown in Figure 9A, H-ferritin-treated DCs were poor stimulators of MLRs compared with CD40L-treated DCs.
We tested whether H-ferritin-treated DCs would increase the suppressive activity of CD25 T regulatory cells by adding purified CD25+ T cells to DCs pretreated with H-ferritin, CD40L, or L-ferritin for 24 hours. The CD25+ T cells were then added to cultures of anti-CD3-stimulated PBLs and their effect on proliferation measured at 3 days. As shown in Figure 9B, exposure to H-ferritin-treated DCs resulted in approximately a 2-fold increase in their ability to suppress lymphocyte proliferation.
The above studies confirm that recombinant H-ferritin induces production of IL-10 from PBLs and that this is responsible for inhibition of anti-CD3-stimulated lymphocyte proliferation.10 The present studies extend these findings by showing that the induction of IL-10 production appears to be due to effects of H-ferritin on adherent cells in blood and monocyte-derived DCs produced from blood. These effects appeared specific for H-ferritin and were not due to contaminants such as endotoxins in the preparation. The latter could not be detected in the preparation of H-ferritin used in the study, and similar effects were not seen with recombinant L-ferritin produced by similar methods. Other sources of nonrecombinant H-ferritin, such as extracts of heart and melanoma cell supernatants, had similar effects as recombinant H-ferritin.10 They further show that the source of IL-10 appeared to be a particular subpopulation of CD4 T cells in blood that constitutively express CD25 and CTLA-4. The latter finding was of much interest because much attention has been
given to a subpopulation of CD4+ T cells that
constitutively express the CD25 component of the IL-2 receptor and the
CTLA-4 T-cell activation receptor for CD80 (B7.1) and CD86
(B7.2).33-37 These cells were shown to suppress autoimmune
disease in several animal models38,39 and facilitated survival of transplants40-42 The mode of action of regulatory T cells is controversial. Suppression
of autoimmune inflammatory bowel disease in mice by regulatory T cells
appeared to be mediated by IL-10 and could not be inhibited by
regulatory T cells from IL-10 knock-out mice.44 Human
self-major histocompatibility complex (MHC)-reactive cells were shown
to have suppressor activity against specific CD4 T cells and
immunoglobulin production when cultured with non-T cells stimulated with pokeweed mitogen (PWM) or CD40L. The effects
were mediated by IL-10 and/or TGF- The processes initiating the induction of regulatory T cells are
largely unknown. Interaction of high-affinity thymocytes with
self-peptides was shown to induce CD4+CD25+
regulatory T cells and appeared distinct from the process of positive
selection or deletion.50 Other factors include chronic antigenic stimulation in mice transgenic for influenza
hemagglutinin,42 chronic exposure to
superantigen,51 exposure to immature DCs,52 and culture of cord blood T cells in IL-10 and IFN- It was examined whether H-ferritin may induce a particular DC phenotype that would explain its ability to induce regulatory T cells. Comparison with DCs exposed to CD40L suggested that there was a relatively greater increase in expression of CD86 (B7.2) and B7-H1 on the H-ferritin-stimulated DCs. These changes appeared important in that mAbs against the CTLA-4 receptor for CD86 and PD-1 for B7-H1 inhibited production of IL-10 from the T cells stimulated with H-ferritin-stimulated DCs. In contrast, mAbs against CD28 resulted in increased production of IL-10. A number of previous studies have shown that activation through the CTLA-4 receptor is needed for induction of regulatory T cells.36,37 The B7-H1 molecule was reported to costimulate T-cell proliferation and to preferentially stimulate IL-10 production.22,54,55 The receptor for B7-H1 appears to be the PD-1 molecule.56,57 Additional ligands for PD-1 have also been recently described,58 but whether H-ferritin selectively up-regulates these ligands has not been studied. We were unable to detect direct effects of H-ferritin on cytokine
production from DCs, which argued against induction of regulatory T
cells by changes in cytokine production by treatment of DCs with
H-ferritin. In contrast, CD40L was shown to increase production of
IL-6, IL-12, and IL-10 by DCs. These results do not, however, exclude a
role for other cytokines not measured in these studies. H-ferritin also
had direct effects on cytokine production by nonadherent PBLs, such as
increases in IFN- On the basis of these results we postulate that production of
H-ferritin by melanoma cells or other cancer cells may induce down-regulation of immune responses by action on APCs, particularly in
areas adjacent to or draining the site of the tumor. Regulatory T cells
generated at these sites could then be expected to suppress CD4+CD25
We thank Raj Ishri from the Department of Medicine (Dermatology), University of Sydney, for assistance in production of DCs. We also thank Mrs. C. Cook for expert secretarial assistance.
Submitted October 1, 2001; accepted December 27, 2001.
Supported by the Hunter Melanoma Foundation and Melanoma and Skin Cancer Institute, New South Wales, Australia.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Peter Hersey, Oncology & Immunology Unit, Rm 443, David Maddison Clinical Sciences Bldg, Cnr King & Watt Streets, Newcastle, NSW 2300 Australia; e-mail: peter.hersey{at}newcastle.edu.au.
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Top
Abstract
Introduction
hence their description as
regulatory T cells. Certain regulatory T cells were reported to also
produce IFN-
.
molecules and inhibits tumor-specific T cell- and natural killer cell-mediated cytotoxicity.
Eur J Immunol.
1996;26:1308-1313